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Colorectal cancer (CRC), one of the most prevalent types of cancer, requires the discovery of new tumor biomarkers for accurate patient prognosis. In this work, the prognostic value of the tRNA fragment i-tRF-GlyGCC in CRC was examined. Total RNA extraction from 211 CRC patient cancer tissue specimens and 83 adjacent normal tissues was conducted. Each RNA extract was subjected to in vitro polyadenylation and reverse transcription. A real-time quantitative PCR assay was used to quantify i-tRF-GlyGCC in all samples. Extensive biostatics analysis showed that i-tRF-GlyGCC levels in CRC tissues were significantly lower than in matched normal colorectal tissues. Additionally, the disease-free survival (DFS) and overall survival (OS) time intervals were considerably shorter in CRC patients with high i-tRF-GlyGCC expression. i-tRF-GlyGCC expression maintained its prognostic value independently of other established prognostic factors, as shown by the multivariate Cox regression analysis. Additionally, survival analysis after TNM stage stratification revealed that higher i-tRF-GlyGCC levels were linked to shorter DFS time intervals in patients with TNM stage II tumors, as well as an increased probability of having a worse OS for patients in TNM stage II. In conclusion, i-tRF-GlyGCC has the potential to be a useful molecular tissue biomarker in CRC, independent of other clinicopathological variables.

In October 2020, the chemistry Nobel Prize was awarded to Emmanuelle Charpentier and Jennifer A. Doudna for the discovery of a new promising genome-editing tool: the genetic scissors of CRISPR-Cas9. The identification of CRISPR arrays and the subsequent identification of cas genes, which together represent an adaptive immunological system that exists not only in bacteria but also in archaea, led to the development of diverse strategies used for precise DNA editing, providing new insights in basic research and in clinical practice. Due to their advantageous features, the CRISPR-Cas systems are already employed in several biological and medical research fields as the most suitable technique for genome engineering. In this review, we aim to describe the CRISPR-Cas systems that have been identified among prokaryotic organisms and engineered for genome manipulation studies. Furthermore, a comprehensive comparison between the innovative CRISPR-Cas methodology and the previously utilized ZFN and TALEN editing nucleases is also discussed. Ultimately, we highlight the contribution of CRISPR-Cas methodology in modern biomedicine and the current plethora of available applications for gene KO, repression and/or overexpression, as well as their potential implementation in therapeutical strategies that aim to improve patients’ quality of life.

Background Treatment of cancer with natural chemotherapeutic agents induces apoptosis along with remarkable alterations in the expression of apoptosis-related genes. Deregulation of microRNA (miRNA) expression is implicated in several human malignancies. Vinca alkaloids compose a class of antimitotic drugs preventing cancer cells from dividing, leading to apoptosis. They are commonly used in clinical practice for breast cancer treatment. Objective The present study focused on the effects of vinca alkaloids (vincristine, vinblastine, and vinorelbine) on miRNA expression of treated breast cancer cells. Methods We investigated the effect of vincristine, vinblastine, and vinorelbine on the expression of oncogenic and tumor-suppressive miRNAs (miR-15a-5p, miR-16-5p, miR-21-5p, miR-25-3p, miR- 29b-3p, miR-125b-5p, miR-148a-3p, miR-214-3p, miR-221-3p, miR-222-3p, and miR-421), as well as on the expression of the apoptosis-related genes BAX, BCL2, TP53, and CDKN1B in BT-20 and SKBR- 3 breast adenocarcinoma cells. Results Treatment of BT-20 cells with vincristine, vinblastine, and/or vinorelbine resulted in upregulation of TP53 expression. However, no alterations in the mRNA levels of the pivotal BCL2 family members BAX and BCL2 were observed. On the other hand, treatment of SK-BR-3 cells with any of these vinca alkaloids led to an increase in the BAX/BCL2 mRNA ratio, implying the activation of the intrinsic apoptotic pathway. No concomitant alteration in TP53 expression was observed in treated SKBR- 3 cells. Regarding the miRNAs examined in this study, miR-222-3p expression exhibited the most remarkable modulations in both treated cell lines. Conclusion This study suggests the possible involvement of miR-222-3p expression in breast cancer cell apoptosis, triggered by vincristine, vinblastine, and vinorelbine.

l-Dopa Decarboxylase (DDC) is a pyridoxal requiring enzyme that catalyzes the decarboxylation of L-3,4-dihydroxyphenylalanine (l-Dopa) to Dopamine (DA). The function of DDC in physiological and pathological biochemical pathways remains poorly understood, while the function and regulation of human DDC isoforms is almost completely elusive. We have shown that Annexin V, a fundamental apoptosis marker, is an inhibitor of l-Dopa decarboxylase activity. Here we show the interaction of both the full-length DDC and the truncated isoform alternative DDC (Alt-DDC) with Annexin V in human tissue and cell lines. Interestingly, DDC isoform expression is enhanced or remains unaffected following staurosporine (STS) treatment, despite increased levels of cytotoxicity and apoptosis. The findings presented here provide novel insights concerning the involvement of DDC in programmed cell death.

Background Bladder cancer (BlCa) heterogeneity and the lack of personalised prognosis lead to patients’ highly variable treatment outcomes. Here, we have analysed the utility of the GAS5 tumour-suppressor lncRNA in improving BlCa prognosis. Methods GAS5 was quantified in a screening cohort of 176 patients. Hedegaard et al. (2016) (n = 476) and TCGA provisional (n = 413) were used as validation cohorts. Survival analysis was performed using recurrence and progression for NMIBC, or death for MIBC. Internal validation was performed by bootstrap analysis, and decision curve analysis was used to evaluate the clinical benefit on disease prognosis. Results GAS5 levels were significantly downregulated in BlCa and associated with invasive high-grade tumours, and high EORTC-risk NMIBC patients. GAS5 loss was strongly and independently correlated with higher risk for NMIBC early relapse (HR = 2.680, p = 0.011) and progression (HR = 6.362, p = 0.035). Hedegaard et al. and TCGA validation cohorts’ analysis clearly confirmed the association of GAS5 loss with NMIBC worse prognosis. Finally, multivariate models incorporating GAS5 with disease established markers resulted in higher clinical benefit for NMIBC prognosis. Conclusions GAS5 loss is associated with adverse outcome of NMIBC and results in improved positive prediction of NMIBC patients at higher risk for short-term relapse and progression, supporting personalised prognosis and treatment decisions.

A bidirectional negative relationship between Hepatitis C virus (HCV) replication and gene expression of the catecholamine biosynthetic enzyme L-Dopa decarboxylase (DDC) was previously shown in the liver and attributed at least to an association of DDC with phosphatidylinositol 3-kinase (PI3K). Here, we report that the biosynthesis and uptake of catecholamines restrict HCV replication in hepatocytes, while HCV has developed ways to reduce catecholamine production. By employing gene silencing, chemical inhibition or induction of the catecholamine biosynthetic and metabolic enzymes and transporters, and by applying the substrates or the products of the respective enzymes, we unravel the role of the different steps of the pathway in viral infection. We also provide evidence that the effect of catecholamines on HCV is strongly related with oxidative stress that is generated by their autoxidation in the cytosol, while antioxidants or treatments that lower cytosolic catecholamine levels positively affect the virus. To counteract the effect of catecholamines, HCV, apart from the already reported effects on DDC, causes the down-regulation of tyrosine hydroxylase that encodes the rate-limiting enzyme of catecholamine biosynthesis and suppresses dopamine beta-hydroxylase mRNA and protein amounts, while increasing the catecholamine degradation enzyme monoamine oxidase. Moreover, the NS4B viral protein is implicated in the effect of HCV on the ratio of the ~50 kDa DDC monomer and a ~120 kDa DDC complex, while the NS5A protein has a negative effect on total DDC protein levels.

Objective Epilepsy is one of the most prevalent neurologic disorders, causing serious psychological problems and reducing quality of life. Although 20 different antiepileptic drugs (AEDs) have been approved by the US Food and Drug Administration (FDA), 30% of patients have drug-resistant epilepsy (DRE). Considering the role of miR-146a and miR-134 in neuroinflammation and dendritic functionality, respectively, the aim of this study was the clinical evaluation of circulating miR-146a and miR-134 as novel noninvasive molecular markers for the prognosis of refractory epilepsy. Methods The study included 162 patients with focal impaired awareness seizures. Total RNA was extracted from serum samples spiked with synthetic cel-miR-39-3p for normalization purposes. First-strand complementary DNA (cDNA) synthesis was performed using microRNA-specific stem-loop primers, and hsa-miR-134/146a levels were quantified by quantitative polymerase chain reaction (qPCR). DRE was used as clinical end point event. Internal validation was performed by bootstrap analysis, and decision curve analysis was used to evaluate the clinical benefit on disease prognosis. Results The circulating levels of both miR-134 and miR-146a were elevated in patients with drug-resistant seizures. The receiver-operating characteristic (ROC) curve and logistic regression analysis demonstrated that patients with increased circulating miR-134/146a levels are at significantly higher risk for developing DRE, independently of temporal lobe sclerosis, epilepsy duration, familial history, age at first seizure, age, body mass index (BMI), smoking behavior, and gender. Finally, decision curve analysis highlighted that the evaluation of circulating miR-134/146a led to superior clinical benefit for DRE prognosis and patients' risk stratification. Significance Elevated serum miR-134/146a levels are associated with a higher risk for AED-resistant epilepsy and could constitute novel noninvasive molecular markers to improve disease early prognosis and support precision medicine.

Certain microRNA molecules (miRNAs) that target genes involved in beta-cell growth and insulin resistance are found deregulated in patients with type-2 diabetes mellitus (T2D) and correlate with its complications. However, the expression profile of miRNAs that regulate genes bearing T2D-related single-nucleotide polymorphisms has been hardly studied. We recently reported that the mRNA patterns of specific T2D-susceptibility genes are impaired in patients, and associate with disease parameters and risk factors. The aim of this study was to explore the levels of miRNAs that target those genes, in peripheral blood of patients versus controls. A panel of 14 miRNAs validated to target the CDKN2A, CDK5, IGF2BP2, KCNQ1, and TSPAN8 genes, was developed upon combined search throughout the DIANNA TarBase v7.0, miRTarBase, miRSearch v3.0-Exiqon, miRGator v3.0, and miRTarget Link Human algorithms. Specifically developed poly(A)polyadenylation(PAP)-reverse transcription(RT)-qPCR protocols were applied in peripheral blood RNA samples from patients and controls. Possible correlations with the disease, clinicopathological parameters and/or risk factors were evaluated. T2D patients expressed decreased levels of let-7b-5p, miR-1-3p, miR-24-3p, miR-34a-5p, miR-98-5p, and miR-133a-3p, compared with controls. Moreover, these levels correlated with certain disease features including insulin and % HbA1c levels in patients, as well as BMI, triglycerides’ levels and family history in controls. A T2D-specific expression profile of miRNAs that target disease-susceptibility genes is for the first time described. Future studies are needed to elucidate the associated transcription-regulatory mechanisms, perchance involved in T2D pathogenesis, and to evaluate the potential of these molecules as possible biomarkers for this disorder.

The human RNASEK gene encodes Ribonuclease κ, an endoribonuclease that belongs to a highly conserved protein family of metazoans. Recent evidence suggests that the mRNA levels of the RNASEK gene possess biomarker attributes in patients with prostate cancer. In the present study, we used 3' RACE and next-generation sequencing (NGS) to detect and identify novel RNASEK transcripts. Computational analysis of the NGS data revealed new alternative splicing events that support the existence of novel RNASEK alternative transcripts. As a result, eight RNASEK splice variants were discovered and their expression profile was analyzed with the use of nested PCR in a wide panel of human cell lines, originating from several cancerous and/or normal human tissues. Based on in silico analysis, six of the eight novel RNASEK transcripts are predicted to encode new protein isoforms, while the remaining two splice variants could be considered as nonsense-mediated mRNA decay (NMD) candidates.

Background Alternative splicing is a key process in carcinogenesis and, from a clinical aspect, holds great promises, as alternatively spliced variants have emerged as an untapped source of diagnostic and prognostic markers. Our aim was to assess the prognostic value of three recently recognized splice variants of the apoptosis-related gene, BCL2L12, in breast cancer (BC). Methods Total RNA was extracted from breast samples (150 BC and 80 tumor-adjacent normal tissues) and, following cDNA synthesis, a variant-specific qPCR was performed for the expressional quantification of BCL2L12 v.1, v.2 and v.4 transcript variants. Extensive statistical analysis, including bootstrap resampling and internal validation, was conducted in order to evaluate the associations of v.1, v.2 and v.4 expression with patients’ clinopathological and survival data. Results All examined BCL2L12 variants were significantly upregulated in BC specimens compared to their non-cancerous counterpart (v.1, pv.2, p=0.009; v.4, p=0.004). Increased BCL2L12 v.4 mRNA expression was associated with markers of unfavorable prognosis namely, advanced tumor grade (p=0.002), ER- (p=0.015)/PR- (pv.4 was significantly overexpressed in women with triple negative BC (TNBC) and HER2-positive tumors compared to those harboring luminal tumors (pBCL2L12 v.2 overexpression, as a continuous variable ([HR]=0.45, 95% CI=0.17–0.82, p=0.010), is a strong and independent marker of favorable prognosis for BC patients. Interestingly, v.2 retains its prognostic value in patients with Grade II/III ([HR]=0.21, 95% CI=0.05–0.57, p=0.006) or HER2-positive/TNBC tumors ([HR]=0.25, 95% CI=0.05–0.74, p=0.042). Conclusions BCL2L12 v.1, v.2, v.4 are aberrantly expressed in BC. Their expressional analysis by cost-effective molecular methods could provide a novel molecular tool for BC management.

Considering the unmet need for novel molecular tumor markers capable of improving prostate cancer (CaP) patients’ management along with the fruitful results regarding the future use of ribonucleases (RNases) as molecular diagnostic and prognostic markers in CaP, we aimed to study the expressional profile of RNase κ in CaP and BPH and to investigate its clinical significance in CaP. Total RNA was extracted from 212 prostatic tissue samples (101 BPH and 111 CaP) and, following cDNA synthesis, quantitative real-time PCR (qPCR) was performed for the expressional quantification of RNase κ. Extensive statistical analysis, including bootstrap resampling, was performed to investigate the differential expression of RNase κ in patients with BPH and CaP and its associations with patients’ clinicopathological and survival data. RNase κ was significantly downregulated (P = 0.002) in CaP patients compared to BPH ones. RNase κ overexpression was associated with decreased risk of CaP development and can discriminate between CaP and BPH independently of serum PSA levels (crude odds ratio = 0.93, P = 0.001). RNase κ upregulation was also associated with less advanced (P = 0.018) and less aggressive (P = 0.001) tumors as well as with longer progression-free survival (PFS) (P = 0.003). Finally univariate bootstrap Cox regression confirmed that RNase κ was associated with favorable prognosis (HR = 0.85, P = 0.002). RNase κ is a biomarker of favorable prognosis in CaP, which is significantly associated with less advanced and aggressive disease, as well as with enhanced PFS.

Breast and ovarian cancers remain a major public health issue, constituting a major cause of female morbidity and mortality worldwide. Even though systemic chemotherapy remains the main course of action for both types of cancer, current research studies aim at the discovery of novel therapeutic targets. Ribonucleases, due to their apparent implication in gene expression regulation, may serve as potential anticancer drugs. Human RNase κ is an endoribonuclease belonging in a recently identified family of proteins. Recent data from expression microarray studies reveal that the RNase κ gene is found either up- or downregulated in a number of human cancers, indicating a possible diagnostic and/or prognostic utility.The aim of this study was to investigate modulations in the expression levels of RNase κ gene, in breast and ovarian cancer cells, as a response to treatment with different chemotherapeutic agents. BT-20 breast cancer cells and SKOV-3 ovarian cancer cells were treated with the cytotoxic drugs paclitaxel, docetaxel, cisplatin, carboplatin, epirubicin and vinorelbine. Gene expression analysis was performed by the comparative CT method also known as 2(-ΔΔCT) method. The results revealed a distinct increase in the expression levels of RNase κ mRNA (up to 9-fold) after treatment with the antineoplastic agent paclitaxel in both cell lines, while treatment with the remaining anticancer drugs did not alter drastically the mRNA levels of RNase κ. Based on the fact that paclitaxel exerts its cytotoxic action by inducing apoptosis, the results could be indicative of a potential implication of RNase κ in apoptosis-related pathways.

Human RNase κ is an endoribonuclease expressed in almost all tissues and organs and belongs to a highly conserved protein family bearing representatives in all metazoans. To gain insight into the role of cysteine residues in the enzyme activity or structure, a recombinant active form of human RNase κ expressed in Pichia pastoris was treated with alkylating agents and dithiothreitol (DTT). Our results showed that the human enzyme is inactivated by DDT, while it remains fully active in the presence of alkylating agents. The unreduced recombinant protein migrates on SDS/PAGE faster than the reduced form. This observation in combination with the above findings indicated that human RNase κ does not form homodimers through disulfide bridges, and cysteine residues are not implicated in RNA catalysis but participate in the formation of intramolecular disulfide bond(s) essential for its ribonucleolytic activity. The role of the cysteine residues was further investigated by expression and study of Cys variants. Ribonucleolytic activity experiments and SDS/PAGE analysis of the wild-type and mutant proteins under reducing and non-reducing conditions demonstrated that Cys7, Cys14 and Cys85 are not essential for RNase activity. On the other hand, replacement of Cys6 or Cys69 with serine led to a complete loss of catalytic activity, indicating the necessity of these residues for maintaining an active conformation of human RNase κ by forming a disulfide bond. Due to the absolute conservation of these cysteine residues, the Cys6-Cys69 disulfide bond is likely to exist in all RNase κ family members.

Cc RNase is the founding member of the recently identified RNase κ family, which is represented by a single ortholog in a wide range of animal taxonomic groups. Although the precise biological role of this protein is still unknown, it has been shown that the recombinant proteins isolated so far from the insect Ceratitis capitata and from human exhibit ribonucleolytic activity. In this work, we report the genomic organization and molecular evolution of the RNase κ gene from various animal species, as well as expression analysis of the ortholog gene in C. capitata. The high degree of amino acid sequence similarity, in combination with the fact that exon sizes and intronic positions are extremely conserved among RNase κ orthologs in 15 diverse genomes from sea anemone to human, imply a very significant biological function for this enzyme. In C. capitata, two forms of RNase κ mRNA (0.9 and 1.5 kb) with various lengths of 3′ UTR were identified as alternative products of a single gene, resulting from the use of different polyadenylation signals. Both transcripts are expressed in all insect tissues and developmental stages. Sequence analysis of the extended region of the longer transcript revealed the existence of three mRNA instability motifs (AUUUA) and five poly(U) tracts, whose functional importance in RNase κ mRNA decay remains to be explored.

A novel protein family, designated hereafter as RNase kappa (kappa) family, has been recently introduced with the characterization of the specific Cc RNase, isolated from the insect Ceratitis capitata. The human ortholog of this family consists of 98 amino acids and shares98% identity with its mammalian counterparts. This RNase is encoded by a single-copy gene found to be expressed in a wide spectrum of normal and cancer tissues. The cDNA of the human ribonuclease has been isolated and subcloned into a variety of prokaryotic expression vectors, but most efforts to express it caused a severe toxic effect. On the other hand, the expression of the human RNase by the use of the methylotrophic yeast Pichia pastoris system resulted in the production of a highly active recombinant enzyme. Using a 30-mer 5'-end-labeled RNA probe as substrate, the purified enzyme seems to preferentially cleave ApU and ApG phosphodiester bonds, while it hydrolyzes UpU bonds at a lower rate. Based on amino acid sequence alignment and substrate specificity data, as well as the complete resistance of the recombinant protein to the placental ribonuclease inhibitor, we concluded that the human RNase kappa is a novel endoribonuclease distinct from other known ribonucleases.

Complementary DNA encoding a protein, designated Cc RNase, was isolated from the insect Ceratitis capitata. Deduced amino acid sequence analysis demonstrates that the Cc RNase has strong sequence homology with other uncharacterized proteins predicted from EST sequences belonging to different animal species, therefore defining a new protein family, which is conserved from Caenorhabditis elegans to humans. Phylogenetic analysis data in addition to extensive homolog searches in all available complete genomes suggested that all family members are true orthologs. Proteins belonging to this family are composed of 95-101 amino acids. The C.capitata orthologous protein was expressed in Escherichia coli. Despite the fact that the amino acid sequence of Cc RNase does not share any significant similarities with other known ribonucleases, our data give strong evidence in support of the assignment of enzymatic activity to the recombinant protein. The expressed molecule exhibits ribonucleolytic activity against poly(C) and poly(U) synthetic substrates, as well as rRNA. It is also demonstrated that expression of Cc RNase in E.coli inhibits growth of the host cells.

A new specific ribonuclease from normal human plasma has been purified to homogeneity, following a five-step purification protocol that included DEAE-Sepharose, CM-Sepharose, and Heparin-Sepharose chromatographies. The purified enzyme was found to be glycosylated and appeared as a single 25-kDa band on a SDS polyacrylamide gel. This RNase is poly(C) preferential, degrading poly(U) at a lower rate. Activity of this RNase toward cleavage of native substrates such as ribosomal RNA was also detected. The human plasma ribonuclease is a thermolabile molecule, exhibiting maximum activity at pH 6.5. Comparison between other known plasma RNases and the human plasma ribonuclease described here indicated a variety of differences in their biochemical and catalytic properties.

Here we report on the conformational changes that are responsible for the appearance of theDicentrarchus labraxencephalitis virus (DlEV) coat protein as a doublet in SDS–PAGE. Wild-type and mutated forms of the coat protein cDNA were expressed inE. coli. The study of the resulting recombinant molecules excluded the possibility of the involvement of a precursor autocatalysis mechanism or a ribosomal frameshifting event in the doublet formation. The appearance of the coat protein doublet was found to be β-mercaptoethanol sensitive. Based on this observation, we carried out substitution of all cysteine residues. The obtained results demonstrated the importance of intramolecular disulfide bonding between cysteines 187 and 201 on coat protein conformational changes.

Subtle alternative splicing leads to the formation of RNA variants lacking or including a small number of nucleotides. To date, the impact of subtle alternative splicing phenomena on protein biosynthesis has been studied in frame-preserving incidents. On the contrary, mRNA isoforms derived from frame-shifting events were poorly studied and generally characterized as non-coding. This work provides evidence for a frame-shifting subtle alternative splicing event which results in the production of a novel protein isoform. We applied a combined molecular approach for the cloning and expression analysis of a human RNase κ transcript (RNase κ-02) which lacks four consecutive bases compared to the previously isolated RNase κ isoform. RNase κ-02 mRNA is expressed in all human cell lines tested end encodes the synthesis of a 134-amino-acid protein by utilizing an alternative initiation codon. The expression of RNase κ-02 in the cytoplasm of human cells was verified by Western blot and immunofluorescence analysis using a specific polyclonal antibody developed on the basis of the amino-acid sequence difference between the two protein isoforms. The results presented here show that subtle changes during mRNA splicing can lead to the expression of significantly altered protein isoforms.

Nodaviruses are widespread causative agents of viral nervous necrosis in fish. Based on the coat protein sequence, fish nodaviruses are categorized into four different genotypes. In this study, we present data on the phylogenetic and antigenic characterization of 12 new isolates, eight European and four of Asian origin, from farmed and wild species of fish. Phylogenetic analysis based on the nucleotide sequence (688 bases) of the coat protein classified the majority of these new isolates to the RGNNV genotype. Geographic or host-species specificities were not revealed by this study. Neutralizing assay experiments, further confirmed the genotypic classification, supporting the possibility that the different nodavirus genotypes can also be serologically distinguishable.

Two overlapping clones encoding for a ribonuclease from six-day-old larvae of the insect Ceratitis capitata (Cc-RNase) have been isolated by immunoscreening a cDNA library and by 5' RACE. The sequence of the Cc-RNase cDNA contains an open reading frame of 414 nucleotides encoding for a precursor protein of 138 amino acids long with a putative signal peptide consisting of 19 amino acids. The calculated M(r) of the mature protein was found to be 13.7 kDa. Multiple alignments of the deduced amino acid Cc-RNase sequence with other ribonucleases revealed an approximate 25% average identity. Despite the low percentage of identity, histidine and lysine residues which are essential for its catalytic activity, were found to be completely conserved. Furthermore, expression of the clone in E. coli resulted in the production of a recombinant product that showed strong immunoreactivity with anti-RNase specific antibodies. These results support the hypothesis that the identified clone encodes for a protein which is a new member of the RNase superfamily.

The coat protein gene from encephalitis virus infecting Dicentrarhus labrax (DIEV) has been cloned by gene amplification, sequenced and expressed in Escherichia coli. DNA sequencing has revealed an open reading frame of 1017 bases encoding a polypeptide of 338 amino acids. The sequence similarities between the DIEV coat protein gene and the same gene in five encephalitis viruses infected other fish species were over 71.5% at the nucleotide level and over 79.5% at the amino acid level. These results indicate that the nodaviruses that cause encephalopathy and retinopathy in fishes are very closed related. E. coli cells harbouring the plasmid containing the DIEV gene can produce the viral coat protein. An efficient purification scheme using a Sepharore-Ni+2 column is presented. This, gives approx. 10 mg of more than 95% pure protein per gr of E. coli culture.

Human RNase κ is an endoribonuclease expressed in almost all tissues and organs and belongs to a highly conserved protein family bearing representatives in all metazoans. To gain insight into the role of cysteine residues in the enzyme activity or structure, a recombinant active form of human RNase κ expressed in Pichia pastoris was treated with alkylating agents and dithiothreitol (DTT). Our results showed that the human enzyme is inactivated by DDT, while it remains fully active in the presence of alkylating agents. The unreduced recombinant protein migrates on SDS/PAGE faster than the reduced form. This observation in combination with the above findings indicated that human RNase κ does not form homodimers through disulfide bridges, and cysteine residues are not implicated in RNA catalysis but participate in the formation of intramolecular disulfide bond(s) essential for its ribonucleolytic activity. The role of the cysteine residues was further investigated by expression and study of Cys variants. Ribonucleolytic activity experiments and SDS/PAGE analysis of the wild-type and mutant proteins under reducing and non-reducing conditions demonstrated that Cys7, Cys14 and Cys85 are not essential for RNase activity. On the other hand, replacement of Cys6 or Cys69 with serine led to a complete loss of catalytic activity, indicating the necessity of these residues for maintaining an active conformation of human RNase κ by forming a disulfide bond. Due to the absolute conservation of these cysteine residues, the Cys6-Cys69 disulfide bond is likely to exist in all RNase κ family members.

Cloning of alternatively polyadenylated transcripts is crucial for studying gene expression and function. Recent transcriptome analysis has mainly focused on large EST clone collections. However, EST sequencing techniques in many cases are incapable of isolating rare transcripts or address transcript variability. In most cases, 3' RACE is applied for the experimental identification of alternatively polyadenylated transcripts. However, its application may result in nonspecific amplification and false positive products due to the usage of a single gene specific primer. Additionally, internal poly(A) stretches primed by oligo(dT) primer in mRNAs with AU-rich 3'UTR may generate truncated cDNAs. To overcome these limitations, we have developed a simple and rapid approach combining SMART technology for the construction of a full length cDNA library and hybrid capture PCR for the selection and amplification of target cDNAs. Our strategy is characterized by enhanced specificity compared to other conventional RT-PCR and 3' RACE procedures.

A ribonuclease from 6 day old larvae of Ceratitis capitata has been purified to homogeneity by a four step procedure. The enzyme appears as a single polypeptide chain of approx. 34 kDa. Poly(U) and poly(C) are the only polyribonucleotides degraded under standard assay conditions. The enzyme degrades the mRNA present in polysomes inducing a shift of polysomes to monosomes. However, no major cleavage could be observed in the mRNA and the ribosomal RNA when the incubation with the ribonuclease took place under protein synthesis conditions of polysomes.

Here we report the development of an accurate and efficient genome walking approach based on ligation-mediated polymerase chain reaction (PCR) and magnetic capture hybrid selection technique. This approach overcomes the nonspecific amplification products that often occur in similar PCR-based methods. Our strategy was successfully applied for the cloning of the promoter region of the Cc RNase gene. This rapid, cost-effective, and sensitive protocol can easily be extended for use in the isolation and amplification of any target sequences for which only partial information is known such as identification of the position of transposable elements and integrated viral DNA sequences.

Cc RNase is the founding member of the recently identified RNase kappa family, which is represented by a single ortholog in a wide range of animal taxonomic groups. Although the precise biological role of this protein is still unknown, it has been shown that the recombinant proteins isolated so far from the insect Ceratitis capitata and from human exhibit ribonucleolytic activity. In this work, we report the genomic organization and molecular evolution of the RNase kappa gene from various animal species, as well as expression analysis of the ortholog gene in C. capitata. The high degree of amino acid sequence similarity, in combination with the fact that exon sizes and intronic positions are extremely conserved among RNase kappa orthologs in 15 diverse genomes from sea anemone to human, imply a very significant biological function for this enzyme. In C. capitata, two forms of RNase kappa mRNA (0.9 and 1.5 kb) with various lengths of 3' UTR were identified as alternative products of a single gene, resulting from the use of different polyadenylation signals. Both transcripts are expressed in all insect tissues and developmental stages. Sequence analysis of the extended region of the longer transcript revealed the existence of three mRNA instability motifs (AUUUA) and five poly(U) tracts, whose functional importance in RNase kappa mRNA decay remains to be explored.

L-Dopa decarboxylase (DDC) has been cloned from several species and was shown to undergo alternative splicing within its 5′-untranslated and coding regions. In this report we describe a novel splice variant of DDC mRNA in human tissue, lacking exons 10 - 15 of the full-length transcript but including an alternative exon 10. The isolated alternative human DDC cDNA (alt-DDC) was cloned from human placenta, and was found to be of the neuronal type. Northern blot analysis indicated that the alt-DDC transcript is expressed in high levels in human kidney. Our results demonstrate the detection of a new alternative splicing event within the coding region of the human DDC mRNA, further suggesting that the single copy human DDC gene undergoes complex processing leading to the formation of multiple mRNA isoforms.

The cDNA encoding the L-dopa decarboxylase (ddc) from the eclosion stage of the insect Ceratitis capitata was isolated by PCR and a molecular cloning strategy. The isolated cDNA clone encoded a protein of 431 amino acids with a calculated molecular weight of 47,843 Da. Northern blot analysis of poly(A)+ RNA showed an approximately 2 kb transcript. The deduced protein sequence shares a high percentage of homology with Ddc protein sequences of other species. Furthermore, the molecular weight of the deduced protein agreed well with that of the purified Ddc from the same insect. Data base search revealed significant and extensive sequence similarities among prokaryotic and eukaryotic PLP-dependent decarboxylases including Ceratitis capitata and bacterial histidine decarboxylase (HDC), strongly suggesting an ancient and common origin for all PLP-dependent decarboxylases.

A specific ribonuclease was detected and purified to homogeneity from six-day-old larvae of the insect Ceratitis capitata and its homogeneity was checked by analysis in polyacrylamide gels in the presence of sodium dodecyl sulfate. The nuclease specifically degrades poly(U) and poly(C) whilst it fails to do so with other single-stranded homopolyribonucleotides. The enzyme has a pH optimum in the region 7–9 and relative molecular mass of about 25000. The effect of this ribonuclease on the integrity of RNAs isolated from six-day-old larvae or rat liver was also studied.

1. 1. A novel inhibitor of in vitro protein synthesis, was purified from six- day old larvae of the insect Ceratitis capitata. 2. 2. The inhibitor was not a protein, showed no RNase activity, was destroyed by alkali and could be recovered quantitatively in the aqueous phase after phenol extraction. 3. 3. Our findings suggest that the inhibitor is a small RNA.