HeT-A and TART, two Drosophila retrotransposons with a bona fide role in chromosome structure for more than 60 million years

Drosophila telomeres have been maintained by retrotransposition for at least 60 MY, which predates the separation of extant species of this genus. Studies of D. melanogaster, D. yakuba, and D. virilis show that, in Drosophila, telomeres are composed of two non-LTR retrotransposons, HeT-A and TART. Far from being static, HeT-A and TART evolve faster than Drosophila euchromatic genes. In spite of their high rate of sequence change, HeT-A and TART maintain their basic structures and unusual individual features. The maintenance of their separate identities suggests that HeT-A and TART cooperate either in the process of retrotransposition onto the chromosome end, or in the formation of telomere chromatin by transposed DNA copies. The telomeric retrotransposons and the Drosophila genome constitute an example of a robust symbiotic relationship between mobile elements and the genome.     

Insights into cereal genomes from two draft genome sequences of rice

Draft genome sequences have been reported for two subspecies of rice. The drafts include the sequences of an estimated 99% of all rice genes and provide major advances in our understanding of the content and complexity of cereal genomes in general and the rice genome in particular.     

Recombination between two TnA transposon sequences oriented as inverse repeats is found less frequently than between direct repeats

Summary Inverse repeats of the transposon Tn2660 in either a ColEl or an R6K replicon, with or without inversions of the parental DNA sequences between the repeats, show no detectable (<2%) evidence of recombination between the repeats after 60 generations of growth in either recA or recA ⁺ hosts. In contrast, attempts made to construct plasmids which carry two direct repeats by in vitro cleavage and ligation in a recA host were unsuccessful, although homologous plasmids with inverse repeats could be constructed, and other plasmids were found consistent with products of recombination between the direct repeats of a transient intermediate structure. It is concluded that in recA or recA ⁺ hosts recombination between direct repeats of a transposon is frequent, whereas recombination between inverse repeats of a homologous structure has not been observed. A model to explain this difference depends upon a mechanism that produces a nick in only one of the pair of strands at the internal resolution site (IRS) sequence of the transposon.     

Comparative expression analysis of two sugarcane polyubiquitin promoters and flanking sequences in transgenic plants

GUS (uidA) reporter gene expression for two sugarcane polyubiquitin promoters, ubi4 and ubi9, was compared to expression from the maize Ubi-1 promoter in stable transgenic rice (only ubi9) and sugarcane (ubi4 and ubi9). Ubi9 drove high-level GUS expression, comparable to the maize Ubi-1 promoter, in both callus and regenerated plants of rice transformed by Agrobacterium. This high level expression was inherited in R1 plants. Expression from ubi4 and ubi9 was quite high in sugarcane callus transformed via particle bombardment. Expression dropped to very low or undetectable levels in the resulting plants; this drop in expression resulted from PTGS. PTGS in regenerated sugarcane plants also occurred with the maize Ubi-1 promoter. In sugarcane callus, ubi4 was HS inducible, but ubi9 was not. This physiological difference corresponds to a MITE insertion that is present in the putative HSEs of ubi9 but not present in ubi4.     

Phylogenetic relationships of major clades of Catostomidae (Teleostei: Cypriniformes) as inferred from mitochondrial SSU and LSU rDNA sequences

Suckers (Family Catostomidae) are holarctic in distribution and include 76 recent species in 14 genera, with 13 genera and 75 species occurring in North and Central America and Siberia. Although this group constitutes a significant component of many aquatic ecosystems, most historic systematic effort has been either alpha- or limited beta-level studies focusing on the two largest tribes within the family, the Catostomini and the Moxostomatini. A recent phylogenetic study based on morphological, biochemical, and early life history characters has advanced current understanding of relationships among catostomid fishes. To further examine phylogenetic relationships among basal lineages of catostomids, we sequenced the entire mitochondrial (mt) SSU and LSU rRNA genes from genera representing all subfamilies and tribes within Catostomidae. Phylogenetic analysis of gene sequences yielded monophyletic Catostomidae, Ictiobinae, and Catostominae and para- or polyphyletic Cycleptinae, with Myxocyprinus as the basal-most taxon and Cycleptus as either the next most-basal taxon or the taxon basal to the Catostominae. Relationships within the Catostominae were generally consistent with those proposed in the above-noted recent phylogenetic study although Thoburnia and Hypentelium were either a clade sister to or a grade group relative to Moxostoma and Scartomyzon. In all trees, Scartomyzon was paraphyletic and embedded within Moxostoma. Phylogenetic affinities of Erimyzon and Minytrema varied depending on data set and character weighting scheme employed. To better reflect phylogenetic relationships resolved in this extensive analysis, we propose the following changes to the classification of catostomids: formation of the new subfamily Myxocyprininae, containing Myxocyprinus from China; restriction of the Cycleptinae to the two species of Cycleptus from North America; restriction of the tribe Moxostomatini to Moxostoma and Scartomyzon; Erimyzon and Minytrema are incertae sedis within Catostominae; and resurrection of the tribe Thoburniini, containing Thoburnia and expanded to include Hypentelium.     

Comparative analysis of protein coding sequences from human,mouse and the domesticated pig

Background  

The availability of abundant sequence data from key model organisms has made large scale studies of molecular evolution an exciting possibility. Here we use full length cDNA alignments comprising more than 700,000 nucleotides from human, mouse, pig and the Japanese pufferfish Fugu rubrices in order to investigate 1) the relationships between three major lineages of mammals: rodents, artiodactyls and primates, and 2) the rate of evolution and the occurrence of positive Darwinian selection using codon based models of sequence evolution.     

Genetic and geographic origin of domesticated peanut as evidenced by 5S rDNA and chloroplast DNA sequences

The history of the cultivated peanut involves natural evolution and human domestication. Despite the economic importance of peanuts and the many studies carried out on their cytology and genetic variability, current knowledge on the origin of the cultigen is still very limited compared with other major crops. In this context, we analyzed the polymorphisms of some non-coding cpDNA regions and the non-transcribed spacer of the nuclear 5S rDNA of the six botanical varieties of the two subspecies of the cultigen, of the wild tetraploid A. monticola, and of the nine diploid species so far proposed as the most probable relatives of the peanut, to gain more insight into the genetic and geographic origin of this legume crop. The analysis showed complete homology in the sequences of all the peanut and A. monticola samples. These results strongly suggest that the six botanical varieties of the cultigen have a single genetic origin and that A. monticola should be regarded as the immediate tetraploid ancestor from which A. hypogaea has arisen upon domestication. Here we provide results from the first sequence-based analysis in which the maternal (A. duranensis) and paternal (A. ipa?nsis) wild diploid species of the AABB tetraploids of Arachis were unequivocally identified. Not only that, but the combination of cpDNA and NTS 5S rDNA identified the population of A. duranensis from Río Seco, Salta, Argentina, and the only known population of A. ipa?nsis from Villa Montes, Tarija, Bolivia, as those to which the genome donors of the peanut could have belonged.     

Isolation and characterization of two phytoalexins from rice as momilactones A and B

Two phytoalexins were isolated as chromatographically homogeneous amorphous solids from UV-irradiated, dark-grown rice coleoptiles. From their mass and ¹H NMR spectra, the compounds were characterized as the known diterpenes, momilactones A and B. The same compounds were also produced in blast-infected, WL 28325-treated rice leaves. They appear to be the first clearly identified cereal phytoalexins.     

Functional relevance of Gedunin as a bona fide ligand of NADPH oxidase 5 and ROS scavenger: An in silico and in vitro assessment in a hyperglycemic RBC model

Clinical evidence suggests that type 2 diabetes therapy can greatly benefit from the suppression of reactive oxygen species generation and the activation or restoration of cellular antioxidant mechanisms. In human, NADPH oxidase (NOX) is the main producer of reactive oxygen species (ROS) that supress the activity of endogenous antioxidant enzymes. In the present study, the antioxidant potential of Gedunin was studied. In silico findings reveal its strong binding affinity with NOX5 C terminal HSP90 binding site that disrupts NOX5 stability and its ability to generate ROS, leading to restoration antioxidant enzymes activities. It was found that Gedunin suppressed hyperglycaemia induced oxidative stress in an in vitro RBC model and markedly reversed glucose induced changes including haemoglobin glycosylation and lipid peroxidation. A significant restoration of activities of cellular antioxidant enzymes; superoxide dismutase, catalase and glutathione peroxidase in the presence of Gedunin revealed its ability to reduce oxidative stress. These results substantiated Gedunin as a bona fide inhibitor of human NOX5 and a ROS scavenging antioxidant with promising therapeutic attributes including its natural origin and inhibition of multiple diabetic targets.     

Molecular evolutionary analysis of the widespread<Emphasis Type="Italic"> piggyBac</Emphasis> transposon family and related "domesticated" sequences

piggyBac is a short inverted-repeat-type DNA transposable element originally isolated from the genome of the moth Trichoplusia ni. It is currently the gene vector of choice for the transformation of various insect species. A few sequences with similarity to piggyBac have previously been identified from organisms such as humans ( Looper), the pufferfish Takifugu rubripes (Pigibaku), Xenopus (Tx), Daphnia (Pokey), and the Oriental fruit fly Bactrocera dorsalis. We have now identified 50 piggyBac-like sequences from publicly available genome sequences and expressed sequence tags (ESTs). This survey allows the first comparative examination of the distinctive piggyBac transposase, suggesting that it might contain a highly divergent DDD domain, comparable to the widespread DDE domain found in many DNA transposases and retroviral integrases which consists of two absolutely conserved aspartic acids separated by about 70 amino acids with a highly conserved glutamic acid about 35 amino acids further away. Many piggyBac-like sequences were found in the genomes of a phylogenetically diverse range of organisms including fungi, plants, insects, crustaceans, urochordates, amphibians, fishes and mammals. Also, several instances of "domestication" of the piggyBac transposase sequence by the host genome for cellular functions were identified. Novel members of the piggyBac family may be useful in genetic engineering of many organisms.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Study of sugarcane pieces as yeast supports for ethanol production from sugarcane juice and molasses

Due to the environmental concerns and the increasing price of oil, bioethanol was already produced in large amount in Brazil and China from sugarcane juice and molasses. In order to make this process competitive, we have investigated the suitability of immobilized Saccharomyces cerevisiae strain AS2.1190 on sugarcane pieces for production of ethanol. Electron microscopy clearly showed that cell immobilization resulted in firm adsorption of the yeast cells within subsurface cavities, capillary flow through the vessels of the vascular bundle structure, and attachment of the yeast to the surface of the sugarcane pieces. Repeated batch fermentations using sugarcane supported-biocatalyst were successfully carried out for at least ten times without any significant loss in ethanol production from sugarcane juice and molasses. The number of cells attached to the support increased during the fermentation process, and fewer yeast cells leaked into fermentation broth. Ethanol concentrations (about 89.73–77.13 g/l in average value), and ethanol productivities (about 59.53–62.79 g/l d in average value) were high and stable, and residual sugar concentrations were low in all fermentations (0.34–3.60 g/l) with conversions ranging from 97.67–99.80%, showing efficiency (90.11–94.28%) and operational stability of the biocatalyst for ethanol fermentation. The results of this study concerning the use of sugarcane as yeast supports could be promising for industrial fermentations. L. Liang and Y. Zhang have contributed equally to this work.     

The N-terminal region of the Oenococcus oeni bacteriophage fOg44 lysin behaves as a bona fide signal peptide in Escherichia coli and as a cis-inhibitory element, preventing lytic activity on oenococcal cells

The function of the N-terminal region of the Oenococcus oeni phage fOg44 lysin (Lys44) as an export signal was investigated. We observed that when induced in Escherichia coli, Lys44 was cleaved between residues 27 and 28 in a SecA-dependent manner. Lys44 processing could be blocked by a specific signal peptidase inhibitor and was severely reduced by modification of the cleavage site. The lethal effect of Lys44 expression observed in E. coli was ascribed to the presence of its N-terminal 27-residue sequence, as its deletion resulted in the production of a nontoxic, albeit active, product. We have further established that lytic activity in oenococcal cells was dependent on Lys44 processing. An active protein with the molecular mass expected for the cleaved enzyme was detected in extracts from O. oeni-infected cells. The temporal pattern of its appearance suggests that synthesis and export of Lys44 in the infected host progress along with phage maturation. Overall, these results provide, for the first time, experimental evidence for the presence of a signal peptide in a bacteriophage lysin. Database searches and alignment of protein sequences support the prediction that other known O. oeni and Lactococcus lactis phages also encode secretory lysins. The evolutionary significance of a putative phage lysis mechanism relying on secretory lytic enzymes is tentatively discussed, on the basis of host cell wall structure and autolytic capacity.     

Cryoprotectant removal temperature as a factor in the survival of frozen rice and sugarcane cells

Removal of cryoprotective additives through use of a room temperature (22 °C) washing step, instead of 0 °C, was found to improve the recovery of sugarcane suspension culture and rice callus tissues. Cultured cells were cryoprotected by gradual addition of a mixture of polyethylene glycol, glucose, and DMSO (PGD) to a final concentration of 10%-8%-10%, w/v, respectively, added at either 0 or 22 °C. After a programmed slow freezing of the cells, they were thawed rapidly and the cryoprotectants were gradually diluted and washed out using a 22 or 0 °C washing medium. Viability of suspension cultured sugarcane cells protected with PGD was greatly diminished when a cold washing solution was used, whether the cells had been frozen (?23 °C) or not. Two mutant lines of rice callus when frozen to ?196 °C in PGD and thawed showed less growth than unfrozen cells, but their growth was improved by washing the thawed cells with a 22 °C solution. With all cultures tested, the addition of PGD at 0 °C and post-thaw washing out at 22 °C gave improved survival. Particularly with the rice lines, optimizing the addition and washing procedures allowed culture survival of liquid nitrogen freezing not otherwise attained.     

Evolution of two major chorion multigene families as inferred from cloned cDNA and protein sequences

Complete or partial sequences are reported from six chorion cDNA clones of the silkmoth Antheraea polyphemus. The proteins encoded belong to the two major chorion protein classes, A and B, each of which is encoded by a multigene family. The sequence comparisons define some major features of the families and suggest how these genes may be evolving. Deletions and insertions might be involved in expanding or contracting internally repetitive regions. Sequence divergence is localized, thus defining sequence domains of distinct evolutionary properties and presumably distinct functions.     

Unbiased functional proteomics strategy for protein kinase inhibitor validation and identification of bona fide protein kinase substrates: application to identification of EEF1D as a substrate for CK2

Protein kinases have emerged as attractive targets for treatment of several diseases prompting large-scale phosphoproteomics studies to elucidate their cellular actions and the design of novel inhibitory compounds. Current limitations include extensive reliance on consensus predictions to derive kinase-substrate relationships from phosphoproteomics data and incomplete experimental validation of inhibitors. To overcome these limitations in the case of protein kinase CK2, we employed functional proteomics and chemical genetics to enable identification of physiological CK2 substrates and validation of CK2 inhibitors including TBB and derivatives. By 2D electrophoresis and mass spectrometry, we identified the translational elongation factor EEF1D as a protein exhibiting CK2 inhibitor-dependent decreases in phosphorylation in (32)P-labeled HeLa cells. Direct phosphorylation of EEF1D by CK2 was shown by performing CK2 assays with EEF1D -FLAG from HeLa cells. Dramatic increases in EEF1D phosphorylation following λ-phosphatase treatment and phospho- EEF1D antibody recognizing EEF1D pS162 indicated phosphorylation at the CK2 site in cells. Furthermore, phosphorylation of EEF1D in the presence of TBB or TBBz is restored using CK2 inhibitor-resistant mutants. Collectively, our results demonstrate that EEF1D is a bona fide physiological CK2 substrate for CK2 phosphorylation. Furthermore, this validation strategy could be adaptable to other protein kinases and readily combined with other phosphoproteomic methods.     

Comparison of dot chromosome sequences from D. melanogaster and D. virilis reveals an enrichment of DNA transposon sequences in heterochromatic domains

Background

Chromosome four of Drosophila melanogaster, known as the dot chromosome, is largely heterochromatic, as shown by immunofluorescent staining with antibodies to heterochromatin protein 1 (HP1) and histone H3K9me. In contrast, the absence of HP1 and H3K9me from the dot chromosome in D. virilis suggests that this region is euchromatic. D. virilis diverged from D. melanogaster 40 to 60 million years ago.

Results

Here we describe finished sequencing and analysis of 11 fosmids hybridizing to the dot chromosome of D. virilis (372,650 base-pairs) and seven fosmids from major euchromatic chromosome arms (273,110 base-pairs). Most genes from the dot chromosome of D. melanogaster remain on the dot chromosome in D. virilis, but many inversions have occurred. The dot chromosomes of both species are similar to the major chromosome arms in gene density and coding density, but the dot chromosome genes of both species have larger introns. The D. virilis dot chromosome fosmids have a high repeat density (22.8%), similar to homologous regions of D. melanogaster (26.5%). There are, however, major differences in the representation of repetitive elements. Remnants of DNA transposons make up only 6.3% of the D. virilis dot chromosome fosmids, but 18.4% of the homologous regions from D. melanogaster; DINE-1 and 1360 elements are particularly enriched in D. melanogaster. Euchromatic domains on the major chromosomes in both species have very few DNA transposons (less than 0.4 %).

Conclusion

Combining these results with recent findings about RNAi, we suggest that specific repetitive elements, as well as density, play a role in determining higher-order chromatin packaging.     

A system to induce the deletion of gemomic sequences using R/RS site-specific recombination and the Ac transposon in transgenic rice plants

Recombinase encoded by the R gene of Zygosaccharomyces rouxii mediates reciprocal recombination between two specific recombination sites (RSs) to induce deletion or inversion of the DNA segment that is flanked by the RSs. The R gene under the control of the CaMV 35 S promoter was introduced into rice (Oryza sativa L.). R/RS-specific deletion was first demonstrated in transgenic rice callus carrying the R gene by transient introduction of a cryptic reporter gene that was designed to confer β-glucuronidase (GUS) expression once deletion between two RSs took place. The rice containing the R gene was subsequently crossed with transgenic rice carrying (I-RS/dAc-I-RS) T-DNA that contained RS sequences within the T-DNA and another RS in a modified Ac element that had been transposed to a new locus by Ac transposase. Deletion of the gemomic sequences flanked by the two RSs was detected by PCR analysis in somatic cells of F₂ plants. These results demonstrate a technical advance in that the R/RS recombination system, in combination with the Ac transposable element, can be used to generate deletion in rice chromosomes. Received: 30 June 2000 / Accepted: 16 October 2000