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Detection of complex splice sites (SSs) and polyadenylation sites (PASs) of eukaryotic genes is essential for the elucidation of gene regulatory mechanisms. Transcriptome-wide studies using high-throughput sequencing (HTS) have revealed prevalent alternative splicing (AS) and alternative polyadenylation (APA) in plants. However, small-scale and high-depth HTS aimed at detecting genes or gene families are very few and limited. We explored a convenient and flexible method for profiling SSs and PASs, which combines rapid amplification of 3′-cDNA ends (3′-RACE) and HTS. Fourteen NAC (NAM, ATAF1/2, CUC2) transcription factor genes of Populus trichocarpa were analyzed by 3′-RACE-seq. Based on experimental reproducibility, boundary sequence analysis and reverse transcription PCR (RT-PCR) verification, only canonical SSs were considered to be authentic. Based on stringent criteria, candidate PASs without any internal priming features were chosen as authentic PASs and assumed to be PAS-rich markers. Thirty-four novel canonical SSs, six intronic/internal exons and thirty 3′-UTR PAS-rich markers were revealed by 3′-RACE-seq. Using 3′-RACE and real-time PCR, we confirmed that three APA transcripts ending in/around PAS-rich markers were differentially regulated in response to plant hormones. Our results indicate that 3′-RACE-seq is a robust and cost-effective method to discover SSs and label active regions subjected to APA for genes or gene families. The method is suitable for small-scale AS and APA research in the initial stage. [ABSTRACT FROM AUTHOR]

Background: Because of the lack of an effective and economical control strategy against malaria (the most devastating infectious disease in developing countries) Transmission-Blocking Vaccines (TBVs) concept has been raised in recent years, promising a more efficient way to malaria control. TBVs aim at interfering and/or blocking pathogen development within the vector, halting transmission to non-infected vertebrate host. Aminopeptidase N (APN) is one of the most potent proteins in parasite development in Anopheles malaria vectors, which is strongly co-localized with human malaria parasites in the mosquito midgut epithelium. Therefore, Aminopeptidase N is one of the best choices for a new TBV. Methods: In this study for the first time we used 3'-RACE to amplify APN gene in Anopheles stephensi (An.stephensi), a major malaria vector in Iran, Indian subcontinent up to China by using different sets of primers including exon junction, conserved and specific region primers. Results: Full length of APN was sequenced stepwise, which could be applied in designing a new regional TBV and act as an essential component of malaria elimination program in An.stephensi distribution areas. Conclusion: Primers design and method modification should be set up exactly in approach based amplifications. From results we came to this conclusion that that 3'-RACE could be applied to amplified key regions which are beyond reach. [ABSTRACT FROM AUTHOR]

Echovirus 18 (EV18) is one of the echovirus serotypes associated with human diseases and in particular aseptic meningitis. To facilitate studies of the molecular epidemiology of EV18 and the evolution of enteroviruses in general, the complete nucleotide (nt) sequence was determined for the echovirus 18 prototype strain (Metcalf, EV18M). Excluding the poly A sequence, the genome consists of 7410 nt divided into a 740 nt 5′ untranslated region (5′ UTR), a 6567 nt long open reading frame coding for a 2189 amino acid (aa) polyprotein and a 103 nt 3′ UTR. Molecular analysis of the EV18M genome showed a typical enterovirus-like organization. Phylogenetic analysis of the structural and non-structural genes revealed a pattern of different relationships to other echo- and coxsackieviruses. Similarity analysis demonstrated that the Hill strain of echovirus 9 is most likely the result of a previous recombination event between ancestors of the echovirus 9 strain Barty (5′ half of the genome) and EV18M (3′ half). Using a maximum likelihood approach, the recombination point was mapped to the 2C gene. [Copyright &y& Elsevier]

SUMMARY A cDNA encoding glutamine synthetase (GS) was characterized from the red alga, Gelidium crinale (Turner) Gaillon, using reverse-transcriptase polymerase chain reaction and the 5′- and 3′-rapid amplification of cDNA ends. Sequence analysis of a 1231-bp GS cDNA transcript included both 5′ and 3′ untranslated regions and a 1056-bp open reading frame encoding a 352 amino acid polypeptide. Comparison with GS sequences from other organisms revealed that the G. crinale cDNA encodes a type-II GS, and the absence of a N-terminal plastid signal sequence suggests that it is a cytosolic isoenzyme. Phylogenetic analyses of GSII amino acid sequences supports the multiple origin of cytosolic and plastid isoenzymes during eukaryotic evolution. [ABSTRACT FROM AUTHOR]

The main objectives of developing vaccines to prevent malaria transmission are malaria control and preventing the reemergence of the disease in endemic regions. Molecular and in silico characterization of a candidate molecule is the first step in the vaccine design process. Determining the sequence and amplification of full-length cDNA copies from the mRNA transcripts is often challenging. The methods in this chapter provide a protocol for the rapid amplification of cDNA ends (RACE) and genome walking. Carboxypeptidase B2 enzyme from A. stephensi (CPBAs-2) was selected as the target molecule and the steps in its characterization and in silico analysis are explained in this chapter., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Detection of complex splice sites (SSs) and polyadenylation sites (PASs) of eukaryotic genes is essential for the elucidation of gene regulatory mechanisms. Transcriptome-wide studies using high-throughput sequencing (HTS) have revealed prevalent alternative splicing (AS) and alternative polyadenylation (APA) in plants. However, small-scale and high-depth HTS aimed at detecting genes or gene families are very few and limited. We explored a convenient and flexible method for profiling SSs and PASs, which combines rapid amplification of 3'-cDNA ends (3'-RACE) and HTS. Fourteen NAC (NAM, ATAF1/2, CUC2) transcription factor genes of Populus trichocarpa were analyzed by 3'-RACE-seq. Based on experimental reproducibility, boundary sequence analysis and reverse transcription PCR (RT-PCR) verification, only canonical SSs were considered to be authentic. Based on stringent criteria, candidate PASs without any internal priming features were chosen as authentic PASs and assumed to be PAS-rich markers. Thirty-four novel canonical SSs, six intronic/internal exons and thirty 3'-UTR PAS-rich markers were revealed by 3'-RACE-seq. Using 3'-RACE and real-time PCR, we confirmed that three APA transcripts ending in/around PAS-rich markers were differentially regulated in response to plant hormones. Our results indicate that 3'-RACE-seq is a robust and cost-effective method to discover SSs and label active regions subjected to APA for genes or gene families. The method is suitable for small-scale AS and APA research in the initial stage., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.