Nucleotide Segment Research Articles

A multiplex PCR assay for the identification of 5 sibling species of the Lindesayi Complex of the genus Anopheles (Diptera: Culicidae) in Bhutan.

Five sibling species of the Lindesayi Complex of the genus Anopheles have been discovered in Bhutan: An. druki Somboon, Namgay & Harbach, An. himalayensis Somboon, Namgay & Harbach, An. lindesayi Giles, An. lindesayi species B, and An. thimphuensis Somboon, Namgay & Harbach. The species are morphologically similar in the adult and/or immature stages. This study aimed to develop a multiplex PCR assay to identify the 5 species. Allele-specific primers were designed for specific nucleotide segments of ITS2 sequences previously reported for each species. The assay provided products of 183 bp for An. druki, 338 bp for An. himalayensis, 126 bp for An. lindesayi, 290 bp for An. lindesayi species B, and 370 bp for An. thimphuensis. The use of the assay produced consistent results. The assay is relatively inexpensive, enables the rapid identification of a large number of specimens, and will foster further studies of the Lindesayi Complex.

Genetic and genealogical (genealogical) study of the genus Nilogovoi (based on the DNA of autosomes)

The article deals with the problem of genetic and genealogical (genealogical) verification of the documentary pedigree of the Nilogovs, reconstructed by the methods of classical genealogy. Thanks to the testing of male and female representatives of the genus Nilogov on the DNA of autosomes, it was possible to genealogically confirm the documentary pedigree of the author of the article to his direct ancestor Semyon Danilovich Nilogov, who lived in the XVIII century. The object of study is the biological pedigree of the Nilogovs, and the subject is the archival and documentary reconstruction of the pedigree of the Nilogovs before the XVIII century. The source base of the work was the collections of such archives as: the State Archive of the Perm Territory, the Komi-Permyatsky District State Archive, the Russian State Archive of Ancient Acts, the archive of the city of Minusinsk; personal archives of several Nilogov families were also used. The novelty of the work is the application of modern genetic and genealogical methodology for the objectification of a specific documentary pedigree by certifying biological kinship in the "descendant – ancestor" chain. Specifically, the genetic relationship of two distant relatives N. A. Nelogov and R. A. Arinicheva was studied at the level of the sixth degree. Thanks to testing of autosomal DNA of these donors, it was possible to identify their common nucleotide segments (text sequences in the form of nitrogenous bases), after which, using the statistical database of genetic kinship, The Shared cM Project 4.0., it was possible to confirm the documented reconstructed pedigree. The introduction of modern methods of molecular genetics (biological science) into scientific genealogy (historical science) is an actual trend of systemic (interdisciplinary) research.

Optimized protocols for the characterization of Cas12a activities.

The CRISPR-associated (Cas) Cas12a is the effector protein for type V-A CRISPR systems. Cas12a is a sequence-specific endonuclease that targets and cleaves DNA containing a cognate short signature motif, called the protospacer adjacent motif (PAM), flanked by a 20 nucleotide (nt) segment that is complementary to the "guide" region of its CRISPR RNA (crRNA). The guide sequence of the crRNA can be programmed to target any DNA with a cognate PAM and is the basis for Cas12a's current use for gene editing in numerous organisms and for medical diagnostics. While Cas9 (type II effector protein) is widely used for gene editing, Cas12a possesses favorable features such as its smaller size and creation of staggered double-stranded DNA ends after cleavage that enhances cellular recombination events. Collected here are protocols for the recombinant purification of Cas12a and the transcription of its corresponding programmable crRNA that are used in a variety of Cas12a-specific in vitro activity assays such as the cis, the trans and the guide-RNA independent DNA cleavage activities with multiple substrates. Correspondingly, protocols are included for the quantification of the activity assay data using ImageJ and the use of MATLAB for rate constant calculations. These procedures can be used for further structural and mechanistic studies of Cas12a orthologs and other Cas proteins.

Identification of flexible Pif1-DNA interactions and their impacts on enzymatic activities.

Flexible regions in biomolecular complexes, although crucial to understanding structure–function relationships, are often unclear in high-resolution crystal structures. In this study, we showed that single-molecule techniques, in combination with computational modeling, can characterize dynamic conformations not resolved by high-resolution structure determination methods. Taking two Pif1 helicases (ScPif1 and BsPif1) as model systems, we found that, besides a few tightly bound nucleotides, adjacent solvent-exposed nucleotides interact dynamically with the helicase surfaces. The whole nucleotide segment possessed curved conformations and covered the two RecA-like domains of the helicases, which are essential for the inch-worm mechanism. The synergetic approach reveals that the interactions between the exposed nucleotides and the helicases could be reduced by large stretching forces or electrostatically shielded with high-concentration salt, subsequently resulting in reduced translocation rates of the helicases. The dynamic interactions between the exposed nucleotides and the helicases underlay the force- and salt-dependences of their enzymatic activities. The present single-molecule based approach complements high-resolution structural methods in deciphering the molecular mechanisms of the helicases.

Development of a Bicistronic Anti-CD19/CD20 CAR Construct Including Optimization to Abrogate Retroviral Recombination Events

Anti-CD19 chimeric antigen receptor (CAR) T cells can eliminate lymphoma, but CD19 loss from lymphoma can cause anti-CD19 CAR T-cell treatment failure. To address CD19 loss, we designed a bicistronic construct encoding 2 CARs. The first CAR in the bicistronic construct was the anti-CD19 CAR Hu19-CD828Z, which incorporated a human anti-CD19 single-chain variable fragment (scFv) along with CD28 and CD3ζ domains (Brudno et al. Nature Medicine 2020). The 2nd CAR in the construct was a novel anti-CD20 CAR incorporating a human anti-CD20 scFv, plus 4-1BB and CD3ζ domains. This bicistronic CAR construct, designated Hu1928-Hu20BB-original, was encoded by the mouse stem cell virus-based splice-gag (MSGV1) gamma-retroviral vector.Repeated segments of identical DNA sequence can lead to recombination events when retroviral vectors are utilized. Recombination events driven by repeated identical nucleotide segments can occur during retroviral vector production in packaging cells. Recombination events can also occur in transduced T cells during reverse transcription when repeated segments of identical nucleotides on the same nucleotide strand anneal, which leads to strand switching by the reverse transcriptase enzyme and deletion of part of the sequence.We assessed T cells transduced with Hu1928-Hu20BB-original for evidence of recombination. Southern blots of DNA from T cells transduced with Hu1928-Hu20BB-original showed a dominant band of the expected CAR transgene length and no aberrant bands after 21 hours of exposure; however, with extended exposure of 8 days, an unexpected band indicating shorter transgene length became visible. This aberrant band was consistent with deletion of part of the transgene sequence due to recombination. To investigate possible recombination by a more sensitive method, we assessed RNA from transduced T cells by single-molecule real-time RNA sequencing (SMRT-RNAseq, PacBio). Deletions of Hu1928-Hu20BB-original RNA sequence between segments of identical sequence were detected by SMRT-RNAseq. These findings were consistent with recombination. The most common recombination events were between the CD8α hinge and transmembrane (HT) domains of the 2 CARs of Hu1928-Hu20BB-original. These HT domains shared regions of identical nucleic acid sequence. Less commonly, recombination events occurred between segments of identical sequence shared by the heavy-chain domains and the light-chain domains of the two CARs in the construct. The attached table summarizes deletions consistent with recombination in Hu1928-Hu20BB-original RNA from transduced T cells of one patient. Similar results were obtained in T cells of 3 other patients. We confirmed deletions consistent with recombination in CAR RNA transcripts by short-read RNA sequencing (Illumina). Digital droplet PCR analysis of DNA from transduced T cells showed that recombination events between the HT domains of Hu1928-Hu20BB-original were present in DNA of transduced T cells.We modified the DNA sequence of Hu1928-Hu20BB-original to reduce repeated segments of identical DNA sequence to form a new CAR designated Hu1928-Hu20BB-standard. When T cells transduced with Hu1928-Hu20BB-standard were studied by SMRT RNAseq, deletions of RNA sequence consistent with recombination were substantially reduced (Table).We modified Hu1928-Hu20BB-standard by lengthening the linker in the anti-CD20 scFv to form a construct designated Hu1928-Hu20BB-long. We compared Hu1928-Hu20BB-standard to Hu1928-Hu20BB-long in vitro, and both specifically recognized CD19 and CD20 in assays of degranulation, cytokine release, and proliferation. In vitro assays showed higher interleukin-2 release by T cells transduced with Hu1928-Hu20BB-long versus Hu1928-Hu20BB-standard in response to 3 different CD20+ target cells (P value range 0.001 to 0.036). We tested Hu1928-Hu20BB-long against tumors established in nod-scid common gamma-chain knockout (NSG) mice. Human T cells expressing Hu1928-Hu20BB-long eliminated tumors of the following tumor cell lines: st486 (CD19+, CD20+), st486-CD19-negative (CD20+, CD19 low to negative expression), and NALM6 (CD19+, CD20 low expression). One million Hu1928-Hu20BB-long T cells per mouse eradicated st486 tumors. These optimized anti-CD19/CD20 bicistronic CAR constructs are promising for clinical treatment of B-cell malignancies. [Display omitted] DisclosuresLam: Kite, a Gilead Company: Patents & Royalties. Kochenderfer: Bristol Myers Squibb: Research Funding; Kite, a Gilead Company: Patents & Royalties: on anti-CD19 CARs, Research Funding.

Searching for DNA Damage: Insights From Single Molecule Analysis.

DNA is under constant threat of damage from a variety of chemical and physical insults, such as ultraviolet rays produced by sunlight and reactive oxygen species produced during respiration or inflammation. Because damaged DNA, if not repaired, can lead to mutations or cell death, multiple DNA repair pathways have evolved to maintain genome stability. Two repair pathways, nucleotide excision repair (NER) and base excision repair (BER), must sift through large segments of nondamaged nucleotides to detect and remove rare base modifications. Many BER and NER proteins share a common base-flipping mechanism for the detection of modified bases. However, the exact mechanisms by which these repair proteins detect their damaged substrates in the context of cellular chromatin remains unclear. The latest generation of single-molecule techniques, including the DNA tightrope assay, atomic force microscopy, and real-time imaging in cells, now allows for nearly direct visualization of the damage search and detection processes. This review describes several mechanistic commonalities for damage detection that were discovered with these techniques, including a combination of 3-dimensional and linear diffusion for surveying damaged sites within long stretches of DNA. We also discuss important findings that DNA repair proteins within and between pathways cooperate to detect damage. Finally, future technical developments and single-molecule studies are described which will contribute to the growing mechanistic understanding of DNA damage detection.

Refactoring the Conjugation Machinery of Promiscuous Plasmid RP4 into a Device for Conversion of Gram-Negative Isolates to Hfr Strains.

Chromosomal exchange and subsequent recombination of the cognate DNA between bacteria was one of the most useful genetic tools (e.g., Hfr strains) for genetic analyses of E. coli before the genomic era. In this paper, yeast assembly has been used to recruit the conjugation machinery of environmentally promiscuous RP4 plasmid into a minimized, synthetic construct that enables transfer of chromosomal segments between donor/recipient strains of P. putida KT2440 and potentially many other Gram-negative bacteria. The synthetic device features [i] a R6K suicidal plasmid backbone, [ii] a mini-Tn5 transposon vector, and [iii] the minimal set of genes necessary for active conjugation (RP4 Tra1 and Tra2 clusters) loaded as cargo in the mini-Tn5 mobile element. Upon insertion of the transposon in different genomic locations, the ability of P. putida-TRANS (transference of RP4-activated nucleotide segments) donor strains to mobilize genomic stretches of DNA into neighboring bacteria was tested. To this end, a P. putida double mutant ΔpyrF (uracil auxotroph) Δedd (unable to grow on glucose) was used as recipient in mating experiments, and the restoration of the pyrF+/edd+ phenotypes allowed for estimation of chromosomal transfer efficiency. Cells with the inserted transposon behaved in a manner similar to Hfr-like strains and were able to transfer up to 23% of their genome at frequencies close to 10–6 exconjugants per recipient cell. The hereby described TRANS device not only expands the molecular toolbox for P. putida, but it also enables a suite of genomic manipulations which were thus far only possible with domesticated laboratory strains and species.

A Multiplex PCR Based on Mitochondrial COI Sequences for Identification of Members of the Anopheles barbirostris Complex (Diptera: Culicidae) in Thailand and Other Countries in the Region.

A multiplex-PCR assay based on mitochondrial cytochrome c oxidase subunit I (COI) sequences was developed for identification of five members of the Barbirostris Complex which occur in Thailand: Anopheles barbirostris s.s., An. dissidens, An. saeungae, An. wejchoochotei and An. barbirostris species A3. Anopheles campestris was not included in the assay due to the lack of unequivocal sequences. Allele-specific primers were designed for specific nucleotide segments of COI sequences of each species. Mismatch method and addition of long GC tail were applied for some primers. The assay provided products of 706 bp for An. barbirostris s.s., 238 bp for An. dissidens, 611 bp for An. saeungae, 502 bp for An. wejchoochotei and 365 bp for An. barbirostris A3. The assay was tested using 111 wild-caught female mosquitoes from Bhutan, Cambodia, Indonesia (Sulawesi) and Thailand. The results of the multiplex PCR were in complete agreement with COI sequencing; however, one of three specimens from Bhutan and all 11 specimens from Indonesia were not amplifiable by the assay due to their distinct COI sequences. This, together with the distinct rDNA sequences of these specimens, suggests the presence of at least two additional new species in the Barbirostris Complex.

Molecular Tagging of NDRG1 using SNAP‐tag Technology

BackgroundSNAP‐tag is a 20kDa versatile protein tag that can be linked to any nucleotide sequence. When fused to a protein, the SNAP‐tag acts as a molecular linker to an array of molecules such as fluorescence probes, biotin, and synthetic compounds. An immediate advantage of the SNAP‐tag as opposed to traditional fluorescence proteins like GFP is temporal control, allowing the user to control the timing of fluorescence expression. SNAP‐tag was fused to NDRG1, N‐myc Down Regulated Gene 1, with the intent to study its localization, expression, and function. NDRG1 is a protein with putative involvement in metastasis suppression, endosomal trafficking, and interaction with surface molecule E‐Cadherin, an important player in mesenchymal to epithelial transition. NDRG1 has been of interest because of its involvement in prostate and breast cancer metastasis.PurposeTo create a NDRG1 and SNAP‐tag fusion protein for use in further study of localization and function of NDRG1MethodsTwo unique SNAP‐tag and NDRG1 fusion sequences, with SNAP at the N terminal in one and at the C terminal in the other, were created, verified, ligated into a hCMV promoter driven pLVx vector, and then transfected into HEK293 cells using Lipofectamine 2000. Cells were incubated in various concentrations of TMR‐Star Red Fluorophore at 37C 5%CO2 for 30 minutes or 1 hour with a parallel control arm of cells transfected with empty vector but were incubated in identical substrate concoctions. The transfected cells, after incubation with the SNAP fluorophore substrate, were verified to express the plasmid and then imaged using Confocal Microscopy. This batch of cells expressing the fusion protein will be used to create a stable cell line, and will be used later on for further study of NDRG1.ResultsThe two constructs (SNAP‐tag attached at the N and C terminals of NDRG1), when transfected into HEK293 cells and subsequently incubated in Optimem + fluorescence TMR‐StarRed substrate achieved equal fluorescence intensity. 1uM of TMR‐Star substrate incubated in 37C and 5%CO2 for 1 hour achieved optimal signal to background ratio and allowed for good imaging conditions. NDRG1‐SNAP expression is seen in the cytoplasm, nucleus, and at the membranes. Intensity is greater in the cytoplasm, but there remain compartments that are void of any fluorescence. Furthermore, faint interactions with cytoskeleton, maybe actin, can be seen. (time lapse video and z‐slices of cells in vitro attached)ConclusionsThe NDRG1‐SNAPtag construct has been successfully synthesized and is able to be viably transfected and subsequently used to study the protein of interest, NDRG1, within HEK293 cells. In this case, fluorescence expression upon incubation of transfected cells with TMR‐StarRed substrate allowed for live cell visualization in vitro, but other small molecules may be conjugated to the SNAP‐tag‐NDRG1 fusion protein in future work. Furthermore, a stable cell line with the fusion sequence integrated pseudo‐randomly into the genome can be generated to study the cell in more in vivo like conditions. Some remaining questions yet to be elucidated are the effect that the SNAP‐tag has on intrinsic NDRG1 function, localization of NDRG1, and on general cell behavior and survival.Support or Funding InformationNCINDRG1 ‐ SNAP nucleotide segment on gel electrophoresis. Approximately 1.7 kb longFigure 1NRDG1‐SNAPtag fusion protein expressed in HEK293T cells. Viewed under fluorescence confocal microscopy.Figure 2

Single-nucleotide polymorphisms in FABP4 gene associated with growth traits in Egyptian sheep.

Aim:The present study was performed to assess the association of single-nucleotide polymorphisms (SNPs) in the fatty acid-binding protein 4 (FABP4) gene with birth weight (BW), final weight (FW), and average daily gain (ADG) in three Egyptian sheep breeds.Materials and Methods:Genomic DNA was extracted from the blood samples of 50 male and female individuals representing Ossimi, Rahmani, and Barki sheep breeds. A 407 bp nucleotide (nt) segment from the first intron of FABP4 was amplified by polymerase chain reaction, sequenced, and analyzed in the different samples.Results:Sequence analysis of the determined segment (407 bp) revealed four SNPs (all transition types) at nt position 372 (CP011894.1:g.57605471) A>G, nt position 211 (CP011894.1:g.57605632) A>G, nt position 143 (CP011894.1:g.57605700) T>C, and nt position 111 (CP011894.1:g.57605732) T>C. The allelic and genotypic frequencies for the identified SNPs in the sheep breeds were calculated. At nt positions 372 and 211, two alleles were identified (A and G). Only two genotypes were present at nt position 372 (AA and AG), while three genotypes were present at nt position 211 (AA, AG, and GG). Two alleles (T and C) and three identified genotypes (TT, TC, and CC) were detected at nt positions 143 and 111. Analysis of the results revealed that AA genotype at nt position 372 is associated with higher estimates for BW, FW, and ADG when compared to all the other genotypes. Very high correlation coefficients were found between the genotypes 143-TT and 111-TT and also between 143-TC and 111-TC. The genotypes 372-AG, 211-GG, 211-AA, 143-TT, 143-CC, 111-TT, 111-TC, and 111-CC were associated with negative effects on BW, FW, and ADG.Conclusion:The detection of four SNPs in a partial sequence of the Egyptian ovine FABP4 gene intron 1 reflected that this gene harbors substantial diversity. In addition, a novel SNP at nt position 372 (CP011894.1:g.57605471) A>G was associated with higher estimates for BW, FW, and ADG.

Structure and function analysis of the essential 3′X domain of hepatitis C virus

The 3′X domain of hepatitis C virus has been reported to control viral replication and translation by modulating the exposure of a nucleotide segment involved in a distal base-pairing interaction with an upstream 5BSL3.2 domain. To study the mechanism of this molecular switch, we have analyzed the structure of 3′X mutants that favor one of the two previously proposed conformations comprising either two or three stem–loops. Only the two-stem conformation was found to be stable and to allow the establishment of the distal contact with 5BSL3.2, and also the formation of 3′X domain homodimers by means of a universally conserved palindromic sequence. Nucleotide changes disturbing the two-stem conformation resulted in poorer replication and translation levels, explaining the high degree of conservation detected for this sequence. The switch function attributed to the 3′X domain does not occur as a result of a transition between two- and three-stem conformations, but likely through the sequestration of the 5BSL3.2-binding sequence by formation of 3′X homodimers.

Quantitative principles of cis-translational control by general mRNA sequence features in eukaryotes

BackgroundGeneral translational cis-elements are present in the mRNAs of all genes and affect the recruitment, assembly, and progress of preinitiation complexes and the ribosome under many physiological states. These elements include mRNA folding, upstream open reading frames, specific nucleotides flanking the initiating AUG codon, protein coding sequence length, and codon usage. The quantitative contributions of these sequence features and how and why they coordinate to control translation rates are not well understood.ResultsHere, we show that these sequence features specify 42–81% of the variance in translation rates in Saccharomyces cerevisiae, Schizosaccharomyces pombe, Arabidopsis thaliana, Mus musculus, and Homo sapiens. We establish that control by RNA secondary structure is chiefly mediated by highly folded 25–60 nucleotide segments within mRNA 5′ regions, that changes in tri-nucleotide frequencies between highly and poorly translated 5′ regions are correlated between all species, and that control by distinct biochemical processes is extensively correlated as is regulation by a single process acting in different parts of the same mRNA.ConclusionsOur work shows that general features control a much larger fraction of the variance in translation rates than previously realized. We provide a more detailed and accurate understanding of the aspects of RNA structure that directs translation in diverse eukaryotes. In addition, we note that the strongly correlated regulation between and within cis-control features will cause more even densities of translational complexes along each mRNA and therefore more efficient use of the translation machinery by the cell.

Unique structure (construction and configuration) and evolution of the array of small serum protein genes of Protobothrops flavoviridis snake.

The nucleotide sequence of Protobothrops flavoviridis (Pf) 30534 bp genome segment which contains genes encoding small serum proteins (SSPs) was deciphered. The genome segment contained five SSP genes (PfSSPs), PfSSP-4, PfSSP-5, PfSSP-1, PfSSP-2, and PfSSP-3 in this order and had characteristic configuration and constructions of the particular nucleotide sequences inserted. Comparison between the configurations of the inserted chicken repeat-1 (CR1) fragments of P. flavoviridis and Ophiophagus hannah (Oh) showed that the nucleotide segment encompassing from PfSSP-1 to PfSSP-2 was inverted. The inactive form of PfSSP-1, named PfSSP-1δ(Ψ), found in the intergenic region (I-Reg) between PfSSP-5 and PfSSP-1 had also been destroyed by insertions of the plural long interspersed nuclear elements (LINEs) and DNA transposons. The L2 LINE inserted into the third intron or the particular repetitive sequences inserted into the second intron structurally divided five PfSSPs into two subgroups, the Long SSP subgroup of PfSSP-1, PfSSP-2 and PfSSP-5 or the Short SSP subgroup of PfSSP-3 and PfSSP-4. The mathematical analysis also showed that PfSSPs of the Long SSP subgroup evolved alternately in an accelerated and neutral manner, whereas those of the Short SSP subgroup evolved in an accelerated manner. Moreover, the ortholog analysis of SSPs of various snakes showed that the evolutionary emerging order of SSPs was as follows: SSP-5, SSP-4, SSP-2, SSP-1, and SSP-3. The unique interpretation about accelerated evolution and the novel idea that the transposable elements such as LINEs and DNA transposons are involved in maintaining the host genome besides its own transposition natures were proposed.

Virus-induced gene silencing and virus-induced flowering in strawberry (Fragaria\u2009\xd7\u2009ananassa) using apple latent spherical virus vectors

Apple latent spherical virus (ALSV) vector is a convenient alternative to genetic transformation in horticultural plants, especially in species recalcitrant to genetic transformation. ALSV, an RNA virus, can infect a wide variety of plant species including major horticultural plants without inducing symptoms. Here, methodologies were developed for infection of ALSV vectors to strawberry seedlings and plantlets cultured in vitro. A seed-propagated F1 hybrid strawberry cultivar 'Yotsuboshi' was aseptically grown on half-strength Murashige–Skoog medium for 1 month and true leaves were inoculated with an ALSV RNA preparation by particle bombardment. ALSV vector infection rates varied from 58 to 100% according to the insertion sequences, in ‘Yotsuboshi’ seedlings. Plantlets (‘Dover’) propagated in vitro could also be infected with ALSV vector at a similar infection rate. For virus-induced gene silencing (VIGS), we prepared an ALSV vector carrying a 201 nucleotide segment of the strawberry phytoene desaturase gene. ‘Yotsuboshi’ and ‘Dover’ plants infected by this vector generated completely white leaves at fifth or sixth true leaves and above. For virus-induced flowering (VIF), we used an ALSV vector expressing the Arabidopsis thaliana flowering locus T gene. Strawberry seedlings infected by this vector started to flower from about 2 months post inoculation and bore fruits with viable seeds. The ALSV vector was no longer detected in any of the seedlings from early-flowered strawberries. Thus, the ALSV vector may be beneficial for examination of gene functions by VIGS in strawberry, and VIF using ALSV vector constitutes an effective new plant breeding technique for the promotion of cross-breeding in strawberry.

PCR amplification of a long rDNA segment with one primer pair in agriculturally important nematodes.

Ribosomal DNA has been a reliable source of taxonomic and phylogenetic markers due to its high copy number in the genome and stable variation with few polymorphisms due to the homogenizing effect of concerted evolution. Typically specific regions are amplified through polymerase chain reaction (PCR) with multiple primer pairs that generate often incomplete and overlapping regions between adjacent segments of 18S, ITS1, 5.8S, ITS2, and 28S rDNA nucleotide sequences when combined in tandem. To improve the efficiency of this effort, a strategy for generating all these molecular sequences at once through PCR amplification of a large ribosomal 3.3 to 4.2 kb DNA target was developed using primer 18S-CL-F3 paired with D3B or a new alternative 28S PCR primer (28S-CL-R) and other well-positioned and ribosomal-specific sequencing primers (including novel primers 18S-CL-F7, 18S-CL-R6, 18S-CL-R7, 18S-CL-F8, 5.8S-CL-F1, 5.8S-CL-R1, 28S-CL-F1, 28S-CL-R3, 28S-CL-F3, 28S-CL-R1, and 28S-CL-F2). The D1 region between ITS2 and 28S boundaries and the flanking sequence between 18S and ITS1 boundaries were fully revealed in this large nucleotide segment. To demonstrate the value of this strategy, the long rDNA segment was amplified and directly sequenced in 17 agriculturally important nematodes from the Tylenchida, Aphelenchida, and Dorylaimida. The primers and their positions may be employed with traditional Sanger sequencing and with next-generation sequencing reagents and protocols.

EVOLUTION OF INFLUENZA A NUCLEOTIDE SEGMENTS THROUGH THE LENS OF DIFFERENT COMPLEXITY MEASURES

Evolution of influenza viruses is a highly complex process that is still poorly understood. Multiyear persistence of similar variants and accumulating evidences of existence of multigenic traits indicates that influenza viruses operate as integrated units and not only as sets of distinct genes. However, there is still no consensus on whether it is the case, and to what extent. One of the main problems is the lack of framework for analyzing and interpreting large body of available high dimensional genomic, clinical and epidemiological data. By reducing dimensionality of data we intend to show whether in addition to gene-centric selective pressure, the evolution of influenza RNA segments is also shaped by their mutual interactions. Therefore, we will analyze how different complexity/entropy measures (Shannon entropy, topological entropy and Lempel–Ziv complexity) can be used to study evolution of nucleotide segments of different influenza subtypes, while reducing data dimensionality. We show that, at the nucleotide level, multiyear clusters of genome-wide entropy/complexity correlations emerged during the H1N1 pandemic in 2009. Our data are the first empirical results that indirectly support the suggestion that a component of influenza evolutionary dynamics involves correlation between RNA segments. Of all used complexity/entropy measures, Shannon entropy shows the best correlation with epidemiological data.

An unexpected RNA distal interaction mode found in an essential region of the hepatitis C virus genome.

The 3’X tail is a functionally essential 98-nt sequence located at the 3′-end of the hepatitis C virus (HCV) RNA genome. The domain contains two absolutely conserved dimer linkage sequence (DLS) and k nucleotide segments involved in viral RNA dimerization and in a distal base-pairing interaction with stem-loop 5BSL3.2, respectively. We have previously shown that domain 3’X forms an elongated structure comprising two coaxially stacked SL1’ and SL2’ stem-loops. This conformation favors RNA dimerization by exposing a palindromic DLS segment in an apical loop, but buries in the upper stem of hairpin SL2’ the k nucleotides involved in the distal contact with 5BSL3.2. Using nuclear magnetic resonance spectroscopy and gel electrophoresis experiments, here we show that the establishment of the complex between domain 3’X and stem-loop 5BSL3.2 only requires a rearrangement of the nucleotides forming the upper region of subdomain SL2’. The results indicate that the interaction does not occur through a canonical kissing loop mechanism involving the unpaired nucleotides of two terminal loops, but rather involves a base-paired stem and an apical loop and may result in a kissing three-way junction. On the basis of this information we suggest how the 3’X tail switches between monomer, homodimer and heterodimer states to regulate the HCV viral cycle.

An ATP-fueled nucleic acid signal amplification strategy for highly sensitive microRNA detection.

Herein, an adenosine triphosphate (ATP)-fueled nucleic acid signal amplification strategy based on toehold-mediated strand displacement (TMSD) and fluorescence resonance energy transfer (FRET) was proposed for highly sensitive detection of microRNA-21. More importantly, the target microRNA-21 could be regenerated with ATP as the fuel rather than a nucleotide segment in conventional approaches, which made the proposed strategy simple and efficient due to the high affinity and strength of the aptamer-target interaction.

Improved 18S small subunit rDNA primers for problematic nematode amplification.

Abstract The 18S small subunit (SSU) ribosomal DNA sequence is one of the most useful molecular loci for identification and phylogeny reconstruction of agriculturally important nematodes. Various pairs of universal primers have been developed in the past to amplify short and long nematode sequences. However, certain nematode taxa were not readily amplified and/or sequenced with the existing primer tools. Frequently, the center region of a roughly 1,000 nucleotide segment would be lost. Therefore new primers were developed based on a very large 276 taxon alignment of 124 agriculturally important nematode species, and tested on problematic nematode taxa such as Aphelenchoides, Bursaphelenchus, Ditylenchus, and Panagrolaimus. New primers and protocols are provided for successful generation of sequences useful in future investigations of nematode systematics.

Deduced probable human leukocyte antigen haplotypes associated with human leukocyte antigen DRB1*04:36 identified by case analysis of Taiwanese individuals.

Objective:Human leukocyte antigen (HLA) DRB1*04:36 is a low-frequency HLA-DRB1 allele. The aim here is to report the ethnicity of DRB1*04:36 and its associated HLA haplotypes among Taiwanese individuals.Materials and Methods:A sequence-based typing method was employed to confirm this low incidence allele. Polymerase chain reaction was performed to amplify exons 2 and 3 of the HLA-A and HLA-B loci and exon 2 of the HLA-DRB1 locus using group-specific primer sets. The amplicons were sequenced in both directions using BigDye Terminator Cycle Sequencing Ready Reaction kits and the manufacturer's protocols. One group of unrelated blood donors used in this study consists of randomized individuals with Taiwanese ethnicity who participate in the Tzu Chi Bone Marrow Donor Registry and the other group are randomized unrelated individuals from mainland China. The family members in the family part of the study are volunteer blood donors.Results:In exon 2, the DNA sequence of DRB1*04:36 is identical to DRB1*04:03:01 except for a nucleotide segment from residue 286 to residue 308. The nucleotide segment from residue 286 to residue 308, incidentally, is identical to that of DRB1*11:01:01:01. These observations suggest that DRB1*04:36 may have been derived through a gene recombination event involving DRB1*04:03:01 and DRB1*11:01:01:01. Our family study indicated that the HLA haplotype in association with DRB1*04:36 can be deduced to be A*24:02-B*39:01-DRB1*04:36. A randomized population study using Taiwanese suggests that additional DRB1*04:36 associated HLA haplotypes seem to exist.Conclusion:The information on the ethnicity of the DRB1**04:36 allele, and the deduced probable HLA haplotypes associated with the low incidence DRB1*04:36 allele that we report here, is of value to HLA testing laboratories for reference purposes. In addition, they can be used by stem cell transplantation donor search coordinators to aid the creation of strategy for finding compatible donors who are part of unrelated bone marrow donor registries when a patient carries this uncommon HLA allele.