End Untranslated Region Research Articles

Kinetic pathway of HIV-1 TAR cotranscriptional folding.

The Trans-Activator Receptor (TAR) RNA, located at the 5'-end untranslated region (5' UTR) of the human immunodeficiency virus type 1 (HIV-1), is pivotal in the virus's life cycle. As the initial functional domain, it folds during the transcription of viral mRNA. Although TAR's role in recruiting the Tat protein for trans-activation is established, the detailed kinetic mechanisms at play during early transcription, especially at points of temporary transcriptional pausing, remain elusive. Moreover, the precise physical processes of transcriptional pause and subsequent escape are not fully elucidated. This study focuses on the folding kinetics of TAR and the biological implications by integrating computer simulations of RNA folding during transcription with nuclear magnetic resonance (NMR) spectroscopy data. The findings reveal insights into the folding mechanism of a non-native intermediate that triggers transcriptional pause, along with different folding pathways leading to transcriptional pause and readthrough. The profiling of the cotranscriptional folding pathway and identification of kinetic structural intermediates reveal a novel mechanism for viral transcriptional regulation, which could pave the way for new antiviral drug designs targeting kinetic cotranscriptional folding pathways in viral RNAs.

METTL14 affects UVB-induced human dermal fibroblasts photoaging via miR-100-3p biogenesis in an m6A-dependent manner.

Exposure to ultraviolet radiation can lead to skin photoaging, which increases the risk of skin tumors. This study aims to investigate how microRNA m6A modification contributes to skin photoaging. This study found that skin fibroblasts exposed to a single UVB dose of 30 mJ/cm2 exhibited characteristics of photoaging. The m6A level of total RNA decreased in photoaged cells with a down-regulated level of METTL14, and overexpression of METTL14 displayed a photoprotective function. Moreover, miR-100-3p was a downstream target of METTL14. And METTL14 could affect pri-miR-100 processing to mature miR-100-3p in an m6A-dependent manner via DGCR8. Furthermore, miR-100-3p targeted at 3' end untranslated region of ERRFI1 mRNA with an inhibitory effect on translation. Additionally, photoprotective effects of overexpression of METTL14 were reversed by miR-100-3p inhibitor or overexpression of ERRFI1. In UVB-induced photoaging of human skin fibroblasts, METTL14-dependent m6A can regulate miR-100-3p maturation via DGCR8 and affect skin fibroblasts photoaging through miR-100-3p/ERRFI1 axis.

MiR-132/212 Impairs Cardiomyocytes Contractility in the Failing Heart by Suppressing SERCA2a.

Compromised cardiac function is a hallmark for heart failure, mostly appearing as decreased contractile capacity due to dysregulated calcium handling. Unfortunately, the underlying mechanism causing impaired calcium handling is still not fully understood. Previously the miR-132/212 family was identified as a regulator of cardiac function in the failing mouse heart, and pharmaceutically inhibition of miR-132 is beneficial for heart failure. In this study, we further investigated the molecular mechanisms of miR-132/212 in modulating cardiomyocyte contractility in the context of the pathological progression of heart failure. We found that upregulated miR-132/212 expressions in all examined hypertrophic heart failure mice models. The overexpression of miR-132/212 prolongs calcium decay in isolated neonatal rat cardiomyocytes, whereas cardiomyocytes isolated from miR-132/212 KO mice display enhanced contractility in comparison to wild type controls. In response to chronic pressure-overload, miR-132/212 KO mice exhibited a blunted deterioration of cardiac function. Using a combination of biochemical approaches and in vitro assays, we confirmed that miR-132/212 regulates SERCA2a by targeting the 3′-end untranslated region of SERCA2a. Additionally, we also confirmed PTEN as a direct target of miR-132/212 and potentially participates in the cardiac response to miR132/212. In end-stage heart failure patients, miR-132/212 is upregulated and correlates with reduced SERCA2a expression. The up-regulation of miR-132/212 in heart failure impairs cardiac contractile function by targeting SERCA2a, suggesting that pharmaceutical inhibition of miR-132/212 might be a promising therapeutic approach to promote cardiac function in heart failure patients.

Co-infection with Border Disease Virus and Brucella melitensis in an Aborted Sheep Foetus

In this study, we investigated the potential roles of BDV and Brucella melitensis infections in a case of sheep abortion. Internal organ specimens from aborted sheep foetus and EDTA whole blood sample from mother of the foetus were collected from a sheep flock in the Konya Province in the Central Anatolia region of Turkey in 2017. The presence of Brucella spp. directly assessed by bacterial isolation and detection of BDV was carried by real time RT-PCR. Genetic characterization of the BDV field isolate was conducted by sequencing the 5’- end untranslated region (UTR) region of BDV. Brucella strain was isolated from the samples of aborted sheep foetus, and it was identified as Brucella melitensis by biochemical characteristics, agglutination with monospecific A and M sera. BDV RNA was detected in EDTA whole blood sample and aborted sheep foetus. Phylogenetic analysis in 5’-UTR region allocated the field isolate of BDV obtained in this study into BDV-7 genotype. To the best of our knowledge, this is the first report on the dual infection of aborted sheep foetus with BDV and Brucella melitensis.

Overview of MicroRNAs in Cardiac Hypertrophy, Fibrosis, and Apoptosis.

MicroRNAs (miRNAs) are non-coding RNAs that play essential roles in modulating the gene expression in almost all biological events. In the past decade, the involvement of miRNAs in various cardiovascular disorders has been explored in numerous in vitro and in vivo studies. In this paper, studies focused upon the discovery of miRNAs, their target genes, and functionality are reviewed. The selected miRNAs discussed herein have regulatory effects on target gene expression as demonstrated by miRNA/3′ end untranslated region (3′UTR) interaction assay and/or gain/loss-of-function approaches. The listed miRNA entities are categorized according to the biological relevance of their target genes in relation to three cardiovascular pathologies, namely cardiac hypertrophy, fibrosis, and apoptosis. Furthermore, comparison across 86 studies identified several candidate miRNAs that might be of particular importance in the ontogenesis of cardiovascular diseases as they modulate the expression of clusters of target genes involved in the progression of multiple adverse cardiovascular events. This review illustrates the involvement of miRNAs in diverse biological signaling pathways and provides an overview of current understanding of, and progress of research into, of the roles of miRNAs in cardiovascular health and disease.

First Report of Catharanthus mosaic virus in Mandevilla in the United States.

Mandevilla (Apocynaceae) is an ornamental tropical vine popular for its bright and attractive flowers. During 2012 to 2013, 12 Mandevilla sp. samples from Minnesota and Florida nurseries were submitted for analysis at the University of Minnesota Plant Disease Clinic. Plants showed mosaic symptoms, leaf deformation, premature leaf senescence, and vine dieback. Filamentous virus particles with modal lengths 700 to 900 nm were observed by transmission electron microscopy (TEM) in partially purified preparations from symptomatic leaves. Partially purified virions were obtained using 30% sucrose cushion centrifuged at 109,000 gmax for 2 h at 10°C (5). No other virus particles were observed in these samples, nor were any observed in non-symptomatic samples. One sample was submitted as potted plant (Mandevilla 'Sunmandeho' Sun Parasol Giant White) and was kept under greenhouse conditions for subsequent analyses. Total RNA (Qiagen) was extracted from this sample, and Potyvirus was detected using the universal primers Poty S (5'-GGN AAY AAY AGY GGN CAR CC-3') and PV1 (5'-20(T)V-3') (1) by reverse transcription (RT)-PCR (3). The amplified product was the expected ~1.7-kb, corresponding to the partial nuclear inclusion body gene, the coat protein (CP) gene, and the 3' end untranslated region. The RT-PCR amplicon was cloned (NEB) and sequenced, and the 1,720-bp consensus sequence was deposited in GenBank (Accession No. KM243928). NCBI BLAST analysis at the nucleotide level revealed highest identity (83%) with an isolate of Catharanthus mosaic virus (CatMV) from Brazil (Accession No. DQ365928). Pairwise analysis of the predicted 256 amino acid CP revealed 91% identity with the CatMV Brazilian isolate (ABI94824) and 68% or less identity with other potyviruses. Two potyviruses are usually considered the same species if their CP amino acid sequences are greater than 80% identical (2). Serological analysis of the infected sample Mandevilla 'Sunmandeho' Sun Parasol Giant White using a CatMV specific antiserum (4) resulted in positive indirect ELISA reactions. CatMV has been previously reported in periwinkle (Catharanthus roseus) in Brazil (4). Based on the analyses by TEM, RT-PCR, nucleotide and amino acid sequence identities, and serological reactivity, we identify this virus as a U.S. Mandevilla isolate of CatMV. To our knowledge, this is the first report of Catharanthus mosaic virus both in the United States and in Mandevilla.

First Report of Nerine yellow stripe virus in Amaryllis in the United States.

Ornamental flower bulbs (including true bulbs, bulbils, corms, tubers, and rhizomes) are increasingly important floriculture crops. Amaryllis is a small genus of flowering bulbs, with two species. The South African native, Amaryllis belladonna, also known as belladonna lily, Jersey lily, naked lady, Amarillo, or March lily, is one of numerous ornamental species with the common name "lily" due to their flower shape and growth habit. Amaryllis are popular for their 6- to 10-inch trumpet shaped colorful flowers that are borne on 1- to 2-foot stalks. In January, 2011, a home gardener in California observed mosaic symptoms on the leaves of A. belladonna growing in her garden. Leaf samples were sent to Agdia Inc. for testing. Samples tested positive for the presence of Potyvirus in a reverse transcription (RT)-PCR screen using universal potyvirus primers (2) yielding the expected ∼1,600-bp product corresponding to the partial nuclear inclusion body (NIb) gene, full-length coat protein (CP) gene, and 3' end untranslated region (UTR). Electron microscopy of symptomatic leaves confirmed the presence of filamentous potyvirus-like particles. The RT-PCR amplicon was cloned and sequenced (2); the 1,616-bp consensus sequence was deposited in GenBank (Accession No. JX865782). NCBI BLAST analysis of the consensus sequence revealed highest identities with isolates of Nerine yellow stripe virus (NeYSV; family Potyviridae, genus Potyvirus). Pair-wise analyses of the 261 amino acid sequence of the predicted CP had 88% sequence identity with a Stenomesson isolate reported from the Netherlands (EU042758); 87% identity with Hymenocallis and Nerine isolates, both also from the Netherlands (EF362622 and EF362621, respectively); and, 86% with two New Zealand isolates infecting Amaryllis or Vallota (FJ618537 and DQ407932, respectively). The five Netherlands and New Zealand isolates are more closely related to each other than to the U.S. isolate as they share 93 to 98% CP identity. When using viral genome sequence relatedness as a criterion for defining potyvirus species, isolates with CP amino acid identity greater than 80% are considered the same species (1). The predicted coat protein gene of the California isolate was sub-cloned into the bacterial expression vector pET44 EK/LIC. Serological analysis of coat protein expressing clones in ELISA and Western Blot analysis using a potyvirus broad-spectrum reacting monoclonal antibody PTY-2 (3) and a NeYSV-specific rabbit antiserum (Applied Plant Research, Lisse, The Netherlands) resulted in positive reactions. NeYSV has previously been reported in the United Kingdom, the Netherlands, Australia, and New Zealand. Based on the results of electron microscopy, RT-PCR, nucleotide and amino acid identity, and serological reactivity, we identify this virus as a U.S. isolate of NeYSV, NeYSV-US. To our knowledge, this is the first report of Nerine yellow stripe virus in the United States. Development of antisera specific to this U.S. isolate is in progress.

Comprehensive Transcriptome Analysis of the Periodontopathogenic Bacterium Porphyromonas gingivalis W83

High-density tiling microarray and RNA sequencing technologies were used to analyze the transcriptome of the periodontopathogenic bacterium Porphyromonas gingivalis. The compiled P. gingivalis transcriptome profiles were based on total RNA samples isolated from three different laboratory culturing conditions, and the strand-specific transcription profiles generated covered the entire genome, including both protein coding and noncoding regions. The transcription profiles revealed various operon structures, 5'- and 3'-end untranslated regions (UTRs), differential expression patterns, and many novel, not-yet-annotated transcripts within intergenic and antisense regions. Further transcriptome analysis identified the majority of the genes as being expressed within operons and most 5' and 3' ends to be protruding UTRs, of which several 3' UTRs were extended to overlap genes carried on the opposite/antisense strand. Extensive antisense RNAs were detected opposite most insertion sequence (IS) elements. Pairwise comparative analyses were also performed among transcriptome profiles of the three culture conditions, and differentially expressed genes and metabolic pathways were identified. With the growing realization that noncoding RNAs play important biological functions, the discovery of novel RNAs and the comprehensive transcriptome profiles compiled in this study may provide a foundation to further understand the gene regulation and virulence mechanisms in P. gingivalis. The transcriptome profiles can be viewed at and downloaded from the Microbial Transcriptome Database website, http://bioinformatics.forsyth.org/mtd.

Hippeastrum mosaic virus diagnosed in Hippeastrum and Eucharis in Brazil.

SUMMARY A potyvirus associated with foliar mosaic symptoms displayed by Hippeastrum (Hip-Br) and two Eucharis samples (Euch-B and Euch-C) from three different cities of Brazil was detected and identified by transmission tests, electron microscopy, reverse-transcription, RT-PCR and sequencing. Transmission electron microscopy revealed the presence of elongated, flexuous viral particles in the extracts of symptomatic leaves, while in thin sections of symptomatic leaves, cytoplasmic aggregates of type II lamellar inclusions (Edwardson’s classification) were observed. The experimental host range was restricted. RT-PCR using universal potyvirus primers amplified DNA products of 1,499 bp corresponding to the partial nuclear inclusion body (NIb) gene, full-length coat protein (CP) gene and 3’end untranslated region (UTR). Pairwise analyses from CP and NIb nucleotide sequences of Hip-Br, Euch-B and Euch-C viruses and other potyviruses revealed high identities with previously reported Alaska (GQ857550) and Rilona (EF203685) Hippeastrum mosaic virus (HiMV) isolates, Amazon lily mosaic virus (ALiMV, AY590143), and Amaryllis potyvirus (AmaPV, AY566239). The phylogeny calculated by a maximum likelihood method showed that Hip-Br, Euch-B and Euch-C formed a monophyletic group supported by 100% bootstrap with HiMV. This is the first report of HiMV in Hippeastrum sp. and Eucharis sp. from Brazil. Our data support the taxonomic position of HiMV and other potyviruses isolated from amaryllis and Amazon lily in a separated cluster of Amazon lily mosaic virus (AB158523).

Transgene‐mediated suppression of dengue viruses in the salivary glands of the yellow fever mosquito, Aedes aegypti

Controlled sex-, stage- and tissue-specific expression of antipathogen effector molecules is important for genetic engineering strategies to control mosquito-borne diseases. Adult female salivary glands are involved in pathogen transmission to human hosts and are target sites for expression of antipathogen effector molecules. The Aedes aegypti 30K a and 30K b genes are expressed exclusively in adult female salivary glands and are transcribed divergently from start sites separated by 263 nucleotides. The intergenic, 5'- and 3'-end untranslated regions of both genes are sufficient to express simultaneously two different transgene products in the distal-lateral lobes of the female salivary glands. An antidengue effector gene, membranes no protein (Mnp), driven by the 30K b promoter, expresses an inverted-repeat RNA with sequences derived from the premembrane protein-encoding region of the dengue virus serotype 2 genome and reduces significantly the prevalence and mean intensities of viral infection in mosquito salivary glands and saliva.

Pestivirus infection in sheep and goats in West Austria

Blood samples from 3112 sheep (185 flocks) and 1196 goats (163 flocks) from the Western region of Austria were tested for pestivirus-specific RNA. In this area, communal Alpine pasturing of sheep, cattle and goats is an important part of farming. The prevalence of sheep persistently-infected (PI) with pestivirus was 0.32% (10 animals) and the PI animals originated from five flocks (2.7% of those investigated). In goats, only one PI animal (0.08%) was detected. Sequence analysis of the 5'-end untranslated region (UTR) revealed that the strains of Border disease virus (BDV) detected were closely related to genotype 3 but the PI animals did not show any clinical signs of Border disease. The goat was PI with bovine viral diarrhoea virus-1 (BVDV-1). On one farm a high abortion rate among sheep had been observed 1year before the study was carried out but the other farms did not show any evidence of reproductive failures. Pestiviruses are endemic in small ruminants in some Alpine regions of Austria and PI healthy animals as described here have a key epidemiological role. A successful BVDV eradication programme in Austria will create highly pestivirus-susceptible cattle populations. Sheep and goats present a high risk for the reintroduction of pestiviruses to cattle herds because they are less likely to be considered to be PI. The results underline the need for the immediate consideration of small ruminants in eradication programmes.

Deletion/insertion polymorphism of the prion protein gene (PRNP) in Polish Red cattle, Polish White-backed cattle and European bison (Bison bonasus L., 1758)

The aim of the present study was to identify deletion/insertion polymorphism of the bovine prion protein (PRNP) gene within the promoter sequence (23 bp indel), intron 1 (12 bp indel) and the 3' end untranslated region (14 bp indel). The experiment was performed on three groups of animals protected under a genetic resources conservation program: 139 Polish Red (PR) cows, 79 Polish White-backed cows and 50 European bison (Bison bonasus L., 1758). White-backed cattle were characterized by a higher frequency of ins/del heterozygotes and a relatively lower frequency of ins/ins homozygotes within the promoter sequence region (23 bp indel), compared to Polish Red cattle. At the polymorphic locus of intron 1 (12 bp indel) the genetic structure of both cattle populations was similar. Monomorphism, expressed by the occurrence of one genotype variant in each of the analyzed sequence regions, was observed in European bison. Five haplotypes were found in Polish White-backed cows, four haplotypes in Polish Red cows and only one in analyzed group of bison. Differences between the observed and expected number of PRNP haplotypes were recorded in Polish Red cattle.

MRNA processing in Antonospora locustae spores

Microsporidia are a group of intracellular parasites characterized by highly reduced and compact genomes. The presence of a high gene density had several consequences for microsporidian genomes, including a high frequency of overlap between transcripts of adjacent genes. This phenomenon is apparently widespread in microsporidia, and strongly correlated with gene density. However, all analyses to date have focused on one or a few transcripts from many loci, so it is unclear how diverse the pool of transcripts at a given locus may be. To address this question, we characterized initiation and termination points from 62 transcripts in gene-dense regions in Antonospora locustae spores using both conventional and fluorescence-based RACE-PCR procedures. In parallel, we investigated the abundance and nature of transcripts along a 6 kb region surrounding the actin locus of A. locustae using northern blotting, RACE-PCR and previously characterised EST sequences. Overall, we confirmed previous suggestions that most transcripts in A. locustae spores overlap with the downstream gene, but that at the 5' end untranslated regions are very short and overlap is rare. From fluorescence-based RACE-PCR we show that transcription of most genes (31 out of 34) initiates at a single position, whereas 35% of loci analyzed with 3' RACE polyadenylate mRNA at several sites. Finally, we identified the presence of previously unsuspected and very large transcripts in A. locustae spores. Those transcripts were found to overlap up to four open reading frames in different strands, adding a novel layer of complexity in the mRNA transcription of this microsporidian species.

Molecular cloning and ontogenesis expression of fatty acid transport protein-1 in yellow-feathered broilers

Fatty acid transport protein-1 (FATP-1) is one of the important transporter proteins involved in fatty acid transmembrane transport and fat deposition. To study the relationship between FATP-1 mRNA expression and fat deposition, chicken ( Gallus gallus) FATP-1 sequence was first cloned by rapid amplification of cDNA ends (RACE). Tissue samples of chest muscle, leg muscle, subcutaneous fat, and abdominal fat were collected from six male and six female broilers each, at 22 days, 29 days, and 42 days, respectively. The tissue specificity and ontogenesis expression pattern of the FATP-1 mRNA of yellow-feathered broilers was studied by real-time reverse transcription polymerase chain reaction (RT-PCR), and the fat deposition laws in different tissues were also compared. A 2,488 bp cDNA sequence of chicken FATP-1 was cloned by RACE (GenBank accession no. DQ352834), including 547 bp 3′ end untranslated region (URT) and 1,941 bp open reading frame (ORF). Chicken FATP-1 encoded 646 amino acid residues, which shared 83.9% and 83.0% identity with those of human and rat, respectively. The results of quantitative PCR demonstrated a constant FATP-1 mRNA expression level in the chest muscle and subcutaneous fat of both male and female broilers at three stages, whereas the expression level of the FATP-1 mRNA in the leg muscle at 42 days was significantly higher than that at 22 days or 29 days. In the abdominal fat of male broilers, the gene expression significantly increased with age, whereas the female broilers showed a dramatic downregulation of FATP-1 expression in abdominal fat at 42 days. This suggested a typical tissue- and gender-specific expression pattern of chicken FATP-1, mediating the specific process of fatty acid transport or utilization in muscle and adipose tissues.

Complete Structure, Genomic Organization, and Expression of Channel Catfish (Ictalurus punctatus, Rafinesque 1818) Matrix Metalloproteinase-9 Gene

In this study, the channel catfish (CC) matrix metalloproteinase-9 (MMP-9) gene was cloned, sequenced, and characterized at both the cDNA and the genomic DNA levels. The complete sequence of the CC MMP-9 cDNA consisted of 2,551 nucleotides, including one open reading frame and 5'- and 3'-end untranslated regions. The open reading frame potentially encoded a 686-amino-acid peptide with a calculated molecular mass (without glycosylation) of approximately 77.4 kDa, which included a signal peptide and potentially heavy O-glycosylation sites. CC MMP-9 did not have the tripeptide Arg-Gly-Asp motif. The degree of conservation of the CC MMP-9 amino acid sequence to human and mouse counterparts was 55%, while to those of other fish species was 67-74%. The full-length CC MMP-9 genomic DNA comprised 5,663 nucleotides, much shorter than human or mouse counterparts. The exon-intron structure followed the splice acceptor/donor consensus rule, and the sequence contained 13 exons. The MMP-9 transcript was constitutively expressed in restrictive CC tissues. This result should provide fundamental information for further exploration of the role of MMP-9 in fish pathophysiology.

Genes associated with resistance to wheat yellow rust disease identified by differential display analysis

This study is focused on the identification of early response genes involved in the resistance mechanism of one of the most important diseases of wheat, yellow rust. The strategy undertaken was to use differential display reverse transcriptase-PCR method (DDRT-PCR) on two of the yellow rust differential lines of wheat, were infected with the virulent and the avirulent Puccinia striiformis f. sp. tritici races together with appropriate control inoculations. Upon infection of the plant materials (resistant and/or susceptible), different time points were evaluated on DDRT-PCR banding profiles. The expression level differences of 33 genes in DDRT-PCR were further analyzed using quantitative real-time PCR (qRT-PCR). Among which only those (14 gene fragments) resulting reproducible induction or repression levels were reported with measured fold changes. Based on the sequence similarity analysis, all but one were absent in NCBI databank, thus they are newly found wheat sequences. Among those, two genes were identified as full ORF, including 5′ and 3′ end untranslated regions (UTR); namely cyclophilin like protein (putative antifungal activity) and ubiquitin-conjugating enzyme (E2) or Rad6. The sequence homology analysis of the cloned gene fragments revealed that the genes detected may have roles in ubiquitinylation, programmed cell death (apoptosis), putative antifungal activities, disease resistance responses, pathogenesis related responses, and including a few with no known function.

Functional characterization of the promoter of the vitellogenin gene, AsVg1, of the malaria vector, Anopheles stephensi

Some genetic strategies for controlling transmission of mosquito-borne diseases call for the introgression of antipathogen effector genes into vector populations. Endogenous mosquito promoter and other cis-acting DNA sequences are needed to direct the expression of the effector molecules to maximize their efficacy. Vitellogenin (Vg)-encoding gene control sequences are candidates for driving tissue-, stage- and sex-specific expression of exogenous genes. One of the Anopheles stephensi Vg genes, AsVg1, was cloned and a full-length cDNA, as well as 850 base pairs adjacent to the 5′-end, were sequenced and characterized. Expression of AsVg1 is restricted to the fat body tissues of blood-fed females, and the amino acid sequence of the conceptual translation product is >85% identical to those of other anopheline Vgs. These characteristics support the conclusion that AsVg1 is a Vg-encoding gene. Functional analyses of the AsVg1 putative cis-regulatory sequences were performed using transgenic mosquitoes. The results showed that DNA fragments encompassing the 850 base pairs immediately adjacent to the 5′-end of the gene and the 3′-end untranslated region are sufficient to direct sex-, stage- and tissue-specific expression of a reporter gene. These data indicate that the AsVg1 promoter is a good candidate for controlling the expression of anti-pathogen effector molecules in this malaria vector mosquito.

Analysis of SD sequences in completed microbial genomes: Non-SD-led genes are as common as SD-led genes

Shine–Dalgarno (SD) sequence has been considered as one of the common features of 5′ end untranslated region (5′UTR) of prokaryotic transcripts. However, more leaderless bacteria and archaea mRNAs are being increasingly reported in recent years. To understand the distribution of SD-led genes and non-SD-led genes, we have analyzed 162 completed prokaryotic genomes leading to various new conclusions and validations of previous smaller scale studies. The fact that the number of the SD-led genes among those genomes varies from 11.6% to 90.8% implies that the populations of non-SD-led genes as well as leaderless genes are significant. We found that there is a strong SD conserved region in genomes with high proportion of SD-led genes. Following a t-test we showed that SD sequence content (SDSC) has no correlation with GC content. We observed that the closely related phylogenetic microbes mostly possess a similar SDSC value, and archaeal nonleading genes possess higher SDSC. This study shows that the 5′UTR of prokaryotic genes are highly diverse, particularly when genomes of distantly related organisms are compared, suggesting that more flexible mechanisms are used for translation initiation process in various prokaryotes.

Two different genes encoding acetylcholinesterase existing in cotton aphid (Aphis gossypii).

Two acetylcholinesterase (AChE) genes, Ace1 and Ace2, have been cloned from cotton aphid, Aphis gossypii Glover, using the rapid amplification of cDNA ends (RACE) technique. To the best of our knowledge, this should be the first direct molecular evidence that multiple AChE genes exist in insects. The Ace1 gene was successfully amplified along its full length of 2371 bp. The open reading frame is 2031 bp long and encodes 676 amino acids (GenBank accession No. AF502082). The Ace2 gene was amplified as a mega-fragment of 2130 bp lacking part of 5'-end untranslated region (UTR). The open reading frame is 1992 bp long and ecodes a protein of 664 amino acids (GenBank accession No. AF502081). Both genes have the conserved amino acids and features shared by the AChE family, but share only 35% identity in amino acid sequence. The Ace1 gene is highly homologous to the AChE gene of Schizaphis graminum (AF321574) with 95% identity, and Ace2 to that of Myzus persicae (AF287291) with 92% identity. Phylogenetic analysis showed that the two cloned AChEs of A. gossypii are different in evolution. The phylogenetic tree generated by the PHYLIP program package inferred that AChE2 of A. gossypii is a more ancestral form of AChE. Homology modeling of structures using Torpedo californica (2ACE_) and Drosophila melanogaster (1Q09:A) native acetylcholinesterase structure as main template indicated that the two AChEs of Aphis gossypii might have different three-dimensional structures. Alternative splicing of Ace1 near the 5'-end resulting in two proteins differing by the presence or absence of a fragment of four amino acids is also reported.

Stage-dependent Localization of a Novel Gene Product of the Malaria Parasite, Plasmodium falciparum

A novel Plasmodium falciparum gene, MB2, was identified by screening a sporozoite cDNA library with the serum of a human volunteer protected experimentally by the bites of P. falciparum-infected and irradiated mosquitoes. The single-exon, single-copy MB2 gene is predicted to encode a protein with an M(r) of 187,000. The MB2 protein has an amino-terminal basic domain, a central acidic domain, and a carboxyl-terminal domain with similarity to the GTP-binding domain of the prokaryotic translation initiation factor 2. MB2 is expressed in sporozoites, the liver, and blood-stage parasites and gametocytes. The MB2 protein is distributed as a approximately 120-kDa moiety on the surface of sporozoites and is imported into the nucleus of blood-stage parasites as a approximately 66-kDa species. Proteolytic processing is favored as the mechanism regulating the distinct subcellular localization of the MB2 protein. This differential localization provides multiple opportunities to exploit the MB2 gene product as a vaccine or therapeutic target.