cDNA Fragments Research Articles

Deciphering Oxidative Stress Responsive Microrna (Mirna) in Hilsa (Tenualosa ilisha) and Rohu (Labeo rohita)

microRNAs (miRNAs) and their target gene expression under oxidative stress play a crucial role in cellular antioxidant regulation. Information on oxidative stress responsive miRNA and their target genes of the fishes of Bangladesh is not reported yet. This study was performed to profile oxidative stress-responsive miRNAs by computational and experimental methods in economically important fish of Bangladesh. Using in silico approach, we could not trace any miRNA of fish of Bangladesh as none has been reported yet in existing databases. We, therefore enlist here the miRNAs that are expressed under different stress conditions in fish applying an extensive literature review. From the list, we selected 10 potential oxidative stressresponsive miRNAs followed by predicting their target genes using miRNA target prediction software (TargetScan Fish). This study decoded the mature sequences of oxidative stress-responsive miRNA (miR-21) in Hilsa (Tenualosa ilisha) and Rohu (Labeo rohita) through specific miRNA primer-based cDNA fragment sequencing. Next, we identified an oxidative stress responsive gene, programmed cell death 4b (pdcd4b) in hilsa genome and showed that the hilsa miR-21 binds within coding sequence region of the predicted hilsa pdcd4b. This study is pioneer in decoding oxidative stress responsive miRNA in fish of Bangladesh using an experimental and bioinformatics approach.

Characterization of the GABAergic system in Asian clam Corbicula fluminea: Phylogenetic analysis, tissue distribution, and response to the aquatic contaminant carbamazepine

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter involved in the neuro-endocrine-immune (NEI) system. In this study, we sequenced the partial length of cDNA fragments of three genes involved in GABA neurotransmitter system of the Asian clam (Corbicula fluminea) (GABAA receptor-associated protein (GABARAP), GABARAPL2 and GABA transporter (GAT-1)). These genes exhibited high amino acid sequence identity compared with other invertebrate orthologs. Expression patterns of the three genes were determined in mantle, gill, gonad, digestive gland and muscle, and the steady state levels of mRNA for each were determined to be highest in gonad and lowest in muscle. To determine their regulation by pharmaceuticals that are present as contaminants in waterways, clams were exposed to carbamazepine (CBZ) for 30 days. CBZ is an agonist for GABA receptors and is an anticonvulsant pharmaceutical that is often detected in aquatic ecosystems. GABARAP and GABARAPL2 mRNA levels were significantly downregulated by 5 and 50 μg/L CBZ in mantle and gill (p < 0.05), while in the gonad and digestive gland, steady state levels (p < 0.05) were decreased with exposure to all three doses. GAT-1 mRNA was upregulated by CBZ (p < 0.05) in the mantle and gill at all three doses tested and in the gonad and digestive system with 5 and 50 μg/L. These data suggest that CBZ disrupt the expression of the GABAergic neurotransmitter system in C. fluminea. Moreover, GABARAP, GABARAPL2 and GAT-1 may be useful biomarkers for the screening of substances that are hazardous to the NEI system of mollusks.

Isolation and Expression Analysis of a WRKY Transcription Factor in Sweet potato

A new WRKY transcription factor gene was isolated by ESTs screening from a cDNA library of suspension cultured cells of Sweet potato (Ipomoea batatas). The 2,285 bp cDNA fragment, IbWRKY, was sequenced, from which a 505 amino acid residue protein was deduced. A search of the protein BLAST database identified significant similarity to other plant WRKY31 protein sequences. RT-PCR analysis showed expression patterns of IbWRKY31 in various intact tissues and suspension cultured cells of Sweet potato, and in leaves exposed to different stresses. The IbWRKY31 gene was highly expressed in suspension cultured cells. In leaf tissues, IbWRKY31 showed strong expression during salicylic acid and methyl jasmonate treatments. Expression of IbWRKY31 was also induced under various abiotic stress and pathogen infection conditions, such as wounding, H2O2, MV, PEG, NaCl, and bacterial pathogen infection. These results suggest that IbWRKY31 is involved in plant responses to various stress conditions, such as abiotic stresses and pathogen infection through a defense signaling pathway.

A novel protein upstream stimulatory factor 2 identified in lamprey, Lethenteron reissneri.

Upstream stimulatory factors are kinds of multi-functional transcription factors, which are expressed in eukaryotes widely, including Upstream stimulatory factor 1 (USFl) and upstream stimulatory factor 2 (USF2). USF protein has a typical basic helix-loop-helix leucine zipper (b-HLH-LZ) structure, which is involved in cell cycle, cell proliferations, glucose and lipid metabolism, and other biochemical processes. Although the USF family is an important regulator of cellular processes, little is known about the USF genes of lampreys, especially their evolutionary relationships, expression profiles, and biological functions. Here, an upstream stimulatory factor 2 (USF2) homolog from lamprey (Lethenteron reissneri) was identified and characterized (designated as L-USF2) because it is closer to USF2 subfamily than to USF1 subfamily. The cDNA fragment of L-USF2 has an open reading frame (ORF) of 765-bp length, encodes 254 amino acids, and contains an HLH domain at the c-terminal of amino acids. Meanwhile, motifs and genetic structure analysis reveal that USF2 gene exons are conserved. Moreover, the 3D structure analysis indicates that L-USF2 adopts the general USF2 folding and has a high structural similarity with H-USF2. The synteny results showed that the L-USF2 adjacent gene changed greatly compared with the jaw vertebrates. By real-time quantitative experiment and Western blot analysis, we found that L-USF2 gene played a significant role in the immune responses. This study not only provides us with a further understanding of the evolution and function of the USF gene family but also provides a basis for exploring its immune responses and immune defenses in lampreys.

Dual Energy Transfer-Based DNA/Graphene Oxide Nanocomplex Probe for Highly Robust and Accurate Monitoring of Apoptosis-Related microRNAs.

Fluorescent labeled single-stranded DNA (ssDNA) molecules physisorbed on graphene oxide (GO) have been extensively explored as a useful sensing platform. However, this approach faces challenges when applied to complex biological samples due to heavy nonspecific desorption of nontarget molecules from GO. To overcome this problem, we introduced a capture DNA (cDNA) fragment with a poly adenine (poly-A) extension into the physisorption system that greatly reduces nonspecific desorption and false positive signal due to strong binding between poly-A and GO. Fluorescence from the dye can be effectively quenched by BHQ, which thus provides a second guarantee of anti-interference to avoid possible nonspecific poly-A DNA displacement. As a proof of concept, we have successfully developed a novel DNA-adsorbing GO nanocomplex probe (DNA-GO nanocomplex probe). This probe has a high anti-interference capability and low background due to the presence of both GO and black hole quencher (BHQ) as a dual-quencher that reduces the background in live cell imaging due to resonance energy transfer (RET). We then employed the DNA-GO nanocomplex probe for simultaneous detection of miR-630 and miR-21 and also for simultaneous in situ dynamic monitoring of intracellular miR-630 and miR-21 in apoptotic cells. We discovered that miR-630 expression was up-regulated during the first 120 min. This simple but powerful protocol has great potential in precise detection and imaging of various substances in complex biological samples with improved accuracy.

The discovery of dystrophin, the protein product of the Duchenne muscular dystrophy gene.

Duchenne muscular dystrophy was a well-established medical and genetic enigma by the 1970s. Why was the new mutation rate so high in all world populations? Why were affected boys doing well in early childhood, but then showed relentless progression of muscle wasting? What was wrong with the muscle? The identification of the first fragments of DMD gene cDNA in 1986, prediction of the entire 3685 amino acid protein sequence, and production of antibodies to dystrophin, both in 1987, provided key tools to understand DMD genetics and molecular pathology. The identification of dystrophin nucleated extensive research on myofiber membrane cytoskeleton, membrane repair, muscle regeneration, and failure of regeneration. This in turn led to molecular therapeutics based on understanding of dystrophin structure and function. This historical perspective describes the events surrounding the initial identification of the dystrophin protein.

Identification of a novel splicing variant of IDS gene in a pedigree affected with type II glycosaminoglycan product storage disease

To analyze variant of IDS gene in a pedigree affected with mucopolysaccharidosis type II (MPS II). The proband was subjected to next generation sequencing and Sanger sequencing to identify potential variants. Suspected variant was analyzed by its co-segregation with the disease in the pedigree. Its impact on mRNA splicing was analyzed by using reverse transcription PCR (RT-PCR). A hemizygous IVS1-3T>G variant was found in the IDS gene in the proband. RT-PCR results revealed two abnormal cDNA fragments of 600 bp and 300 bp. The 600 bp fragment had inserted 216 nucleotides at the 3' end of intron 1, while the 300 bp fragment had lost 109 nucleotides at the 5' end of exon 2, which resulted in two truncated proteins comprising 38 and 92 amino acids, respectively, instead of the normal product (550 amino acids). The proband and his mother were respectively hemizygous and heterozygous for the variant. The same variant was not found among 100 normal controls. The IVS1-3T>G variant of the IDS gene probably underlies the MPS II in this pedigree by causing reduction or elimination of the IDS protein.

The Transfection of HepG2 cells with Truncated β-Catenin Coding Expression Vector

β-catenin is an effector protein in Wnt signaling. β-catenin mutations are reported in the development of many diseases such as autism, colorectal carcinoma, developmental delay, intellectual disability, neurodegeneration, skin, hair and facial anomalies. Exon 3 deletion mediated truncations of the β-catenin associated with these diseases. Therefore understanding the functions of wild type and exon 3 deleted forms of β-catenin may provide an enhancement in the treatment of many diseases. However, to conduct controlled experiments, there could be a demand for the expression vectors that code for wild type and exon 3 deleted forms of β-catenin and originated from the same organism. Since it has long been known that HepG2 cells are heterozygous for β-catenin, in this study, it was found worthy of constructing the expression vectors from the total RNA of HepG2 cells. Then the utility of truncated β-catenin coding pcDNA3.1/CTNNB1 expression vector for upregulation of truncated β-catenin in HepG2 cells was examined. To this end, RNA was isolated from HepG2 cells, cDNA fragments were amplified by polymerase chain reaction (PCR), expression vectors were constructed then sequenced from 5’-prime regions. Following the BLAST analysis, it was concluded that both truncated and wild type β-catenin coding pcDNA3.1/CTNNB1 expression vectors were successfully cloned in E. coli cells. Interestingly, when the parental HepG2 cells were transfected with exon 3 deleted expression vector, β-catenin protein levels were not affected. Moreover, cellular morphology and population doubling time were not significantly altered.

Characterization of the cathepsin D in Procambarus clarkii and its biological role in innate immune responses

Cathepsin D belongs to aspartic protease family, produced in the rough endoplasmic reticulum, and then transported to lysosomes, where it participates in various physiological processes. Despite its importance, only a few reports available on the functional role of cathepsin D in crustaceans. Herein, we cloned a cDNA fragment of cathepsin D from the hepatopancreas of the red swamp crayfish, Procambarus clarkii (Pc-cathepsin D) for the first time. It included 1158 base pairs open reading frame, encoding a protein of 385 amino acids. Multiple alignment analysis confirmed the presence of aspartic proteinase active sites and N glycosylation sites. Pc-cathepsin D mRNA expression was high in the gills followed by gut, heart, hepatopancreas of P. clarkii. At different time points post-infection with lipopolysaccharides, peptidoglycan, or polyinosinic polycytidylic acid, Pc-cathepsin D mRNA expression significantly enhanced compared with the control group. Knockdown of the Pc-cathepsin D by double-stranded RNA, strikingly, changed the expression of all the tested P. clarkii immune-associated genes, including Pc-Toll, Pc-lectin, Pc-cactus, Pc-anti-lipopolysaccharide factor, Pc-phospholipase, and Pc-sptzale. Altogether, these results suggest that Pc-cathepsin D is needed to confer innate immunity against microbial pathogens by modulating the expression of crucial transcripts that encode immune-associated genes.

Complete genomic sequence and comparative analysis of rice stripe virus from Sichuan Province, China

Rice stripe virus (RSV) causes viral disease and yield losses in rice. Leaves of rice plants showing yellowing, stripes and curling were collected in Sichuan Province, China and tested for rice strip virus (RSV) by RT-PCR using specific primers. Nine of the nineteen symptomatic samples were positive for RSV. The complete sequence of RSV RNA1 to RNA4 was determined by assembling cDNA fragments obtained from isolate Huid04 in Huidong County, Sichuan Province (GenBank accession Nos. KX611337 to KX611340). Phylogenetic analyses of RNA-dependent RNA polymerase sequences showed a close relationship of RSV isolate Hui0d4 and isolates DaL08 and KunM08 from Yunnan Province, China. This is the first complete genomic sequence of an RSV isolate from Sichuan Province, China.

First report of Lihan Tick virus (Phlebovirus, Phenuiviridae) in ticks, Colombia

BackgroundTick-borne phenuivirus (TBPVs) comprise human and animal viruses that can cause a variety of clinical syndromes ranging from self-limiting febrile illness to fatal haemorrhagic fevers.ObjectiveDetect Phlebovirus (Family Phenuiviridae) in ticks collected from domestic animals in Córdoba, Colombia.MethodsWe collected 2365 ticks from domestic animals in three municipalities of the Department of Cordoba, Colombia in 2016. Ticks were identified and pooled by species for RNA extraction. A nested real-time PCR with specific primers for Phlebovirus and a specific probe for Heartland virus (HRTV) formerly a Phlebovirus, now a Banyangvirus were performed. Also, a conventional nested PCR, with the same specific primers was used to detect other Phleboviruses, with positive reactions indicated by an amplified cDNA fragment of approximately 244 bp determined by gel electrophoresis. These bands were gel-purified and sequenced by the Sanger method.ResultsUsing real-time RT-PCR, no positive results for HRTV were found. However, using conventional nested PCR 2.2% (5/229 pools) yielded a product of 244 bp. One positive sample was detected in a pool of Dermacentor nitens ticks collected from a horse, and the four remaining positive pools were from Rhipicephalus microplus collected from cattle. The five positive nucleotide sequences had identities of 93 to 96% compared to a section of the L-segment of Lihan Tick virus, a Phlebovirus originally detected in R. microplus ticks in China. The strongest identity (96–99%) was with Lihan Tick virus detected in R. microplus ticks from Brazil.ConclusionsThis is the first report of viral detection in ticks in Colombia. We detected a Colombian strain of Lihan Tick virus. We recommend expanding the sampling area and carrying out more eco-epidemiological studies related to epidemiological surveillance of viruses on ticks in Colombia.

Isoform repertoire of Blood mononuclear cells from SLE patients

Abstract Systemic Lupus Erythematosus (SLE) is characterized by numerous immunological alterations, including increased expression of immune activators, such as IFNa, along with alterations in cell composition, such as expansion of plasma cells that produce autoantibodies. Alternative Splicing (AS) is a biological process that regulates post-transcriptional alterations of the RNA. Pathogenic AS events have been associated to several diseases. Furthermore, the description of SLE autoantibodies that target components of the spliceosome such as anti-Sm/RNP, anti-Ro/La, and anti-dsDNA has led us to hypothesize that SLE is associated to abnormal AS events. RNA-seq uses standard Short-Read technologies which involve RNA or cDNA fragmentation for the generation of 150 bp sequencing libraries. Since most mammalian mRNAs are 1–2 kb long, using RNA-seq to identify novel alternatively spliced isoforms is extremely challenging. However, emerging Long-Read sequencing technologies allow full-length mRNA sequencing, de novo transcriptome assembly, and isoform profiling. Our lab has built a computational pipeline that combines Long-Read sequencing with RNA-seq data to identify and quantify novel transcripts in peripheral blood mononuclear cells (PBMCs) from SLE patients compared to healthy controls. From our computational analysis, we have observed that SLE patients display differential exon-inclusion and exclusion events in transcripts coding for immune activating and regulatory proteins, respectively. We conclude that SLE might be associated with pathogenic AS events that might place the immune balance under double jeopardy, through activation of immune activating receptors and inhibition of immune inhibiting receptors.

KRAS-retroviral fusion transcripts and gene amplification in arsenic-transformed, human prostate CAsE-PE cancer cells

CAsE-PE cells are an arsenic-transformed, human prostate epithelial line containing oncogenic mutations in KRAS compared to immortalized, normal KRAS parent cells, RWPE-1. We previously reported increased copy number of mutated KRAS in CAsE-PE cells, suggesting gene amplification. Here, KRAS flanking genomic and transcriptomic regions were sequenced in CAsE-PE cells for insight into KRAS amplification. Comparison of DNA-Seq and RNA-Seq showed increased reads from background aligning to all KRAS exons in CAsE-PE cells, while a uniform DNA-Seq read distribution occurred in RWPE-1 cells with normal transcript expression. We searched for KRAS fusions in DNA and RNA sequencing data finding a portion of reads aligning to KRAS and viral sequence. After generation of cDNA from total RNA, short and long KRAS probes were generated to hybridize cDNA and KRAS enriched fragments were PacBio sequenced. More KRAS reads were captured from CAsE-PE cDNA versus RWPE-1 by each probe set. Only CAsE-PE cDNA showed KRAS viral fusion transcripts, primarily mapping to LTR and endogenous retrovirus sequences on either 5′- or 3′-ends of KRAS. Most KRAS viral fusion transcripts contained 4 to 6 exons but some PacBio sequences were in unusual orientations, suggesting viral insertions within the gene body. Additionally, conditioned media was extracted for potential retroviral particles. RNA-Seq of culture media isolates identified KRAS retroviral fusion transcripts in CAsE-PE media only. Truncated KRAS transcripts suggested multiple retroviral integration sites occurred within the KRAS gene producing KRAS retroviral fusions of various lengths. Findings suggest activation of endogenous retroviruses in arsenic carcinogenesis should be explored.

Identification and characterization of human PEIG-1/GPRC5A as a 12-O-tetradecanoyl phorbol-13-acetate (TPA) and PKC-induced gene

Homo sapiens orphan G protein-coupling receptor PEIG-1 was first cloned and characterized by applying differential display to T84 colonic carcinoma cells incubated in the presence of phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (GenBank AF506289.1). Later, Lotan's laboratory found the same gene product in response to retinoic acid analogues, naming it with the symbol RAIG1. Now the official HGNC symbol is GPRC5A. Here, we report the extension of its original cDNA fragment towards the 5' and 3' end. In addition, we show that TPA (100 ng/ml, 162 nM) strongly stimulated GPRC5A mRNA in T84 colonic carcinoma cells, with maximal expression at 4 h and 100 ng/ml (162 nM). Western blots showed several bands between 35 and 50 kDa, responding to TPA stimulation. Confocal microscopy confirmed its TPA upregulation and the location in the plasma membrane. The PKC inhibitor Gö 6983 (10 μM), and the Ca2+ chelator BAPTA-AM (150 μM), strongly inhibited its TPA induced upregulation. The PKA inhibitor H-89 (10 μM), and the MEK1/2 inhibitor U0126 (10 μM), also produced a significant reduction in the TPA response (~50%). The SGK1 inhibitor GSK650394 stimulated GPRC5A basal levels at low doses and inhibit its TPA-induced expression at concentrations ≥10 μM. The IL-1β autocrine loop and downstream signalling did not affect its expression. In conclusion, RAIG1/RAI3/GPRC5A corresponds to the originally reported PEIG-1/TIG1; the inhibition observed in the presence of Gö 6983, BAPTA and U0126, suggests that its TPA-induced upregulation is mediated through a PKC/Ca2+ →MEK1/2 signalling axis. PKA and SGK1 kinases are also involved in its TPA-induced upregulation.

Identification and in silico structural and functional analysis of a trypsin-like protease from shrimp Macrobrachium carcinus.

Macrobrachium carcinus (Linnaeus, 1758) is a species of freshwater shrimp widely distributed from Florida southwards to southern Brazil, including southeast of Mexico. In the present work, we identified a putative trypsin-like protease cDNA fragment of 736 nucleotides from M. carcinus hepatopancreas tissue by the 3′RACE technique and compared the deduced amino acid sequence to other trypsin-related proteases to describe its structure and function relationship. The bioinformatics analyses showed that the deduced amino acid sequence likely corresponds to a trypsin-like protease closely related to brachyurins, which comprise a subset of serine proteases with collagenolytic activity found in crabs and other crustacea. The M. carcinus trypsin-like protease sequence showed a global sequence identity of 94% with an unpublished trypsin from Macrobrachium rosenbergii (GenBank accession no. AMQ98968), and only 57% with Penaeus vannamei trypsin (GenBank accession no. CAA60129). A detailed analysis of the amino acid sequence revealed specific differences with crustacean trypsins, such as the sequence motif at the beginning of the mature protein, activation mechanism of the corresponding zymogen, amino acid residues of the catalytic triad and residues responsible for substrate specificity.

Identification of the Ipomoea nil cDNA clone encoding protein with one transmembrane domain by differential display PCR

A modified differential display procedure was used to compare Ipomoea nil gene expression under flower inductive and non-inductive light/dark conditions and a 170 bp cDNA fragment was displayed. Screening of the cDNA library from I. nil cotyledons led to the isolation of a 577 bp cDNA clone with high nucleotide and amino acids homology to One Helix Protein (OHP) genes from Arabidopsis thaliana and Deschampsia antartica. The membrane spanning helix of InOHP is 91% identical to the MSH of the A. thaliana OHP. MSH is located in the C-terminal part of InOHP protein and corresponds to third MSH of LHC proteins. InOHP transcripts are numerous in leaves of plants when grown under continuous light and are also present in grown under flower inductive condition but their quantity is lower. Southern analysis showed that InOHP is member of a gene family involved in photoprotection of photosystems against excessive light

An Infectious cDNA Clone of SARS-CoV-2

SummaryThe ongoing pandemic of COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), underscores the urgency to develop experimental systems for studying this virus and identifying countermeasures. We report a reverse genetic system for SARS-CoV-2. Seven complimentary DNA (cDNA) fragments spanning the SARS-CoV-2 genome were assembled into a full-genome cDNA. RNA transcribed from the full-genome cDNA was highly infectious after electroporation into cells, producing 2.9 × 106 plaque-forming unit (PFU)/mL of virus. Compared with a clinical isolate, the infectious-clone-derived SARS-CoV-2 (icSARS-CoV-2) exhibited similar plaque morphology, viral RNA profile, and replication kinetics. Additionally, icSARS-CoV-2 retained engineered molecular markers and did not acquire other mutations. We generated a stable mNeonGreen SARS-CoV-2 (icSARS-CoV-2-mNG) by introducing this reporter gene into ORF7 of the viral genome. icSARS-CoV-2-mNG was successfully used to evaluate the antiviral activities of interferon (IFN). Collectively, the reverse genetic system and reporter virus provide key reagents to study SARS-CoV-2 and develop countermeasures.

Cloning, Molecular Characterization and Expression Patterns of DMRTC2 Implicated in Germ Cell Development of Male Tibetan Sheep.

The double sex and mab-3-related transcription factors like family C2 (DMRTC2) gene is indispensable for mammalian testicular function and spermatogenesis. Despite its importance, what expression and roles of DMRTC2 possesses and how it regulates the testicular development and spermatogenesis in sheep, especially in Tibetan sheep, remains largely unknown. In this study, DMRTC2 cDNA from testes of Tibetan sheep was firstly cloned by the RT-PCR method, and its molecular characterization was identified. Subsequently, the expression and localization patterns of DMRTC2 were evaluated by quantitative real-time PCR (qPCR), Western blot, and immunofluorescence. The cloning and sequence analysis showed that the Tibetan sheep DMRTC2 cDNA fragment contained 1113 bp open reading frame (ORF) capable of encoding 370 amino acids, and displayed high identities with some other mammals, which shared an identical DM domain sequence of 47 amino acids ranged from residues 38 to 84. qPCR and Western blot results showed that DMRTC2 was expressed in testes throughout the development stages while not in epididymides (caput, corpus, and cauda), with higher mRNA and protein abundance in Tibetan sheep testes of one- and three-year-old (post-puberty) compared with that of three-month-old (pre-puberty). Immunofluorescence results revealed that immune staining for DMRTC2 protein was observed in spermatids and spermatogonia from post-puberty Tibetan sheep testes, and gonocytes from pre-puberty Tibetan sheep testes. Together, these results demonstrated, for the first time, in sheep, that DMRTC2, as a highly conserved gene in mammals, is essential for sheep spermatogenesis by regulating the proliferation or differentiation of gonocytes and development of spermatids in ram testes at different stages of maturity.

First report of Sugarcane mosaic virus in achira (Canna edulis Ker.) in Nariño, Colombia

Achira (Canna edulis Ker.) is a cultivated species for handcrafted food products and starch production. In Colombia is estimated an achira cultivated area of 800 ha; in the department of Nariño there has been a disturbance of viral etiology, known by farmers as Streak Virus, due to its symptoms in the leaves, but without previous records in the area. The disease causes losses in performance, although they have not been established precisely. In order to clarify the nature of this pathology and the identity of the pathogen associated with the problem, an investigation was carried out at the University of Nariño, by means of molecular tests of PCR and RT-PCR, sequencing, serology and electron microscopy, of foliar samples collected in the producing areas. The most outstanding symptoms in affected tissues were yellow mosaic, mottled, chlorotic streak and ribs discoloration, among others. There were no cytoplasmic inclusions similar to those produced by Potyvirus, nor viral particles were observed, nor serology positive results, but it was possible to achieve the amplification of a cDNA fragment, with specific primers for Potyvirus and 98% of homology of the sequences with Sugarcane mosaic virus. This is the first SCMV report in achira in Nariño, Colombia.

Highly degraded RNA can still provide molecular information: An in vitro approach.

The long-term survival of RNA in postmortem tissues is a tricky topic. Many aged/forensic specimens show, in fact, high rates of null/inconclusive PCR-based results, while reliable outcomes were sometimes achieved from archaeological samples. On the other hand, several data show that the RNA is a molecule that survives even to several physical-chemical stresses. In the present study, a simple protocol, which was already developed for the prolonged hydrolysis of DNA, was applied to a RNA sample extracted from blood. This protocol is based on the heat-mediated (70°C) hydrolysis for up to 36 h using ultrapure water and di-ethyl-pyro-carbonate-water as hydrolysis medium. Measurable levels of depurination were not found even if microfluidic devices showed a progressive pattern of degradation. The reverse transcription/quantitative PCR analysis of two (60bp long) housekeeping targets (glyceraldehyde-3-phosphate dehydrogenase and porphobilinogen deaminase) showed that the percentage of amplifiable target (%AT) decreased in relation to the duration of the damaging treatment (r2 >0.973). The comparison of the %AT in the degraded RNA and in the DNA samples that underwent the same damaging treatment showed that the %AT is always higher in RNA, reaching up to three orders of magnitude. Lastly, even the end-point PCR of blood-specific markers gave reliable results, which is in agreement with the body fluid origin of the sample. In conclusion, all the PCR-based results show that RNA maintains the ability to be retro-transcribed in short cDNA fragments even after 36 h of incubation at 70°C in mildly acidic buffers. It is therefore likely that the long-term survival of RNA samples depends mainly on the protection against RNAase attacks rather than on environmental factors (such as humidity and acidity) that are instead of great importance for the stability of DNA. As a final remark, our results suggest that the RNA analysis can be successfully performed even when DNA profiling failed.