Reverse Transcriptase PCR Amplification Research Articles

Saliva as an Alternative Specimen for Molecular COVID-19 Testing in Community Settings and Population-Based Screening.

PurposeWith the easing of restriction measures, repeated community-based sampling for tracking new COVID-19 infections is anticipated for the next 6 to 12 months. A non-invasive, self-collected specimen like saliva will be useful for such public health surveillance. Investigations on the use of saliva for SARS-CoV-2 RT-PCR have largely been among COVID-19 in-pa\ ients and symptomatic ambulatory patients with limited work in a community-based screening setting. This study was carried out to address this paucity of data and reported discrepancies in diagnostic accuracy for saliva samples.Patients and MethodsFrom 29th June to 14th July 2020, adults presenting for COVID-19 testing at a community-based screening facility in Dubai, United Arab Emirates were recruited. Clinical data, nasopharyngeal swab in universal transport media and drooling saliva in sterile containers were obtained. Reverse transcriptase PCR amplification of SARS-CoV-2 RdRp and N genes was used to detect the presence of the SARS-CoV-2 virus.ResultsOf the 401 participants, 35 (8.7%) had viral detection in at least one specimen type and the majority (n=20/35; 57.1%) were asymptomatic. Both swab and saliva were positive in 19 (54.2%) patients, while 7 (20.0%) patients had swab positive/saliva negative results. There were 9 (25.7%) patients with saliva positive/swab negative result and this included 5 asymptomatic COVID-19 patients undergoing repeat screening. Using the swab as the reference gold standard, the sensitivity and specificity of saliva were 73.1% (95% CI 52.2–88.4%) and 97.6% (95% CI 95.5–98.9%) while the positive and negative predictive values were 67.9% (95% CI 51.5–80.8%) and 98.1% (95% CI 96.5–99.0%), respectively.ConclusionThe findings suggest good diagnostic accuracy for saliva and feasibility of utilization of specimen without transport media for SARS-CoV-2 RT-PCR. Saliva represents a potential specimen of choice in community settings and population-based screening.

Molecular cloning, tissue expression and polymorphism analysis of the Caveolin-3 gene in ducks

ABSTRACT 1. Duck meat is considered a delicacy, but choosing the best meat is problematic. Caveolin-3 (CAV-3) is a muscle-specific protein marker in animals. The goal of the current study was to detect the characteristics of CAV-3 gene in ducks. 2. Full-length CAV-3 was acquired from ducks (Anas platyrhynchos) using reverse transcriptase PCR and rapid amplification of cDNA ends. DNAMAN software was used for homology comparisons. Quantitative reverse transcription-PCR, PCR-single-strand conformation polymorphism, and sequencing were used to determine CAV-3 expression and polymorphism of a single nucleotide, respectively. The study examined four types of ducks, including Jinding, Chaohu, Cherry Valley, and Gaoyou ducks. 3. The study acquired 1066 bp of CAV-3 cDNA sequences, including a 456 bp complete open reading frame encoding 151 amino acids. Both coding sequences (CDSs) and translated amino acids exhibited highest homology with Gallus gallus (CDS homology 91.67%, amino acids 94.04%), followed by mammalian species (CDS homology 79.0%, amino acids 78.0%). Single nucleotide polymorphism analysis revealed five mutations in exons (A489G, G501A, A557G, T563A, and A602G), and a C805T mutation in an intron. Among amplified polymorphic loci detected using primer 2, allele frequency was higher for A (489A501G507A563T602A) than B (489G501G507G563T602C) or C (489G501A507G563A602C). The highest occurred in Cherry Valley ducks (0.7587). Using primer 4, the M allele frequency was higher than that of the N allele. CAV-3 was most highly expressed in the heart, followed by skeletal muscles. Additionally, CAV-3 had higher expression in heart and breast muscle of overfed Muscovy ducks than control ducks, but no difference was seen in thigh muscle. 4. CAV-3 in ducks had the highest homology with Gallus gallus CAV-3, and it could be used as a marker for muscle quality in ducks.

Abstract 1243: Effect of antigen retrieval on immuno-based RNA microdissection methods

Abstract Introduction: Clinical applications and research studies routinely utilize immunohistochemistry (IHC) approaches on tissue sections. The aim is to selectively target antigens (proteins) in the cells of a tissue section by exploiting the principle of antibody binding, thus enabling target specificity. The antigen retrieval (AR) process employed at the beginning of most IHC protocols increases epitope availability and improves staining characteristics; however, the procedure can damage nucleic acids, in this case RNA, to an unknown extent. Experimental Procedures: To better understand the effects of AR on RNA quality and quantity, model system samples (lung cancer patient derived xenograft tissue) were subjected to a commonly employed AR method [heat induced epitope retrieval (HIER)] and the effects on RNA were assessed by Qubit, Fragment Analyzer, and digital droplet PCR (ddPCR). The ddPCR experiments were performed following reverse transcriptase (RT) product generation and construction of cDNA, which then served as input to the ddPCR assay. Absolute quantitation of gene expression levels and expressed mutations were evaluated by ddPCR. Data Summary: The results showed that HIER resulted in optimal staining characteristics, but induced significant damage to RNA, producing extensive fragmentation and decreased yields. In general, fragmentation showed an inflection point at ~200 nt transcript length. Variation in the HIER protocol mitigated but did not eliminate the negative effects. Apart from the AR procedure, the IHC process itself also resulted in a significant decreased yield of RNA. Of note, in these experiments DAB chromogen was used exclusively in the IHC process. However, in spite of the observed deleterious effects on RNA, none of the AR methods combined with IHC negatively affected RT product generation and PCR amplification of small amplicons. Regarding the detection of RT product under different conditions, the size of the amplicon was found to make a difference, with the detection of longer amplicons being more difficult. In addition to a number of successful human and mouse gene expression measurements, KRAS gene mutations were successfully identified in the clinical cases under all conditions. Conclusions: The data indicate that RNA recovered from histology slides after standard AR and IHC processing can be successfully employed for genomic applications utilizing RT product formation (cDNA) for gene expression and detection of expressed mutations, especially if the detection methods are based on relatively short length nucleic acids. Studies that require larger RNA fragments such as long-read sequencing should avoid the use of HIER. Citation Format: Donald J. Johann, Meeiyueh Liu, Adam Roberge, Sarah Laun, Michael Emmert-Buck, Michael Tangrea. Effect of antigen retrieval on immuno-based RNA microdissection methods [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1243.

Molecular characterization and tissue expression profile of the Dnmts gene family in pig

DNA methyltransferases (Dnmts) comprise a family of proteins which involved in the establishment and maintenance of DNA methylation patterns. In pig, the molecular characterization and tissue expression profile of Dnmt gene family are not clear. To solve this problem, reverse transcriptase PCR and rapid amplification of cDNA ends were used to clone the sequences of the porcine Dnmt2 and Dnmt3b genes. Furthermore, the mRNA expression profiles of Dnmt1, Dnmt2, Dnmt3a and Dnmt3b genes from 54 adult tissues and 2 entire fetuses of Rongchang pig were analyzed by quantitative real-time PCR (qRT-PCR). As a result, the lengths of porcine Dnmt2 and Dnmt3b gene cDNAs were 1 227 and 2 559 bp with cytosine-C5 specific DNA methylase domain, respectively. The four Dnmt genes were highly expressed in longissimus dorsi muscle (P<0.01). Dnmt1 is highly expressed in heart (P<0.01) and Dnmt 2 shows its preference in liver and seminal vesicle tissue (P<0.01). Dnmt3a and Dnmt3b are highly expressed in the two fetus stages (P<0.01). All these results suggested that each gene has its specific expression profile, and deeper study is required to dig more details between the methylation level and Dnmt family mRNA expressions in different tissues.

Identification and Characterization of the Diverse Stress-Responsive R2R3-RMYB Transcription Factor from Hibiscus sabdariffa L.

Various regulatory proteins play a fundamental role to manage the healthy plant growth under stress conditions. Differential display reverse transcriptase PCR and random amplification of cDNA ends (RACE) was used to explore the osmotic stress-responsive transcripts. We identified and characterized the salt stress-responsive R2R3 type RMYB transcription factor from Hibiscus sabdariffa which has an open reading frame of 690 bp, encoding 229 long chain amino acids. In silico analysis confirmed the conserved R2 and R3 domain as well as an NLS-1 localization site. The deduced amino acids of RMYB shared 83, 81, 80, 79, 72, 71, and 66% homology with Arabidopsis thaliana, Glycine max, Oryza sativa, Zea maize, Malus domestica, Populus tremula × Populus alba, and Medicago sativa specific MYB family, respectively. We observed the gene upregulation in stem, leaf, and root tissue in response to abiotic stress. Furthermore, RMYB gene was cloned into plant expression vector under CaMV35S promoter and transformed to Gossypium hirsutum: a local cotton cultivar. Overexpression of RMYB was observed in transgenic plants under abiotic stresses which further suggests its regulatory role in response to stressful conditions. The RMYB transcription factor-overexpressing in transgenic cotton plants may be used as potential agent for the development of stress tolerant crop cultivars.

Improved detection of artifactual viral minority variants in high-throughput sequencing data.

High-throughput sequencing (HTS) of viral samples provides important information on the presence of viral minority variants. However, detection and accurate quantification is limited by the capacity to distinguish biological from artificial variation. In this study, errors related to the Illumina HiSeq2000 library generation and HTS process were investigated by determining minority variant frequencies in an influenza A/WSN/1933(H1N1) virus reverse-genetics plasmid pool. Errors related to amplification and sequencing were determined using the same plasmid pool, by generation of infectious virus using reverse genetics followed by in duplo reverse-transcriptase PCR (RT-PCR) amplification and HTS in the same sequence run. Results showed that after “best practice” quality control (QC), within the plasmid pool, one minority variant with a frequency >0.5% was identified, while 84 and 139 were identified in the RT-PCR amplified samples, indicating RT-PCR amplification artificially increased variation. Detailed analysis showed that artifactual minority variants could be identified by two major technical characteristics: their predominant presence in a single read orientation and uneven distribution of mismatches over the length of the reads. We demonstrate that by addition of two QC steps 95% of the artifactual minority variants could be identified. When our analysis approach was applied to three clinical samples 68% of the initially identified minority variants were identified as artifacts. Our study clearly demonstrated that, without additional QC steps, overestimation of viral minority variants is very likely to occur, mainly as a consequence of the required RT-PCR amplification step. The improved ability to detect and correct for artifactual minority variants, increases data resolution and could aid both past and future studies incorporating HTS. The source code has been made available through Sourceforge (https://sourceforge.net/projects/mva-ngs).

Phylogenetic Characterization of a Novel Insect-Specific Flavivirus Detected in a Culex Pool, Collected from Assam, India

Objective: We report the phylogenetic characterization of a unique flavivirus sequence detected in a wild Culex tritaeniorhynchus mosquito pool, collected from the northeast Indian state of Assam. Methods: DNA and RNA were extracted from field-collected mosquito pools. Extracts were subjected to PCR and reverse transcriptase PCR amplification using universal and type-specific primers for direct detection of flavivirus-specific viral nucleic acids. An amplified flavivirus nonstructural protein 5 (NS5) genetic region was sequenced and BLAST searched, and phylogenetic analyses performed with reference sequences retrieved from GenBank. Results: Phylogenetic analyses revealed the sequence to be related to insect-specific flaviviruses (ISFs) of the genus Flavivirus, family Flaviviridae. Despite being related to the Palm Creek virus (PCV; an ISF very recently reported from Northern Australia), the present sequence (provisionally named Assam virus) was found to be highly divergent from PCV and other ISF sequences available in GenBank. The partial NS5 sequence analysis demonstrated low nucleotide sequence identity (66-77%) with known ISFs reported from other parts of the globe. Conclusion: Findings of this study suggest the presence of a candidate novel ISF - the first to be reported from India.

Rotavirus G2P[4] Detection in Fresh Vegetables and Oysters in Mexico City

Rotaviruses are the principal cause of dehydration caused by diarrhea in children younger than 2 years of age. Although these viral infections have mainly been associated with ingestion of fecally contaminated food and water, few studies have addressed the presence of the virus in food that is consumed raw or slightly cooked. In this work, 30 oyster samples and 33 vegetable samples were examined for the presence of rotavirus genotypes to evaluate their potential to produce gastrointestinal infections. The rotaviruses were identified by reverse transcriptase PCR amplification of the VP7 gene. G and P genotyping was also performed by reverse transcriptase PCR, with a detection sensitivity of up to 15 PFU/ml. Rotaviruses were found in 17 (26.9%) of 63 samples (10 oysters and 7 vegetables). The G2 genotype was found in 11 (64.7%) of 17 of the rotavirus strains, and 16 (94.1%) of 17 had the P[4] genotype. The combined genotypes found most frequently were G2P[4] (10 [58.82%] of 17), GNTP[4] (6 [35.29%] of 17), and G2P[NT] (1 [5.8%] of 17).

Cloning and expression characteristics of the pig Stra8 gene.

Stra8 (Stimulated by Retinoic Acid 8) is considered a meiotic gatekeeper gene. Using reverse transcriptase PCR and rapid amplification of cDNA ends (RACE), the complete sequence of the pig Stra8 gene was cloned. Bioinformatics analyses of this sequence were performed. Using semi-quantitative methods, the expression characteristics of Stra8 in Testis, cauda epididymis, body epididymis, caput epididymis, seminal vesicles, prostate gland, Cowper’s gland, heart, liver, spleen, lung, kidney, stomach, hypothalamus, pituitary gland, cerebrum, cerebellum, and hippocampus of adult Meishan boar and sow tissues were examined. The expression pattern in the testis of 2-, 30-, 60-, 90-, and 150-day old Meishan boars were analyzed using real-time PCR. We constructed a eukaryotic expression vector for the Stra8 gene and used it to transfect NIH-3T3 cells and third generation pig spermatogonial stem cells (SSCs) cultured in vitro. Testes weight and sperm count in the cauda epididymis were evaluated at various time points. The results showed that the length of the pig Stra8 gene cDNA was 1444 bp encoding 366 amino acids with one typical helix-loop-helix (HLH) domain. It is testes-specific expression. Expression was first detected in boar testis starting at day 2, and its expression significantly (p < 0.05) increased with age and body weight. When NIH-3T3 cells and pig SSCs were transfected with the eukaryotic expression vector EGFP (enhanced green fluorescent protein)-N1-pStra8, it was expressed in the cytoplasm of NIH-3T3 cells. However, in SSCs, Stra8 was expressed predominantly in cytoplasm and few in nucleus. Our data suggest that perhaps Stra8 acts as a transcription factor to initiate meiosis in young boar.

Molecular Identification and Expressive Characterization of an Olfactory Co-Receptor Gene in the Asian Honeybee,Apis cerana cerana

Olfaction recognition process is extraordinarily complex in insects, and the olfactory receptors play an important function in the process. In this paper, a highly conserved olfactory co-receptor gene, AcerOr2 (ortholog to the Drosophila melanogaster Or83b), cloned from the antennae of the Asian honeybee, Apis cerana cerana Fabricius (Hymenoptera: Apidae), using reverse transcriptase PCR and rapid amplification of cDNA ends. The full-length sequence of the gene was 1763 bp long, and the cDNA open reading frame encoded 478 amino acid residues, including 7 putative transmembrane domains. Alignment analysis revealed that AcerOr2 shares high homology (> 74%) with similar olfactory receptors found in other Hymenoptera species. The amino acid identity with the closely related species Apis mellifera reached 99.8%. The developmental expression analysis using quantitative real-time reverse transcriptase PCR suggested that the AcerOr2 transcript was expressed at a relatively low level in the larval stage, whereas it was expressed broadly in the pupal and adult stages, with a significantly high level on the days just before and after eclosion. In situ hybridization showed that AcerOr2 mRNA was expressed in sensilla placodea and on the basal region of the worker antennal cuticle, in accordance with the previous conclusions that the conserved genes are expressed in most olfactory receptor neurons.

Lix1 Knockout Mouse Does Not Exhibit Spinal Muscular Atrophy Phenotype

Feline spinal muscular atrophy (SMA) is an autosomal recessive juvenile onset lower motor neuron disease caused by an ∼ 140 kb deletion that disrupts expression of 2 genes, limb expression 1 (LIX1) and leucyl/cystinyl aminopeptidase (LNPEP). A previously generated Lnpep knockout (KO) mouse did not demonstrate a neuromuscular phenotype. Little is known about LIX1, except that it is evolutionarily conserved and highly expressed in spinal cord motor neurons. To determine whether loss of LIX1 alone is responsible for the feline SMA phenotype, a Lix1 intron 1 gene trap KO mouse line was obtained from Lexicon Genetics, Inc. Mating of F(1) heterozygotes produced offspring in the expected Mendelian ratios. KO and normal littermates were studied through 2 years of age by hanging latency, rotarod, inked footprint analysis, and histological methods. Disruption of Lix1 expression did not affect survival nor result in any neuromuscular phenotype. Reverse transcriptase-PCR amplification of spinal cord RNA identified a Lix1 alternative transcript beginning in intron 4 and containing exons 5 and 6. The alternative transcript appeared to be rodent specific, and its expression was not disrupted in Lix1 KO mice. Expression of the alternative transcript may have compensated for the loss of Lix1 in the KO mice and thus protected against motor neuron degeneration.

Comparison of extraction techniques for amniotic fluid supernatant demonstrates improved yield of cell‐free fetal RNA

Comparison of extraction techniques for amniotic fluid supernatant demonstrates improved yield of cell‐free fetal RNA

NS1 Detection in Addition to Reverse Transcriptase PCR and Transcription-Mediated Amplification of Dengue Virus RNA in Acutely Ill Patients

We comment on the potential utility of transcription-mediated amplification (TMA) of dengue virus (DENV) RNA in acutely ill patients between 1 and 5 days of the appearance of symptoms. The superiority of TMA to reverse transcriptase PCR (RT-PCR) ([1][1]) would be evident not only in a comparison

Studying bacteria in respiratory specimens by using conventional and molecular microbiological approaches

BackgroundDrawing from previous studies, the traditional routine diagnostic microbiology evaluation of samples from chronic respiratory conditions may provide an incomplete picture of the bacteria present in airways disease. Here, the aim was to determine the extent to which routine diagnostic microbiology gave a different assessment of the species present in sputa when analysed by using culture-independent assessment.MethodsSix different media used in routine diagnostic microbiology were inoculated with sputum from twelve patients. Bacterial growth on these plates was harvested and both RNA and DNA extracted. DNA and RNA were also extracted directly from the same sample of sputum. All nucleic acids served as templates for PCR and reverse transcriptase-PCR amplification of "broad range" bacterial 16S rRNA gene regions. The regions amplified were separated by Terminal Restriction Fragment Length Polymorphism (T-RFLP) profiling and compared to assess the degree of overlap between approaches.ResultsA mean of 16.3 (SD 10.0) separate T-RF band lengths in the profiles from each sputum sample by Direct Molecular Analysis, with a mean of 8.8 (SD 5.8) resolved by DNA profiling and 13.3 (SD 8.0) resolved by RNA profiling. In comparison, 8.8 (SD 4.4) T-RF bands were resolved in profiles generated by Culture-derived Molecular Analysis. There were a total of 184 instances of T-RF bands detected in the direct sputum profiles but not in the corresponding culture-derived profiles, representing 83 different T-RF band lengths. Amongst these were fifteen instances where the T-RF band represented more than 10% of the total band volume (with a mean value of 23.6%). Eight different T-RF band lengths were resolved as the dominant band in profiles generated directly from sputum. Of these, only three were detected in profiles generated from the corresponding set of cultures.ConclusionDue to their focus on isolation of a small group of recognised pathogens, the use of culture-dependent methods to analyse samples from chronic respiratory infections can provide a restricted understanding of the bacterial species present. The use of a culture-independent molecular approach here identifies that there are many bacterial species in samples from CF and COPD patients that may be clinically relevant.

The CB 2 cannabinoid receptor regulates human sperm cell motility

To analyze the expression and distribution of cannabinoid receptors in human sperm cells and evaluate the effects of activation of receptors by specific agonists and antagonists, with a special emphasis on the CB(2) receptor. We performed expression assays for CB(1) and CB(2) by reverse transcriptase PCR, Western blot, and immunofluorescence techniques in spermatozoa and performed motility analysis after incubation of semen samples with cannabinoid agonists and CB(2) antagonist SR144528. Academic research laboratory. Semen from 50 normozoospermic, healthy human donors. Spermatozoa isolated from semen by two consecutive swim-ups were used for all techniques. Reverse transcriptase PCR amplification gels, immunoblots, indirect immunofluorescence antibody assays, and percentage of motile sperm. We have verified the presence of CB(1) and CB(2) receptors in human spermatozoa. The distribution of both of these receptors was distinct. Incubation with selective cannabinoid receptor agonists induced a significant reduction in the proportion of rapidly progressive motile spermatozoa, and whereas the CB(1) agonist increased the proportion of immobile sperm cells, the CB(2) receptor agonist increased the slow/sluggish progressive sperm cell population. The effect of the CB(2) agonist was antagonized by the CB(2)-specific antagonist. The functional CB(2) cannabinoid receptor is present in human spermatozoa and regulates the sperm motility in a more distinct manner than CB(1).

Human Endogenous Retrovirus K (HML-2) Elements in the Plasma of People with Lymphoma and Breast Cancer

Actively replicating endogenous retroviruses entered the human genome millions of years ago and became a stable part of the inherited genetic material. They subsequently acquired multiple mutations, leading to the assumption that these viruses no longer replicate. However, certain human tumor cell lines have been shown to release endogenous retroviral particles. Here we show that RNA from human endogenous retrovirus K (HERV-K) (HML-2), a relatively recent entrant into the human genome, can be found in very high titers in the plasma of patients with lymphomas and breast cancer as measured by either reverse transcriptase PCR or nucleic acid sequence-based amplification. Further, these titers drop dramatically with cancer treatment. We also demonstrate the presence of reverse transcriptase and viral RNA in plasma fractions that contain both immature and correctly processed HERV-K (HML-2) Gag and envelope proteins. Finally, using immunoelectron microscopy, we show the presence of HERV-K (HML-2) virus-like particles in the plasma of lymphoma patients. Taken together, these findings demonstrate that elements of the endogenous retrovirus HERV-K (HML-2) can be found in the blood of modern-day humans with certain cancers.

Targeted Skipping of a Single Exon Harboring a Premature Termination Codon Mutation: Implications and Potential for Gene Correction Therapy for Selective Dystrophic Epidermolysis Bullosa Patients

This study examined the feasibility of antisense oligoribonucleotide (AON) therapy for dystrophic epidermolysis bullosa (DEB). AON was designed to induce skipping of a targeted exon containing a premature termination codon mutation, resulting in restoration of the open reading frame. We targeted exon 70 of COL7A1, as a recurrent mutation 5818delC in Japanese DEB patients was localized to exon 70. We found that one AON induced effective skipping of normal exon 70 containing 16 amino acids. Attachment and migration analyses showed that recombinant collagen without contribution of exon 70 was similar in effect to normal type VII collagen. Next, we synthesized mutation-specific AON by deleting cytosine at 5818. Introduction of this AON into DEB keratinocytes harboring 5818delC showed that the AON induced skipping of exon 70 in the abnormal 5818delC allele. Furthermore, 6.2% of DEB keratinocytes started to express type VII collagen in vitro after application of the mutation-specific AON. Injection of the AON into rat model grafted with DEB keratinocytes and fibroblasts induced a low amount of type VII collagen expression. We conclude that skipping of targeted exons using mutation-specific AON may show potential for future gene therapy for DEB patients.

Detection of common and private mutations in the COL6A1 gene of patients with Bethlem myopathy

Dominant mutations in COL6A1, COL6A2, and COL6A3, the three genes encoding collagen type VI, a ubiquitous extracellular matrix protein, are associated with Bethlem myopathy (BM) and Ullrich scleroatonic muscular dystrophy. The authors devised a method to screen the entire coding sequence of the three genes by reverse transcriptase-PCR amplification of total RNA from skin fibroblasts and direct sequencing of the resulting 25 overlapping cDNA fragments covering 107 exons. Four splicing and four missense mutations were identified in 16 patients with BM, six of which are novel mutations in COL6A1. Both common and private mutations are localized in the alpha1 (VI) chain between the regions corresponding to the 3' end of the NH2-globular domain and the 5' end of the triple helix, encoded by exons 3 through 14. The clustering of the mutations in a relatively narrow area of the three collagen type VI chains in patients with Bethlem myopathy (BM) suggests that mutations in different regions could result in different phenotypes or in no phenotype at all. Moreover, the detection of mutations in only 60% of the patients suggests the existence of at least another gene associated with BM. The authors propose the direct sequencing of COL6 cDNAs as the first mutation screening analysis in BM, given the high number of exon-skipping events.

Receptor Activity-modifying Protein (RAMP) Isoform-specific Regulation of Adrenomedullin Receptor Trafficking by NHERF-1

Receptor activity-modifying proteins (RAMPs 1-3) are single transmembrane accessory proteins critical to various G-protein coupled receptors for plasma membrane expression and receptor phenotype. A functional receptor for the vasodilatory ligand, adrenomedullin (AM), is comprised of RAMP2 or RAMP3 and calcitonin receptor-like receptor (CRLR). It is now known that RAMP3 protein-protein interactions regulate the recycling of the AM2 receptor. The major aim of this study was to identify other interaction partners of RAMP3 and determine their role in CRLR-RAMP3 trafficking. Trafficking of G-protein-coupled receptors has been shown to be regulated by the Na+/H+ exchanger regulatory factor-1 (NHERF-1), an adaptor protein containing two tandem PSD-95/Discs-large/ZO-1 homology (PDZ) domains. In HEK 293T cells expressing the AM2 receptor, the complex undergoes agonist-induced desensitization and internalization. However, in the presence of NHERF-1, although the AM receptor (CRLR/RAMP3) undergoes desensitization, the internalization of the receptor complex is blocked. Overlay assays and mutational analysis indicated that RAMP3 and NHERF-1 interact via a PDZ type I domain on NHERF-1. The internalization of the CRLR-RAMP complex was not affected by NHERF-1 when CRLR was co-expressed with RAMP1 or RAMP2. Mutation of the ezrin/radixin/moesin (ERM) domain on NHERF-1 indicated that NHERF-1 inhibits CRLR/RAMP3 complex internalization by tethering the complex to the actin cytoskeleton. When examined in a primary culture of human proximal tubule cells endogenously expressing the CRLR-RAMP3 complex and NHERF-1, the CRLR-RAMP complex desensitizes but is unable to internalize upon agonist stimulation. Knock-down of either RAMP3 or NHERF-1 by RNA interference technology enabled agonist-induced internalization of the CRLR-RAMP complex. These results, using both endogenous and overexpressed cellular models, indicate a novel function for NHERF-1 and RAMP3 in the internalization of the AM receptor and suggest additional regulatory mechanisms for receptor trafficking.

Sequence analysis of murine leukemia virus envelope gene from inoculated mice

Introduction of amino acids substitutions in murine leukemia virus genome is a powerful method to determine the relative importance of various viral factors in pathogenesis. However, introduction of such amino acids substitution could result in viruses at a selective disadvantage, and eventual selection of revertants. It is thus essential to verify if the mutation is maintained stably in replicating virus and in infected tumor cells. In the present study, viral nucleic acid sequences from diseased animals were determined using different approaches. Small blood samples were found adequate for direct RNA extraction and reverse transcriptase-PCR amplification followed by automated DNA sequencing. Alternatively, replication-competent viruses were recovered specifically by applying blood samples onto permissive cells; viral RNA is then extracted from tissue culture medium and similarly sequenced. Tissue samples were also used to amplify viral sequences from tumors DNA while small pieces of tumors tissues were applied onto permissive cells to isolate replicating viruses. The combined experimental approach was used to show sequence conservation using a mutant altered in the intracytoplasmic region of viral envelope glycoprotein. No difference was observed between viruses recovered directly from the animal and those amplified onto cultured cells.