Some biological and genomic properties of rice tungro bacilliform badnavirus and rice tungro spherical waikavirus from Nepal

A survey of rice fields during the main growing seasons in 81 locations from 21 districts of the Southern Terai region of Nepal indicated that rice tungro was primarily restricted to the Hardinath (Janakpur) and Parwanipur (Bara) regions. The tungro incidence in Hardinath ranged from 17% to 51% and in Parwanipur from 6% to 61% causing about 89% grain yield loss in Hardinath. Both rice tungro bacilliform badnavirus (RTBV) and rice tungro spherical picornavirus (RTSV) were found in tungro isolates collected from Hardinath and Parwanipur. These isolates were transmitted by Nephotettix virescens and leaf extracts reacted to antisera against RTBV and RTSV. In a dot blot hybridisation assay, leaf extracts of 12 weed species collected from the tungro-affected area in Hardinath and Parwanipur also reacted with RTBV DNA probes. On mass inoculation of 15 popular rice cultivars most became more than 50% infected and only cv. Radha 9 had low (22.2%) infection. RTBV DNA and the coat protein region of RTSV from the Hardinath isolate were cloned and partially characterised. A comparative analyses by restriction endonuclease digestion, cross hybridisation, the polymerase chain reaction and partial sequencing indicated that the Nepalese RTBV DNA clone and the cDNA clones of the RTSV RNA were more similar to the various tungro isolates from the Indian subcontinent than to those from the Philippines.     

对虾白斑杆状病毒DNA聚合酶调控序列的克隆

为了揭示对虾白斑杆状病毒的致病机理,将该病毒基因组中推测的DNA聚合酶上游调控序列克隆进荧光素酶报告基因载体中,以便寻找一个能表达该病毒基因的细胞系统.     

Inheritance of tolerance to rice tungro bacilliform virus (RTBV) in rice (Oryza sativa L.)

Summary From a large number of rice varieties tested, no variety was identified as resistant to tungro bacilliform virus (RTBV). Only in Utri Merah was the RTBV multiplication restrictive, whereas other varieties such as Kataribhog and Pankhari 203 were identified as tolerant. These varieties were crossed with a susceptible variety. TN1, to study the inheritance of restrictive multiplication and tolerance to RTBV. After 3 weeks of inoculation with RTBV, F₁; F₂, and F₃ progenies were assessed by enzyme-linked immunosorbent assay (ELISA). The RTBV concentration in all F₁ populations was intermediate between parents. The frequency distribution of F₂ seedlings with various levels of RTBV concentration indicated that the RTBV tolerance is controlled by multiple genes. The RTBV concentrations in F₁ and F₂ progenies from the Utri Merah x TN1 cross revealed that restrictive multiplication of RTBV in Utri Merah is a polygenic character. The continuous variation observed in F₂ populations from crosses between tolerant varieties and Utri merah indicated no allelic relationships between tolerant and restrictive multiplication traits.Part of PhD thesis submitted by senior author to University of Kebangsaan Malaysia, Bangi     

Sequence-specific and methylation-dependent and -independent binding of rice nuclear proteins to a rice tungro bacilliform virus vascular bundle expression element

Nuclear proteins from rice (Oryza sativa) were identified that bind specifically to a rice tungro bacilliform virus promoter region containing a vascular bundle expression element (VBE). One set of proteins of 29, 33, and 37 kDa, present in shoot and cell suspension extracts but hardly detectable in root extracts, bound to a site containing the sequence AGAAGGACCAGA within the VBE, which also contains two CpG and one CpNpG potential methylation motifs. Binding by these proteins was determined to be cytosine methylation-independent. However, a novel protein present in all analyzed extracts bound specifically to the methylated VBE. A region of at least 49 nucleotides overlapping the VBE and complete cytosine methylation of the three Cp(Np)G motifs was required for efficient binding of this methylated VBE-binding protein (MVBP).     

The rice tungro bacilliform virus gene II product interacts with the coat protein domain of the viral gene III polyprotein

Rice tungro bacilliform virus (RTBV) is a plant pararetrovirus whose DNA genome contains four genes encoding three proteins and a large polyprotein. The function of most of the viral proteins is still unknown. To investigate the role of the gene II product (P2), we searched for interactions between this protein and other RTBV proteins. P2 was shown to interact with the coat protein (CP) domain of the viral gene III polyprotein (P3) both in the yeast two-hybrid system and in vitro. Domains involved in the P2-CP association have been identified and mapped on both proteins. To determine the importance of this interaction for viral multiplication, the infectivity of RTBV gene II mutants was investigated by agroinoculation of rice plants. The results showed that virus viability correlates with the ability of P2 to interact with the CP domain of P3. This study suggests that P2 could participate in RTBV capsid assembly.     

DNA excision repair and transcription: implications for genome evolution

The past two years have seen a substantial increase in knowledge regarding the enzymology of DNA excision repair. These data support a growing body of information which suggests that transcribed nucleotide sequences are preferentially subject to excision repair. It is possible that these mechanisms, or related ones, are relevant to the molecular evolution of sequences that appear not to evolve according to models which do not take into account regional sequence differences in the extent of DNA repair.     

Duplication and DNA segmental loss in the rice genome: implications for diploidization

* Large-scale duplication events have been recently uncovered in the rice genome, but different interpretations were proposed regarding the extent of the duplications. * Through analysing the 370 Mb genome sequences assembled into 12 chromosomes of Oryza sativa subspecies indica, we detected 10 duplicated blocks on all 12 chromosomes that contained 47% of the total predicted genes. Based on the phylogenetic analysis, we inferred that this was a result of a genome duplication that occurred c. 70 million years ago, supporting the polyploidy origin of the rice genome. In addition, a segmental duplication was also identified involving chromosomes 11 and 12, which occurred c. 5 million years ago. * Following the duplications, there have been large-scale chromosomal rearrangements and deletions. About 30-65% of duplicated genes were lost shortly after the duplications, leading to a rapid diploidization. * Together with other lines of evidence, we propose that polyploidization is still an ongoing process in grasses of polyploidy origins.     

Organisation and properties of repeated DNA sequences in rice genome

Reassociation of high molecular weight rice DNA has revealed the occurrence of long stretches of repeated DNA which are not interrupted by single copy DNA even at a fragment length as high as 20 kilo base pairs (kbp). Majority of these repeated sequences are unusually G + C rich and show significant variations in their thermal stability. Homology studies indicate that short repeats may have evolved from long repeats in total repetitive DNA while they may be of different origin in highly repetitive DNA fraction. Restriction enzyme analysis shows the occurrence of Ava I and EcoR V repeat families.     

Periodicity in DNA coding sequences: implications in gene evolution

In this paper we have employed Fourier analysis of DNA coding and non-coding sequences in an attempt to identify possible patterns in gene sequences. It was found that while intronic sequences show a rather random pattern, coding sequences show periodicities and in particular a periodicity of 3. We were able to reconstruct such patterns by assuming a gene having one codon occurring in about 40% of the sequence. This could indicate that the predominant presence of codons all starting from the same base could confer the observed periodicities. Indeed, it was found that proteins do obey this rule. Implications of this finding in gene evolution are discussed.     

Ancient repeated sequences in the pea and mung bean genomes and implications for genome evolution

Essentially all of the sequences in the pea (Pisum sativum) genome which reassociate with single copy kinetics at standard (T_m -25°C) criterion follow repetitive kinetics at lower temperatures (about T_m-35°C). Analysis of thermal stability profiles for presumptive single copy duplexes show that they contain substantial mismatch even when formed at standard criterion. Thus most of the sequences in the pea genome which are conventionally defined as single copy are actually fossil repeats — that is, they are members of extensively diverged (mutuated) and thus presumably ancient families of repeated sequences. Coding sequences as represented by a cDNA probe prepared from poly-somal poly(A) + mRNA reassociate with single copy kinetics regardless of criterion and do not form mismatched duplexes. The coding regions thus appear to be composed of true single copy sequences but they cannot represent more than a few percent of the pea genome. Ancient diverged repeats are present, but not a prominent feature of the smaller mung bean (Vigna radiata) genome. An extension of a simple evolutionary model is proposed in which these and other differences in genome organization are considered to reflect different rates of sequence amplification or genome turnover during evolution. The model accounts for some of the differences between typical plant and animal genomes.     

New insertion sequences of Sulfolobus: functional properties and implications for genome evolution in hyperthermophilic archaea

Analyses of complete genomes indicate that insertion sequences (ISs) are abundant and widespread in hyperthermophilic archaea, but few experimental studies have measured their activities in these hosts. As a way to investigate the impact of ISs on Sulfolobus genomes, we identified seven transpositionally active ISs in a widely distributed Sulfolobus species, and measured their functional properties. Six of the seven were found to be distinct from previously described ISs of Sulfolobus, and one of the six could not be assigned to any known IS family. A type II 'Miniature Inverted-repeat Transposable Element' (MITE) related to one of the ISs was also recovered. Rates of transposition of the different ISs into the pyrEF region of their host strains varied over a 250-fold range. The Sulfolobus ISs also differed with respect to target-site selectivity, although several shared an apparent preference for the pyrEF promoter region. Despite the number of distinct ISs assayed and their molecular diversity, only one demonstrated precise excision from the chromosomal target region. The fact that this IS is the only one lacking inverted repeats and target-site duplication suggests that the observed precise excision may be promoted by the IS itself. Sequence searches revealed previously unidentified partial copies of the newly identified ISs in the Sulfolobus tokodaii and Sulfolobus solfataricus genomes. The structures of these fragmentary copies suggest several distinct molecular mechanisms which, in the absence of precise excision, inactivate ISs and gradually eliminate the defective copies from Sulfolobus genomes.     

Adeno-associated virus Rep78 protein and terminal repeats enhance integration of DNA sequences into the cellular genome.

Two adeno-associated virus (AAV) elements are necessary for the integration of the AAV genome: Rep78/68 proteins and inverted terminal repeats (ITRs). To study the contribution of the Rep proteins and the ITRs in the process of integration, we have compared the integration efficiencies of three different plasmids containing a green fluorescent protein (GFP) expression cassette. In one plasmid, no viral sequences were present; a second plasmid contained AAV ITRs flanking the reporter gene (integration cassette), and a third plasmid consisted of an integration cassette plus a Rep78 expression cassette. One day after transfection of 293 cells, fluorescent cells were sorted by flow cytometry and plated at 1 cell per well. Two weeks after sorting, colonies were monitored for stable expression of GFP. Transfection with the GFP plasmid containing no viral sequences resulted in no stable fluorescent colonies. Transfection with the plasmid containing the integration cassette alone (GFP flanked by ITRs) produced stable fluorescent colonies at a frequency of 5.3% +/- 1.0% whereas transfection with the plasmid containing both the integration cassette and Rep78 expression cassette produced stable fluorescent colonies at a frequency of 47% +/- 7.5%. Southern blot analysis indicated that in the presence of Rep78, integration is targeted to the AAVSI site in more than 50% of the clones analyzed. Some clones also showed tandem arrays of the integrated GFP cassette. Both head-to-head and head-to-tail orientations were detected. These findings indicate that the presence of AAV ITRs and the Rep78 protein enhance the integration of DNA sequences into the cellular genome and that the integration cassette is targeted to AAVS1 in the presence of Rep78.     

Organization and integration sites in the human genome of endogenous retroviral sequences belonging to HERV-E family

The present paper reports the characterization of HERV-E endogenous retroviral sequences in the human genome by using three complementary approaches. Firstly, we identified genomic clones containing HERV-E by BLAST screening of human DNA databases by using the entire sequence of a characterized HERV-E clone (M10976) as a query. The genomic structure and integration sites of the HERV-E elements were characterized. Secondly, new integration sites of HERV-E elements were revealed by a retroviral LTR-arbitrary primer-PCR (RELAP-PCR) technique. BLAST analysis of the PCR products identified a subgroup that shows low identity (75%) to the original clone M10976 and slightly higher identity (80%) to a closely related HERV-E (Ac. n. K02166). Finally, we performed FISH mapping, which revealed sites of integration of HERV-E not previously identified at the cytogenetic level. A preliminary analysis of genes mapping in the same bands as HERV-E integration sites was performed: several loci relevant to physiological and/or pathological processes were detected. Our findings may provide clues to identify HERV-E integration sites adjacent to genes with important biological roles.