Divergence of 3′ ends as a driver of short interspersed nuclear element (SINE) evolution in the Salicaceae

Short interspersed nuclear elements (SINEs) are small, non‐autonomous and heterogeneous retrotransposons that are widespread in plants. To explore the amplification dynamics and evolutionary history of SINE populations in representative deciduous tree species, we analyzed the genomes of the six following Salicaceae species: Populus deltoides, Populus euphratica, Populus tremula, Populus tremuloides, Populus trichocarpa, and Salix purpurea. We identified 11 Salicaceae SINE families (SaliS‐I to SaliS‐XI), comprising 27 077 full‐length copies. Most of these families harbor segmental similarities, providing evidence for SINE emergence by reshuffling or heterodimerization. We observed two SINE groups, differing in phylogenetic distribution pattern, similarity and 3′ end structure. These groups probably emerged during the ‘salicoid duplication’ (~65 million years ago) in the Salix–Populus progenitor and during the separation of the genus Salix (45–65 million years ago), respectively. In contrast to conserved 5′ start motifs across species and SINE families, the 3′ ends are highly variable in sequence and length. This extraordinary 3′‐end variability results from mutations in the poly(A) tail, which were fixed by subsequent amplificational bursts. We show that the dissemination of newly evolved 3′ ends is accomplished by a displacement of older motifs, leading to various 3′‐end subpopulations within the SaliS families.     

A novel tetrameric short tandem repeat located in the 3' flanking region of the human ABO-secretor gene (FUT2) and association between FUT2 and FUT2/01 loci

We found a novel polymorphic short tandem repeat (FUT2/01), 3.8 kb downstream of the coding region of FUT2. Seventeen length and 33 sequence variants were identified in 300 individuals representing three major human populations. Africans (Xhosa) and Europeans were characterized by high microvariation, and Japanese were characterized by a simple repeat structure. All exhibited high haplotype diversity.     

Presence of poly(A) in a flavivirus: significant differences between the 3' noncoding regions of the genomic RNAs of tick-borne encephalitis virus strains.

A poly(A) tail was identified on the 3' end of the prototype tick-borne encephalitis (TBE) virus strain Neudoerfl. This is in contrast to the general lack of poly(A) in the genomic RNAs of mosquito-borne flaviviruses analyzed so far. Analysis of several closely related strains of TBE virus, however, revealed the existence of two different types of 3' noncoding (NC) regions. One type (represented by strain Neudoerfl) is only 114 nucleotides long and carries a 3'-terminal poly(A) structure. This was also found in several TBE virus strains isolated from different geographic regions over a period of almost 30 years. The other type (represented by strain Hypr) is 461 nucleotides long and not polyadenylated. The sequence homology between the two types of TBE virus 3' NC regions terminates at a specific position 81 nucleotides after the stop codon. The second type of 3' NC region more closely resembles the common flavivirus pattern, including the potential for the formation of a 3'-terminal hairpin structure. However, it lacks primary sequence elements that are conserved among other flavivirus genomes.     

Specific interaction of HeLa cell proteins with coxsackievirus B3 3'UTR: La autoantigen binds the 3' and 5'UTR independently of the poly(A) tail

Coxsackievirus B3 (CVB3) is a positive, single-stranded RNA virus. The secondary structure of the 3' untranslated region (3'UTR) of CVB3 RNA consists of three stem-loops and is followed by a poly(A) tail sequence. These stem-loop structures have been suggested to participate in the regulation of viral replication through interaction with cellular proteins that are yet to be identified. In this study, by competitive UV cross-linking using mutated 3'UTR probes we have demonstrated that the poly(A) tail is essential for promoting HeLa cell protein interactions with the 3'UTR because deletion of this sequence abolished most of the protein interactions. Unexpectedly, mutations that disrupted the tertiary loop-loop interactions without affecting the stem-loops did not apparently affect these protein interactions, indicating that secondary structure rather than the high-order structure may play a major role in recruiting these RNA binding proteins. Among the observed 3'UTR RNA binding proteins, we have confirmed a 52 kDa protein as the human La autoantigen by using purified recombinant protein and a polyclonal La antibody. This protein can interact with both the 3' and 5'UTRs independently of the poly(A) tail. Further analysis by two-stage UV cross-linking, we found that the 3' and 5'UTR sequences may share the same binding site on the La protein.     

The light switch effect upon the association of [Ru(1,10-phenanthroline)2dipyrido[3,2-a:2',3'-c]phenazine]2+ with single stranded poly(dA) and poly(dT)

Large enhancement in the luminescence intensity of the Delta- and Lambda-Ru(phenanthroline)(2)dipyrido[3,2-a:2',3'-c]phenazine](2+) ([Ru(phen)(2)DPPZ](2+)) complexes upon their association with single stranded poly(dA) and poly(dT) is reported in this work. As the mixing ratio ([[Ru(phen)(2)DPPZ](2+)]/[DNA base]) increases, the luminescence intensity increase in a sigmoidal manner, indicating that the enhancement involves some cooperativity. At a high mixing ratio, the luminescence properties are affected by the nature of the DNA bases and not by the absolute configuration of the [Ru(phen)(2)DPPZ](2+) complex, indicating that the single stranded poly(dA) and poly(dT) do not recognize the configuration of the metal complex. In the case of the Lambda-[Ru(phen)(2)DPPZ](2+)-poly(dT) complex, the manner of the enhancement is somewhat different from the other Ru(II) complex-polynucelotide combinations: the luminescence intensity reached a maximum at an intermediate mixing ratio of 0.32, and gradually decreased as the mixing ratio increased. In contrast to other complexes at high mixing ratios, an upward bending curve was found in the Stern-Volmer plot, which indicates that the micro-environment of the Lambda-[Ru(phen)(2)DPPZ](2+) is heterogeneous. In the Delta-[Ru(phen)(2)DPPZ](2+)-poly(dT) complex case, formation of this highly luminescent species at an intermediate mixing ratio is far less effective.     

Comparison between precipitin and ELISA tests in the bloodmeal detection of Aedes aegypti (Linnaeus) and Aedes fluviatilis (Lutz) mosquitoes experimentally fed on feline, canine and human hosts.

The identification of arthropod bloodmeals is important in many epidemiological studies, as, the understanding of the life cycle of vectors and the pathogens they transmit, as well as helping to define arthropods' control strategies. The precipitin test has been used for decades, but ELISA is slowly becoming more popular. To compare the two tests for sensitivity, specificity and accuracy to detect small insect bloodmeals, Aedes aegypti or Ae. fluviatilis mosquitoes were fed either on feline, canine or human hosts. Mosquitoes were frozen at 6, 12, 24, 48 or 72 h after feeding. Precipitin test showed better specificity and accuracy and ELISA test showed higher sensitivity. Better results with both tests were achieved when mosquitoes were frozen within 48 h from feeding.     

Host protein interactions with the 3' end of bovine coronavirus RNA and the requirement of the poly(A) tail for coronavirus defective genome replication

RNA viruses have 5' and 3' untranslated regions (UTRs) that contain specific signals for RNA synthesis. The coronavirus genome is capped at the 5' end and has a 3' UTR that consists of 300 to 500 nucleotides (nt) plus a poly(A) tail. To further our understanding of coronavirus replication, we have begun to examine the involvement of host factors in this process for two group II viruses, bovine coronavirus (BCV) and mouse hepatitis coronavirus (MHV). Specific host protein interactions with the BCV 3' UTR [287 nt plus poly(A) tail] were identified using gel mobility shift assays. Competition with the MHV 3' UTR [301 nt plus poly(A) tail] suggests that the interactions are conserved for the two viruses. Proteins with molecular masses of 99, 95, and 73 kDa were detected in UV cross-linking experiments. Less heavily labeled proteins were also detected in the ranges of 40 to 50 and 30 kDa. The poly(A) tail was required for binding of the 73-kDa protein. Immunoprecipitation of UV-cross-linked proteins identified the 73-kDa protein as the cytoplasmic poly(A)-binding protein (PABP). Replication of the defective genomes BCV Drep and MHV MIDI-C, along with several mutants, was used to determine the importance of the poly(A) tail. Defective genomes with shortened poly(A) tails consisting of 5 or 10 A residues were replicated after transfection into helper virus-infected cells. BCV Drep RNA that lacked a poly(A) tail did not replicate, whereas replication of MHV MIDI-C RNA with a deleted tail was detected after several virus passages. All mutants exhibited delayed kinetics of replication. Detectable extension or addition of the poly(A) tail to the mutants correlated with the appearance of these RNAs in the replication assay. RNAs with shortened poly(A) tails exhibited less in vitro PABP binding, suggesting that decreased interactions with the protein may affect RNA replication. The data strongly indicate that the poly(A) tail is an important cis-acting signal for coronavirus replication.     

Monitoring of resistance to the pyrethroid cypermethrin in Brazilian Aedes aegypti (Diptera: Culicidae) populations collected between 2001 and 2003

Resistance to cypermethrin of different Aedes aegypti Brazilian populations, collected at two successive periods (2001 and 2002/2003), was monitored using the insecticide-coated bottles bioassay. Slight modifications were included in the method to discriminate between mortality and the knock down effect. Although this pyrethroid was recently started to be used in the country to control the dengue vector, a decrease in susceptibility was noted between both periods analyzed, particularly in the city of Rio de Janeiro. The results indicate that resistance is due at least in part to a target site alteration.     

Translational control of dosage compensation in Drosophila by Sex-lethal: cooperative silencing via the 5' and 3' UTRs of msl-2 mRNA is independent of the poly(A) tail.

Translational repression of male-specific-lethal 2 (msl-2) mRNA by Sex-lethal (SXL) controls dosage compensation in Drosophila. In vivo regulation involves cooperativity between SXL-binding sites in the 5' and 3' untranslated regions (UTRs). To investigate the mechanism of msl-2 translational control, we have developed a novel cell-free translation system from Drosophila embryos that recapitulates the critical features of mRNA translation in eukaryotes: cap and poly(A) tail dependence. Importantly, tight regulation of msl-2 translation in this system requires cooperation between the SXL-binding sites in both the 5' and 3' UTRs, as seen in vivo. However, in contrast to numerous other developmentally regulated mRNAs, the regulation of msl-2 mRNA occurs by a poly(A) tail-independent mechanism. The approach described here allows mechanistic analysis of translational control in early Drosophila development and has revealed insights into the regulation of dosage compensation by SXL.     

Electrophoretic evidence concerning the relationship between Haplometrana and Glypthelmins (Digenea: Plagiorchiiformes)

Analysis of genetic data from a multilocus allozyme study, using cellulose acetate gel electrophoresis, of 2 trematode species, Glypthelmins californiensis Stafford, 1905, and Haplometrana intestinalis Lucker, 1931, from Rana aurora and Rana pretiosa, yielded a Nei's genetic distance coefficient between parasites of 0.70 +/- 0.10. This amount of genetic divergence is characteristic of comparisons between congeneric rather than intergeneric trematode species and supports the conclusion, corroborated by morphology and life history, that H. intestinalis is more closely related to some members of Glypthelmins than to any other species.     

Pstl repeat: a family of short interspersed nucleotide element (SINE)-like sequences in the genomes of cattle, goat, and buffalo.

The PstI family of elements are short, highly repetitive DNA sequences interspersed throughout the genome of the Bovidae. We have cloned and sequenced some members of the PstI family from cattle, goat, and buffalo. These elements are approximately 500 bp, have a copy number of 2 x 10(5) - 4 x 10(5), and comprise about 4% of the haploid genome. Studies of nucleotide sequence homology indicate that the buffalo and goat PstI repeats (type II) are similar types of short interspersed nucleotide element (SINE) sequences, but the cattle PstI repeat (type I) is considerably more divergent. Additionally, the goat PstI sequence showed significant sequence homology with bovine serine tRNA, and is therefore likely derived from serine tRNA. Interestingly, Southern hybridization suggests that both types of SINEs (I and II) are present in all the species of Bovidae. Dendrogram analysis indicates that cattle PstI SINE is similar to bovine Alu-like SINEs. Goat and buffalo SINEs formed a separate cluster, suggesting that these two types of SINEs evolved separately in the genome of the Bovidae.     

Parity and gonotrophic discordance of females of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in the city of São Paulo,SP, Brazil

The objective of this study was to assess the parity, presence of blood in the stomach, and the gonotrophic discordance of females of Aedes aegypti and Aedes albopictus captured in two areas of the city of São Paulo. The captures were undertaken monthly, by aspiration, in the period from January, 2015 to August, 2017. All the females of the two species had their midguts and ovaries dissected to determine the presence of blood and the parity/stage of maturation. With regard to parity, 27% and 34% of the females of Ae. aegypti and Ae. albopictus, respectively, were parous or were in advanced stages of the development of their ovaries (33% and 27%, respectively). The larger part of the females of Ae. aegypti and Ae. albopictus contained blood in their stomachs (77% and 60%, respectively), beyond which 36% and 27% of the females of Ae. aegypti and Ae. albopictus, respectively, were in gonotrophic discordance. Our results indicate favorable conditions in the study areas because of the presence of parous females. Moreover, this frequent and multiple contact of Ae. aegypti and Ae. albopictus females with vertebrate hosts, such as humans, increases the possibility of transmitting the viruses they may be carrying.