A densovirus of German cockroach Blatella germanica: detection,nucleotide sequence and genome organization

A new Blattella germanica densovirus (BgDNV, Parvoviridae: Densovirinae, Densovirus) was found. Virus DNA and cockroach tissues infected with BgDNV were examined by electron microscopy. Virus particles about 20 nm in diameter were observed both in the nucleus and in the cytoplasm of infected cells. Virus DNA proved to be a linear molecule sized about 1.2 microns. The complete BgDNV genome was sequenced and analyzed. Five ORF were detected: two coded for structural capsid proteins and were on one DNA strand, and three coded for regulatory proteins and were on the other strand. Potential promoters and polyadenylation signals were identified. Structural analysis was performed for terminal inverted repeats containing extended palindromes. The genome structure of BgDNV was compared with that of other Parvoviridae.     

Restriction map of the Casphalia extranea densovirus genome

A physical map of the Casphalia extranea densovirus genome (CeDNV) was constructed. The size of the intact viral genome was estimated to be 4.9 kilobases or 1.6 MDa (single strand). The double-stranded CeDNV genomic DNA was cleaved with 26 restriction endonucleases and 20 restriction sites were mapped on the genome. The CeDNV DNA restriction map was compared to those of other densoviruses. Southern blotting hybridization experiments failed to reveal any homology between the genomes of CeDNV and Junoniacoenia densovirus (JcDNV).     

Genetic transformation of Brassica campestris var. rapa protoplasts with an engineered cauliflower mosaic virus genome

Summary A hybrid Cauliflower Mosaic Virus (CaMV) genome containing a selectable marker gene was constructed by replacing the gene VI coding region with the aminoglycoside (neomycin) phosphotransferase type II [APH(3)II] gene from Tn5. This modified viral genome was tested for its infectivity both in planta and in a protoplast transformation system of Brassica campestris var. rapa. Stable, genetically transformed cell lines of B. campestris var. rapa were obtained after transformation. DNA of the hybrid CaMV genome was found to be integrated into high molecular weight plant genomic DNA. Transformation was achieved only when the hybrid genome was supplied together with wild type viral DNA. A possible complementation of the modified CaMV genome with the wild type viral DNA as a helper molecule in planta and in the protoplast system is discussed.     

Structure comparisons of Aedes albopictus densovirus with other parvoviruses

Parvoviridae is a family of the smallest viruses known with a wide variety of hosts. The capsid structure of the Aedes albopictus C6/36 cell densovirus (C6/36 DNV) at 1.2-nm resolution was obtained by electron cryomicroscopy (cryoEM) and three-dimensional (3D) image reconstruction. Structure comparisons between the C6/36 DNV and other parvoviruses reveal that the degree of structural similarity between C6/36 DNV and the human parvovirus B19 is higher than that between C6/36 DNV and other insect parvoviruses. The amino acid sequence comparisons of structural and non-structural proteins also reveal higher levels of similarity between C6/36 DNV and parvovirus B19 than those between C6/36 DNV and other parvoviruses. These findings indicate that C6/36 DNV is closely related to the human virus B19, and the former might evolve from the human species other than from other insect viruses.     

Genome specific,highly repeated sequences of Hordeum vulgare: cloning,sequencing and squash dot test

Summary Highly repeated sequences of nuclear DNA from barley Hordeum vulgare (L.) variety Erfa were cloned. Several clones containing barley specific repeated DNA were analysed by sequence analysis and Southern blot hybridization. The investigated repeats differ from each other in their length, sequence and redundancy. Their length ranges from 36 bp to about 180 bp. The repeats are AT-rich and differ widely in their redundancy within the barley genome. Southern analysis showed that the repeats belong to different repetition complexes. The possibility for utilizing these clones as probes for simple and fast genome analysis is demonstrated in squash dot experiments.     

A comparative study of the mitochondrial genome organization in in vitro cultures of diploid, tetraploid, and hexaploid Triticum species

Southern-blot hybridizations of total DNA to mitochondrial DNA (mtDNA) probes were used to investigate the extent of mtDNA variability in cultures derived from immature embryos of diploid (Triticum monococcum, genomic formula: AA, T. tauschii, genomic formula: DD), allotetraploid (T. durum cv Creso, genomic formula: AABB), and allohexaploid (T. aestivum, genomic formula: AABBDD) wheat species. Similar distinct changes in mtDNA organization were observed in in vitro cultures of the derived tetraploid and the hexaploid species with related genomes. The tetraploid and hexaploid species share the B genome and mtDNA variability in in vitro culture is known to be under nuclear control. These results suggest that a study of B genome diploids and other polyploid combinations would now shed light on whether or not mtDNA variability in tissue cultures is under B-genome control.     

Effect of the direction of DNA replication on mutagenesis by N-methyl-N''-nitro-N-nitrosoguanidine in adapted cells of Escherichia coli

Summary The methylating agent N-methyl-N-nitro-N-nitrosoguanidine preferentially induces G:C to A:T transitions at DNA base pairs with the G in one particular strand of the cI gene in a lambda prophage, in this case the non-transcribed straind, in Escherichia coli cells in which the adaptive response is induced. The same preference is found for the cI gene inserted in the genome in the inverse orientation, so the differential effect is not caused by the direction of motion of the DNA replicating fork.     

Structure of Penaeus stylirostris Densovirus,a Shrimp Pathogen

Penaeus stylirostris densovirus (PstDNV), a pathogen of penaeid shrimp, causes significant damage to farmed and wild shrimp populations. In contrast to other parvoviruses, PstDNV probably has only one type of capsid protein that lacks the phospholipase A2 activity that has been implicated as a requirement during parvoviral host cell infection. The structure of recombinant virus-like particles, composed of 60 copies of the 37.5-kDa coat protein, the smallest parvoviral capsid protein reported thus far, was determined to 2.5-Å resolution by X-ray crystallography. The structure represents the first near-atomic resolution structure within the genus Brevidensovirus. The capsid protein has a β-barrel “jelly roll” motif similar to that found in many icosahedral viruses, including other parvoviruses. The N-terminal portion of the PstDNV coat protein adopts a “domain-swapped” conformation relative to its twofold-related neighbor similar to the insect parvovirus Galleria mellonella densovirus (GmDNV) but in stark contrast to vertebrate parvoviruses. However, most of the surface loops have little structural resemblance to any of the known parvoviral capsid proteins.The Parvoviridae family is a family of small DNA viruses that is divided into two subfamilies, the Parvovirinae that infect vertebrates and the Densovirinae that infect invertebrates. Penaeus stylirostris densovirus (PstDNV), also known as infectious hypodermal and hematopoietic necrosis virus (IHHNV), belongs to the Densovirinae subfamily and was first reported as a highly lethal disease of juvenile shrimp in 1983 (22). The virus has significant commercial impact on the shrimp farming industry, causing mass mortality and severe deformations in penaeid shrimp during catastrophic epidemics in marine aquaculture facilities worldwide (14). PstDNV is closely related to the mosquito brevidensoviruses (35), which have the potential to be used as biological control agents of mosquito-borne diseases, such as malaria (30), dengue, chikungunya, and yellow fever (8).The single-stranded DNA genome of parvoviruses is encapsidated within a nonenveloped, icosahedral protein shell of less than 280 Å in external diameter. The capsid consists of 60 structurally equivalent subunits that are composed of the major viral coat protein and a few copies of N-terminally extended variants of the major capsid protein. A phospholipase A2 (PLA2) activity in the unique N-terminal extension of the largest minor capsid protein plays a crucial role during parvoviral host cell infection (7, 12, 13, 20, 46). The structures of the major capsid protein of several vertebrate parvoviruses have previously been determined to near-atomic resolution (1, 18, 23, 37, 41, 43, 44). However, the only high-resolution structure available for the invertebrate subfamily is that of the insect parvovirus Galleria mellonella densovirus (GmDNV) (36). The central motif of parvoviral capsid proteins is an eight-stranded, antiparallel β-barrel “jelly roll” fold. The surface of the virion, however, is formed by large insertions connecting the strands of the β-barrel, thereby creating features that govern antigenicity, receptor binding, and most intersubunit contacts. Surface characteristics common to most parvoviruses are protrusions at or around the icosahedral threefold axes, depressions on the twofold axes, and canyons surrounding the fivefold axes. At each fivefold apex, a cylindrical pore connects the interior of the virus particle with its exterior surroundings. In full virions, these pores are occupied by a glycine-rich motif in the N-terminal region of the major capsid protein, presumably positioning the N-terminal peptide for externalization. The general surface topology of GmDNV is smoother, probably due to smaller loop insertions. The structure of some of these insertions has diverged from vertebrate parvoviruses beyond recognition (4, 36). The N-terminal portions of twofold-related subunits in GmDNV have swapped their positions relative to those of the vertebrate parvoviruses. A cryo-electron microscopy (cryo-EM) study of Aedes albopictus densovirus, a brevidensovirus, has shown that its surface features are different from GmDNV and the mammalian parvoviruses, in particular in having prominent protrusions at the fivefold axes (9).Although it has been reported that PstDNV contains four structural proteins, as determined by SDS-polyacrylamide gel electrophoresis (3), these data do not fit the coding sequence (35). The 4.1-kb DNA genome of PstDNV (3) encodes in the 3′ half of the plus strand just one structural protein of 329 amino acids, as of now the smallest reported parvoviral capsid protein, and in the 5′ half of the plus strand two nonstructural proteins (666 and 363 amino acids) (35). Having only a single type of capsid protein is an unusual feature for viruses in the Parvoviridae family, where capsids are generally reported to contain two or more coat protein variants. A stretch of 11 amino acids in the N-terminal region of the capsid protein (17-DAHNEDEEHAE-27) is reminiscent of the PLA2 catalytic site (35), but it lacks important conserved motifs of PLA2s. Consequently, but curiously, PstDNV does not have the enzymatic activity that has previously been described as a requirement for parvoviral infectivity.We report here the three-dimensional (3D) crystal structure of recombinant, empty virus-like particles (VLPs) of the shrimp parvovirus PstDNV at 2.5-Å resolution. The loops connecting the strands of the structurally conserved jelly roll motif differ considerably in structure and length from other parvoviruses. The near-atomic resolution structure might provide the basis for the design of capsid binding antiviral compounds that may protect shrimp against parvoviral infection (16, 32, 42). Furthermore, the structure might aid the targeting of monoclonal antibodies to gain functional data about the role of the Brevidensovirus capsid protein during the infection cycle. Such information in turn may permit the design of densovirus-based delivery systems for drugs or pest control agents in aquacultural facilities. The small dimensions of PstDNV VLPs can be advantageous for their possible use as nanoparticles for antigen presentation and transport of immune stimulatory substances or interfering RNAs (21, 26). Additionally, the small size of the PstDNV capsid protein makes the system attractive as a model for studying assembly mechanisms of icosahedral virus capsids.     

Structural Organization of the Human Genome: Distribution of Nucleotides,AluRepeats, and Exons in Chromosomes 21 and 22

Analysis of DNA sequences of the human chromosomes 21 and 22 performed using a specially designed MegaGene software allowed us to obtain the following results. Purine and pyrimidine nucleotide residues are unevenly distributed along both chromosomes, displaying maxima and minima (waves) with a period of about 3 Mbp. Distribution of G+C along both chromosomes has no distinct maxima and minima, however, chromosome 21 contains considerably less G+C than chromosome 22. Both exons and Alurepeats are unevenly distributed along chromosome 21: they are scarce in its left part and abundant in the right part, while MIR elements are quite monotonously spread along this chromosome. The Alurepeats show a wave-like distribution pattern similar for both repeat orientations. The number of the Alurepeats of opposite orientations was equal for both studied chromosomes, and this may be considered a new property of the human genome. The positive correlation between the exon and Aludistribution patterns along the chromosome, the concurrent distribution of Alurepeats in both orientations along the chromosome, and the equal copy numbers for Aluin direct and inverted orientations within an individual chromosome point to their important role in the human genome, and do not fit the notion that Alurepeats belong to parasitic (junk) DNA.     

Transformation of cultivated tomato by a binary vector in Agrobacterium rhizogenes: transgenic plants with normal phenotypes harbor binary vector T-DNA,but no Ri-plasmid T-DNA

Summary Cultivated tomato was genetically transformed using two procedures. In the first procedure, punctured cotyledons were infected with disarmed Agrobacterium tumefaciens strain LBA4404 or with A. rhizogenes strain A4, each containing the binary vector pARC8. The chimeric neomycin phosphotransferase (NPT II) gene on pARC8 conferred on transformed plant cells the ability to grow on medium containing kanamycin. Transformation reproducible yielded kanamycin-resistant transformants in different tomato genotypes. NPT II activity was detected in transformed calli and in transgenic plants. All of these plants were phenotypically normal, fertile and set seeds. Using the second procedure, inverted cotyledons, we recovered transformed tomato plants from A. rhizogenes-induced hairy roots. In this case, all of the transgenic plants exhibited phenotypes similar to hairy root-derived plants reported for other species. Southern blot analysis on these plants revealed that the plant DNA hybridized with both probes representing pARC8-T-DNA, and the T-DNAs of the A4 Ri-plasmid. However, southern analysis on those phenotypically normal transgenic plants from the first procedure revealed that only the pARC8-T-DNA was present in the plant genome, thus indicating that the pARC8-T-DNA integrated into the plant genome independently of the pRi A4-T-DNA. Genetic analysis of these phenotypically normal transgenic plants for the kanamycin-resistance trait showed Mendelian ratios, 31 and 11, for selfed (R1) and in crossed progeny, respectively.     

Peribacteroid membrane nodulin gene induction by Bradyrhizobium japonicum mutants

Seventeen translation products from Glycine max root mRNA precipitated with antiserum prepared against a peribacteroid membrane preparation from effective root nodules. Messenger RNA from fix ⁺ nodules coded for these 17 products plus 7 other nodule-specific polypeptides which bound to the antiserum. Of these 7 nodulins only 4 were present when nodules were infected with Bradyrhizobium japonicum 110 rif 15 2960, which induces the plant to produce empty peribacteroid membranes. In nodules infected with B. japonicum strains inducing either very short-lived or defective peribacteroid membrane, only 5 or 6, respectively, of these nodulins could be detected.From these results we hypothesize that the microsymbiont is responsible for the production of at least 4 different signals leading to peribacteriod membrane formation by the plant.     

Surrogate transformation of perennial ryegrass, Lolium perenne, using genetically modified Acremonium endophyte.

Summary Conditions have been developed for transforming protoplasts of the perennial ryegrass endophyteAcremonium strain 187BB. Unlike most other ryegrass endophytes, this strain does not produce the lolitrem B neurotoxin and is therefore suitable as a host for surrogate introduction of foreign genes into grasses. Transformation frequencies of 700–800 transformants/g DNA were obtained for both linear and circular forms of pAN7-1, a hygromycin (hph) resistant plasmid. Up to 80% of the linear transformants were stable on further culturing but only 25% of the circular transformants retained hygromycin resistance. Integration of pAN7-1 into the genome was confirmed by Southern blotting and probing of genomic digests of transformant DNA. Both single and tandemly repeated copies of the plasmid were found in the genome and both the number and sites of integration varied among the transformants. At least 13 chromosomes were identified in 187BB using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Probing of Southern blots of these gels confirmed that pAN7-1 had integrated into different chromosomes. The -glucuronidase (GUS) gene,uidA, was also introduced into 187BB by co-transformation of pNOM-2 with pAN7-1. GUS activity was detected by growing the transformants on plates containing 5-bromo-4-chloro-3-indolyl -D-glucuronic acid and by enzyme assays of mycelial extracts. Severalhph- anduidA-containing transformants were reintroduced into ryegrass seedlings and expression of GUS visualized in vivo, demonstrating that 187BB can be used as a surrogate host to introduce foreign genes into perennial ryegrass. Molecular analysis of fungal isolates from the leaf sheath confirmed that the pattern of pAN7-1 and pNOM-2 hybridizing fragments was identical to that observed in the fungus used as inoculum.     

Protein polymorphism in sugarcane revealed by two-dimensional gel analysis

Summary In order to identify molecular markers for the analysis of the sugarcane genome, proteins extracted from apical segments of shoot tissues were resolved by a combination of equilibrium (IEF) and nonequilibrium (NEPHGE) two-dimensional polyacrylamide gel electrophoresis. A number of taxa of the Saccharum complex group (Saccharum species and the related genera of Andropogoneae) with presumed contributions to the sugarcane genome were surveyed. Protein profiles were compared to a reference map consisting of 1,482 protein spots from the noble cane,Saccharum officinarum L. Fifty-three polypeptides, representing about 3.6% of the total resolved spots, showed interspecific variation, whereas 78 polypeptides, about 5.3% of the total, showed intergeneric variation. Of the total polymorphic protein spots, qualitative (presence/absence) variation was more prevalent among the wild than among the cultivated species of the genusSaccharum, but the quantitative (spot intensity) variation was similar for both groups. The population of protein spots showing qualitative and quantitative variations was similar among the related genera of Andropogoneae. These polymorphic proteins can be used in genetic and evolutionary studies of the sugarcane genome.     

Genomic organization of multiple genes coding for rhodotorucine A,a lipopeptide mating pheromone of the basidiomycetous yeast Rhodosporidium toruloides

Rhodotorucine A, a lipopeptide mating pheromone, is secreted from mating type A cells of Rhodosporidium toruloides and induces sexual differentiation of the opposite mating type a cells. Genome of A-type cells contains three homologous genes (RHA1, RHA2, and RHA3) encoding rhodotorucine A. Genomic Southern blot analysis using RHA1 DNA as a probe showed that RHA1 strongly hybridize with A-type genomic DNA but weakly with a-type, suggesting that the sequences of RHA genes were dissimilar in the opposite a-type genome. The range of dissimilar regions in a-type genome was searched using RHA-flanking DNA segments as probes. The result suggests that a-type genome lacks sequences coding for rhodotorucine A and its 5 upstream but contains its 3 non-coding sequences. The absence of mating pheromone genes in the opposite mating type genome suggests that the expression of mating-type-specific genes in R. toruloides is not controlled trans-criptionally, as shown in the yeast Saccharomyces cerevisiae.     

Virus infections in three marine brown algae: Feldmannia irregularis,F. simplex,and Ectocarpus siliculosus

Culture studies with healthy and virus-infected isolates of Ectocarpus siliculosus, Feldmannia simplex and F. irregularis gave the following results: