Molecular Characterization of Viruses Causing the Cassava Brown Streak Disease Epidemic in Eastern Africa

Cassava brown streak disease (CBSD) was described for the first time in Tanganyika (now Tanzania) about seven decades ago. Tanganyika (now Tanzania) about seven decades ago. It was endemic in the lowland areas of East Africa and inland parts of Malawi and caused by Cassava brown streak virus (CBSV; genus Ipomovirus; Potyviridae). However, in 1990s CBSD was observed at high altitude areas in Uganda. The causes for spread to new locations were not known.The present work was thus initiated to generate information on genetic variability, clarify the taxonomy of the virus or viruses associated with CBSD in Eastern Africa as well as to understand the evolutionary forces acting on their genes. It also sought to develop a molecular based diagnostic tool for detection of CBSD-associated virus isolates. Comparison of the CP-encoding sequences of CBSD-associated virus isolates collected from Uganda and north-western Tanzania in 2007 and the partial sequences available in Genbank revealed occurrence of two genetically distinct groups of isolates. Two isolates were selected to represent the two groups. The complete genomes of isolates MLB3 (TZ:Mlb3:07) and Kor6 (TZ:Kor6:08) obtained from North-Western (Kagera) and North-Eastern (Tanga) Tanzania, respectively, were sequenced. The genomes were 9069 and 8995 nucleotides (nt), respectively. They translated into polyproteins that were predicted to yield ten mature proteins after cleavage. Nine proteins were typical in the family Potyviridae, namely P1, P3, 6K1, CI, 6K2, VPg, NIa-Pro, NIb and CP, but the viruses did not contain HC-Pro. Interestingly, genomes of both isolates contained a Maf/HAM1-like sequence (HAM1h; 678 nucleotides, 25 kDa) recombined between the NIb and CP domains in the 3’-proximal part of the genomes. HAM1h was also identified in Euphorbia ringspot virus (EuRSV) whose sequence was in GenBank. The HAM1 gene is widely spread in both prokaryotes and eukaryotes. In yeast (Saccharomyces cerevisiae) it is known to be a nucleoside triphosphate (NTP) pyrophosphatase. Novel information was obtained on the structural variation at the N-termini of polyproteins of viruses in the genus Ipomovirus. Cucumber vein yellowing virus (CVYV) and Squash vein yellowing virus (SqVYV) contain a duplicated P1 (P1a and P1b) but lack the HC-Pro. On the other hand, Sweet potato mild mottle virus (SPMMV), has a single but large P1 and has HC-Pro. Both virus isolates (TZ:Mlb3:07 & TZ:Kor6:08) characterized in this study contained a single P1 and lacked the HC-Pro which indicates unique evolution in the family Potyviridae. Comparison of 12 complete genomes of CBSD-associated viruses which included two genomes characterized in this study, revealed genetic identity of 69.0–70.3% (nt) and amino acid (aa) identities of 73.6–74.4% at polyprotein level. Comparison was also made among 68 complete CP sequences, which indicated 69.0-70.3 and 73.6-74.4 % identity at nt and aa levels, respectively. The genetic variation was large enough for dermacation of CBSD-associated virus isolates into two distinct species. The name CBSV was retained for isolates that were related to CBSV isolates available in database whereas the new virus described for the first time in this study was named Ugandan cassava brown streak virus (UCBSV) by the International Committee on Virus Taxonomy (ICTV). The isolates TZ:Mlb3:07 and TZ:Kor6:08 belong to UCBSV and CBSV, respectively. The isolates of CBSV and UCBSV were 79.3-95.5% and 86.3-99.3 % identitical at nt level, respectively, suggesting more variation amongst CBSV isolates. The main sources of variation in plant viruses are mutations and recombination. Signals for recombination events were detected in 50% of isolates of each virus. Recombination events were detected in coding and non-coding (3’-UTR) sequences except in the 5’UTR and P3. There was no evidence for recombination between isolates of CBSV and UCBSV. The non-synonomous (dN) to synonomous (dS) nucleotide substitution ratio (ω) for the HAM1h and CP domains of both viruses were ≤ 0.184 suggesting that most sites of these proteins were evolving under strong purifying selection. However, there were individual amino acid sites that were submitted to adaptive evolution. For instance, adaptive evolution was detected in the HAM1h of UCBSV (n=15) where 12 aa sites were under positive selection (P< 0.05) but not in CBSV (n=12). The CP of CBSV (n=23) contained 12 aa sites (p<0.01) while only 5 aa sites in the CP gene of UCBSV were predicted to be submitted to positive selection pressure (p<0.01). The advantages offered by the aa sites under positive selection could not be established but occurrence of such sites in the terminal ends of UCBSV-HAMIh, for example, was interpreted as a requirement for proteolysis during polyprotein processing. Two different primer pairs that simultaneously detect UCBSV and CBSV isolates were developed in this study. They were used successfully to study distribution of CBSV, UCBSV and their mixed infections in Tanzania and Uganda. It was established that the two viruses co-infect cassava and that incidences of co-infection could be as high as 50% around Lake Victoria on the Tanzanian side. Furthermore, it was revealed for the first time that both UCBSV and CBSV were widely distributed in Eastern Africa. The primer pair was also used to confirm infection in a close relative of cassava, Manihot glaziovii (Müller Arg.) with CBSV. DNA barcoding of M. glaziovii was done by sequencing the matK gene. Two out of seven M. glaziovii from the coastal areas of Korogwe and Kibaha in north eastern Tanzania were shown to be infected by CBSV but not UCBSV isolates. Detection in M. glaziovii has an implication in control and management of CBSD as it is likely to serve as virus reservoir. This study has contributed to the understanding of evolution of CBSV and UCBSV, which cause CBSD epidemic in Eastern Africa. The detection tools developed in this work will be useful in plant breeding, verification of the phytosanitary status of materials in regional and international movement of germplasm, and in all diagnostic activities related to management of CBSD. Whereas there are still many issues to be resolved such as the function and biological significance of HAM1h and its origin, this work has laid a foundation upon which the studies on these aspects can be based.

A Deterministic Optimization Approach to Protein Sequence Design Using Continuous Models

Determining the sequence of amino acid residues in a heteropolymer chain of a protein with a given conformation is a discrete combinatorial problem that is not generally amenable for gradient-based continuous optimization algorithms. In this paper we present a new approach to this problem using continuous models. In this modeling, continuous "state functions" are proposed to designate the type of each residue in the chain. Such a continuous model helps define a continuous sequence space in which a chosen criterion is optimized to find the most appropriate sequence. Searching a continuous sequence space using a deterministic optimization algorithm makes it possible to find the optimal sequences with much less computation than many other approaches. The computational efficiency of this method is further improved by combining it with a graph spectral method, which explicitly takes into account the topology of the desired conformation and also helps make the combined method more robust. The continuous modeling used here appears to have additional advantages in mimicking the folding pathways and in creating the energy landscapes that help find sequences with high stability and kinetic accessibility. To illustrate the new approach, a widely used simplifying assumption is made by considering only two types of residues: hydrophobic (H) and polar (P). Self-avoiding compact lattice models are used to validate the method with known results in the literature and data that can be practically obtained by exhaustive enumeration on a desktop computer. We also present examples of sequence design for the HP models of some real proteins, which are solved in less than five minutes on a single-processor desktop computer Some open issues and future extensions are noted.

A hypothesis on a specific sequence-dependent conformation of DNA and its relation to the binding of the lac-repressor protein

The structure and properties of the double-helical form of the alternating copolymer poly(dA-dT) are considered. Different lines of evidence are interpreted in terms of a structure in which every second phosphate-diester linkage has a conformation different from that of the normal B form. A rationale for this “alternating-B” structure is given which provides an explanation for the effects of chemical modifications of the T residues on the binding of the poly(dA-dT)· poly(dA-dT) to the lac repressor of Escherichia coli.

Defective in Cuticular Ridges (DCR) of Arabidopsis thaliana, a Gene Associated with Surface Cutin Formation, Encodes a Soluble Diacylglycerol Acyltransferase

A key step in the triacylglycerol (TAG) biosynthetic pathway is the final acylation of diacylglycerol (DAG) by DAG acyltransferase. In silico analysis has revealed that the DCR (defective in cuticular ridges) (At5g23940) gene has a typical HX4D acyltransferase motif at the N-terminal end and a lipid binding motif VX(2)GF at the middle of the sequence. To understand the biochemical function, the gene was overexpressed in Escherichia coli, and the purified recombinant protein was found to acylate DAG specifically in an acyl-CoA-dependent manner. Overexpression of At5g23940 in a Saccharomyces cerevisiae quadruple mutant deficient in DAG acyltransferases resulted in TAG accumulation. At5g23940 rescued the growth of this quadruple mutant in the oleate-containing medium, whereas empty vector control did not. Lipid particles were localized in the cytosol of At5g23940-transformed quadruple mutant cells, as observed by oil red O staining. There was an incorporation of 16-hydroxyhexadecanoic acid into TAG in At5g23940-transformed cells of quadruple mutant. Here we report a soluble acyl-CoA-dependent DAG acyltransferase from Arabidopsis thaliana. Taken together, these data suggest that a broad specific DAG acyltransferase may be involved in the cutin as well as in the TAG biosynthesis by supplying hydroxy fatty acid.

A new classification and linear sequence of extant gymnosperms

A new classification and linear sequence of the gymnosperms based on previous molecular and morphological phylogenetic and other studies is presented. Currently accepted genera are listed for each family and arranged according to their (probable) phylogenetic position. A full synonymy is provided, and types are listed for accepted genera. An index to genera assists in easy access to synonymy and family placement of genera.

A linear sequence of extant families and genera of lycophytes and ferns

Throughout the history of the classification of extant ferns (monilophytes) and lycophytes, familial and generic concepts have been in great flux. For the organisation of lycophytes and ferns in herbaria, books, checklists, indices and spore banks and on the internet, this poses a problem, and a standardized linear sequence of these plants is therefore in great need. We provide here a linear classification to the extant lycophytes and ferns based on current phylogenetic knowledge; this provides a standardized guide for organisation of fern collections into a more natural sequence. Two new families, Diplaziopsidaceae and Rhachidosoraceae, are here introduced.

Structural features of B-DNA dodecamer crystal structures: Influence of crystal packing versus base sequence

We have analyzed the set of inter and intra base pair parameters for each dinucleotide step in single crystal structures of dodecamers, solved at high and medium resolution and all crystallized in P2(1)2(1)2(1) space group. The objective was to identify whether all the structures which have either the Drew-Dickerson (DD) sequence d[CGCGAATTCGCG] with some base modification or related sequence (non-DD), would display the same sequence dependent structural variability about its palindromic sequence, despite the molecule being bent at one end because of similar crystal lattice packing effect. Most of the local doublet parameters for base pairs steps G2-C3 and G10-C11 positions, symmetrically situated about the lateral twofold, were significantly correlated between themselves. In non-DD sequences, significant correlations between these positional parameters were absent. The different range of local step parameter values at each sequence position contributed to the gross feature of smooth helix axis bending in all structures. The base pair parameters in some of the positions, for medium resolution DD sequence, were quite unlike the high-resolution set and encompassed a higher range of values. Twist and slide are the two main parameters that show wider conformational range for the middle region of non-DD sequence structures in comparison to DD sequence structures. On the contrary, the minor and major groove features bear good resemblance between DD and non-DD sequence crystal structure datasets. The sugar-phosphate backbone torsion angles are similar in all structures, in sharp contrast to base pair parameter variation for high and low resolution DD and non-DD sequence structures, consisting of unusual (epsilon =g(-), xi =t) B-II conformation at the 10(th) position of the dodecamer sequence. Thus examining DD and non-DD sequence structures packed in the same crystal lattice arrangement, we infer that inter and intra base pair parameters are as symmetrically equivalent in its value as the symmetry related step for the palindromic DD sequence about lateral two-fold axis. This feature would lead us to agree with the conclusion that DNA conformation is not substantially affected by end-to-end or lateral inter-molecular interaction due to crystal lattice packing effect. Non-DD sequence structures acquire step parameter values which reflect the altered sequence at each of the dodecamer sequence position in the orthorhombic lattice while showing similar gross features of DD sequence structures

A Heat and Light Mediated Synthetic Sequence Serendipitous Synthesis of Methyl Ester of Tris-Norpterosin-E

Synthesis of methyl ester of 3-oxo-indan-5-acetic acid (3), an analogue of the natura1 product pterosin-E (4), starting from cyclopentadiene (1) and p-benzoquinone (2) using a sequence of six ground and excited state reactions, is described.

2(4)-SEMA as a sensitive and offset compensated SLF sequence

Separated Local Field (SLF) spectroscopy is a powerful tool for the determination of structure and dynamics of oriented systems such as membrane proteins oriented in lipid bilayers and liquid crystals. Of many SLF techniques available, Polarization Inversion Spin Exchange at Magic Angle (PISEMA) has found wide application due to its many favorable characteristics. However the pulse sequence suffers from its sensitivity to proton resonance frequency offset. Recently we have proposed a new sequence named 2(4)-SEMA (J. Chem. Phys. 132 (2010) 134301) that overcomes this problem of PISEMA. The present work demonstrates the advantage of 2(4)-SEMA as a highly sensitive SLF technique even for very large proton offset. 2(4)-SEMA has been designed for obtaining reliable dipolar couplings by switching the magic-angle spin-lock for protons over four quadrants as against the use of only two quadrants in PISEMA. It is observed that for on-resonance condition, 2(4)-SEMA gives rise to signal intensity comparable to or slightly higher than that from PISEMA. But under off-resonance conditions, intensities from 2(4)-SEMA are several fold higher than those from PISEMA. Comparison with another offset compensated pulse sequence, SAMPI4, also indicates a better intensity profile for 2(4)-SEMA. Experiments carried out on a single crystal of N-15 labeled N-acetyl-DL-valine and simulations have been used to study the relative performance of the pulse sequences considered. (C) 2010 Elsevier Inc. All rights reserved.

Functional regulation of the Neurofibromatosis 2 tumor suppressor merlin

Neurofibromatosis 2 (NF2) is an autosomal dominant disorder manifested by the formation of multiple benign tumors of the nervous system. Affected individuals typically develop bilateral vestibular schwannomas which lead to deafness and balance disorders. The syndrome is caused by inactivation of the NF2 tumor suppressor gene, and mutation or loss of the NF2 product, merlin, is sufficient for tumorigenesis in both hereditary and sporadic NF2-associated tumors. Merlin belongs to the band 4.1 superfamily of cytoskeletal proteins, which also contain the related ezrin, radixin, and moesin (ERM) proteins. The ERM members provide a link between the cell cytoskeleton and membrane by connecting membrane-associated proteins to actin filaments. By stabilizing complexes in the cell cortex, the ERMs modulate morphology, growth, and migration of cells. Despite their structural homology, overlapping subcellular distribution, direct molecular association, and partial overlap of molecular interactions, merlin and ezrin exert opposite effects on cell proliferation. Merlin suppresses cell proliferation, whereas ezrin expression is linked to oncogenic activity. We hypothesized that the regions which differ between the proteins might explain merlin s specificity as a tumor suppressor. We therefore analyzed the regions, which are most diverse between merlin and ezrin; the N-terminal tail and the C-terminus. To determine the properties of the C-terminal region, we studied the two most predominant merlin isoforms together with truncation variants similar to those found in patients. We also focused on the evolutionally conserved C-terminal residues, E545-E547, that harbor disease causing mutations in its corresponding DNA sequence. In addition to inhibiting cell proliferation, merlin regulates cytoskeletal organization. The morphogenic properties of merlin may play a role in tumor suppression, since patient-derived tumor cells demonstrate cytoskeletal abnormalities. We analyzed the mechanisms of merlin-induced extension formation and determined that the C-terminal region of amino acids 538-568 is particularly important for the morphogenic activity. We also characterized the role of C-terminal merlin residues in the regulation of proliferation, phosphorylation, and intramolecular associations. In contrast to previous reports, we demonstrated that both merlin isoforms are able to suppress cell proliferation, whereas C-terminally mutated merlin constructs showed reduced growth inhibition. Phosphorylation serves as a mechanism to regulate the tumor suppressive activity of merlin. The C-terminal serine 518 is phosphorylated in response to both p21-activated kinase (PAK) and protein kinase A (PKA), which inactivates the growth inhibitory function of merlin. However, at least three differentially phosphorylated forms of the protein exist. In this study we demonstrated that also the N-terminus of merlin is phosphorylated by AGC kinases, and that both PKA and Akt phosphorylate merlin at serine 10 (S10). We evaluated the impact of this N-terminal tail phosphorylation, and showed that the phosphorylation state of S10 is an important regulator of merlin s ability to modulate cytoskeletal organization but also regulates the stability of the protein. In summary, this study describes the functional effect of merlin specific regions. We demonstrate that both S10 in the N-terminal tail and residues E545-E547 in the C-terminus are essential for merlin activity and function.

Local variability and base sequence effects in DNA crystal structures.

The importance and usefulness of local doublet parameters in understanding sequence dependent effects has been described for A- and B-DNA oligonucleotide crystal structures. Each of the two sets of local parameters described by us in the NUPARM algorithm, namely the local doublet parameters, calculated with reference to the mean z-axis, and the local helical parameters, calculated with reference to the local helix axis, is sufficient to describe the oligonucleotide structures, with the local helical parameters giving a slightly magnified picture of the variations in the structures. The values of local doublet parameters calculated by NUPARM algorithm are similar to those calculated by NEWHELIX90 program, only if the oligonucleotide fragment is not too distorted. The mean values obtained using all the available data for B-DNA crystals are not significantly different from those obtained when a limited data set is used, consisting only of structures with a data resolution of better than 2.4 A and without any bound drug molecule. Thus the variation observed in the oligonucleotide crystals appears to be independent of the quality of their crystallinity. No strong correlation is seen between any pair of local doublet parameters but the local helical parameters are interrelated by geometric relationships. An interesting feature that emerges from this analysis is that the local rise along the z-axis is highly correlated with the difference in the buckle values of the two basepairs in the doublet, as suggested earlier for the dodecamer structures (Bansal and Bhattacharyya, in Structure & Methods: DNA & RNA, Vol. 3 (Eds., R.H. Sarma and M.H. Sarma), pp. 139-153 (1990)). In fact the local rise values become almost constant for both A- and B-forms, if a correction is applied for the buckling of the basepairs. In B-DNA the AA, AT, TA and GA basepair sequences generally have a smaller local rise (3.25 A) compared to the other sequences (3.4 A) and this seems to be an intrinsic feature of basepair stacking interaction and not related to any other local doublet parameter. The roll angles in B-DNA oligonucleotides have small values (less than +/- 8 degrees), while mean local twist varies from 24 degrees to 45 degrees. The CA/TG doublet sequences show two types of preferred geometries, one with positive roll, small positive slide and reduced twist and another with negative roll, large positive slide and increased twist.(ABSTRACT TRUNCATED AT 400 WORDS)

Sequence Independent Recombination Triple Helices: A Molecular Dynamics Study

Recent experimental studies have shown that the Rec-A mediated homologous recombination reaction involves a triple helical intermediate, in which the third strand base forms hydrogen bonds with both the bases in the major groove of the Watson-Crick duplex. Such 'mixed' hydrogen bonds allow formation of sequence independent triplexes. DNA triple helices involving 'mixed' hydrogen bonds have been studied, using model building, molecular mechanics (MM) and molecular dynamics (MD). Models were built for a tripler comprising all four possible triplets viz., G.C*C, C.G*G, A.T*T and T.A*A. To check the stability of all the 'mixed' hydrogen bonds in such triplexes and the conformational preferences of such tripler structures, MD studies were carried out starting from two structures with 30 degrees and 36 degrees twist between the basepairs. It was observed that though the two triplexes converged towards a similar structure, the various hydrogen bonds between the WC duplex and the third strand showed differential stabilities. An MD simulation with restrained hydrogen bonds showed that the resulting structure was stable and remained close to the starting structure. These studies help us in defining stable hydrogen bond geometries involving the third strand and the WC duplex. It was observed that in the C.G*G triplets the N7 atom of the second strand is always involved in hydrogen bonding. In the G.C*C triplets, either N3 or O2 in the third strand cytosine can interchangeably act as a hydrogen bond acceptor.

Molecular Mechanics Studies of Homopolymeric and Mixed Sequence Py.Pu*Pu Triple Helices,

DNA triple helices containing two purine strands and one pyrimidine strand (C.G*G and T.A*A) have been studied, using model building followed by energy minimisation, for different orientations of the third strand resulting from variation in the hydrogen bonding between the Watson-Crick duplex and the third strand and the glycosidic torsion angle in the third strand. Our results show that in the C.G*G case the structure with a parallel orientation of the third strand, resulting from Hoogsteen hydrogen bonds between the third strand and the Watson-Crick duplex, is energetically the most favourable while in the T.A*A case the antiparallel orientation of the third strand, resulting from reverse Hoogsteen hydrogen bonds, is energetically the most favourable. These studies when extended to the mixed sequence triplexes, in which the second strand is a mixture of G and A, correspondingly the third strand is a mixture of G and APT, show that though the parallel orientation is still energetically more favourable, the antiparallel orientation becomes energetically comparable with an increasing number of thymines in the third strand. Structurally, for the mixed triplexes containing G and T in the third strand, it is seen that the basepair non-isomorphism between the C.G*G and the T.A*T triplets can be overcome with some changes in the base pair parameters without much distortion of either the backbone or the hydrogen bonds.

Analysis of sequence dependent variations in secondary and tertiary structure of tRNA molecules

The double helical regions of the five tRNA(Phe) and two tRNA(Asp) crystal structures have been analyzed using the local basepair step parameters. The sequence dependent effects in the mini double helices of tRNA are very similar to those observed in the crystal structures of oligonucleotides in the A-form, the purine-pyrimidine and purine-purine steps have small roll angles when compared to the fiber models of A-DNA as well as A-RNA, while the pyrimidine-purine doublet steps have large roll angles. The orientation of the basepairs in the D-stem is unusual and invariant i.e. they are different from the other three stems but are very similar in all the five tRNA(Phe) crystal structures, presumably due to tertiary interaction of the Watson-Crick basepairs with other bases, with all bases being highly conserved. The origin of the differences between the tertiary structures of tRNA(Phe) and tRNA(Asp) from yeast has also been investigated. It is found that even though the angle between the acceptor arm and the D-stem is very similar in the two structures, the angle subtended by the acceptor arm and the anticodon arm is smaller in the tRNA(Phe) structure (by more than 10 degrees). This is due to differences in the orientation of the two mini helices constituting the anticodon arm, which are inclined to each other by approximately 25 degrees in tRNA(Phe) and 16 degrees in tRNA(Asp). In addition, the acceptor arm, the D-stem and the anticodon stem are nearly coplanar in tRNA(Phe), while in tRNA(Asp) the anticodon stem projects out of the plane defined by the acceptor arm and the anticodon stem. These two features together lead to a larger separation between the acceptor and anticodon ends in tRNA(Asp) and indicate that the junction between the D-stem and the anticodon stem is quite variable, with features characteristic of a ball-and-socket type joint and determined for each tRNA molecule by the base sequence at the junction.