Chloroplast and Mitochondrial Comparison

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Sequence similarities between the viral genomes and the rice nuclear genome

In this analysis we confirmed a previous report that the Rice tungro bacilliform virus (RTBV) is integrated into the rice nuclear genome. Nagano et al., (2000) reported a highly variable region on chromosome 6 with homology (54 – 59%) to RTBV that was also highly variable in copy number between rice cultivars. Kunii et al., (2004) reported on 29 segments homologous to RTBV. Previous studies focused on the characterization of specific fragments through PCR, sequencing and Southern Blot Analysis rather than a whole genome comparison. Using this approach this study identified additional fragments that have not yet been reported. Up to 40 copies of some regions of the Rice tungro bacilliform virus were found to be present in the rice genome, indicating either multiple integrations or subsequent duplication of some regions. If indeed the RTBV genome has been inserted more than 40 times over the course of time, it would seem that it has been accompanied by extensive elimination. Even so most of the genome is still represented in the rice nuclear genome at various copy levels. It is interesting that we find similar fragments of RTBV only in one other plant species namely Vitis vinifera. We could find no reports of RTBV infecting Vitis, but is seems likely that it does or that there is a as yet unidentified/ un-sequenced counterpart of RTBV associated with Vitis.
Furthermore this study provides evidence of possible integration events between rice and five different RNA viruses and the rice genome were also identified. Fifty-one fragments that show similarity to rice related RNA viruses have been identified. The integration of RNA viruses into the plant genome has not yet been reported (Harper et al., 2002), except for the finding that grapevine genomic DNA carries the entire gene of a potyviral coat protein (CP) and the potyviral 3′UTR. Potyviral-homologous sequences were also found in tobacco DNA, albeit in a rearranged form (Tanne and Sela, 2005). The sequence fragments identified in this study show between 40 – 70% identity with the viral genome and range in length between 184 and 1800 bps. This variation in homology would seem to indicate different times of integration during the evolution of the rice genome. Only fragments of the viral genomes are present. Since many of the RNA viruses of rice have genomes containing several fragments rather than circular genomes, partial integration would be more likely. With animal and bacterial retroviruses the early steps of replication involve reverse transcription of the viral RNA genome to make a cDNA copy followed by the integration of that cDNA copy into a chromosome of the host cell. The integration reaction requires specific sequences at the ends of the viral cDNA, which bind the viral-encoded integrase and other proteins to form pre-integration complexes (Schröder et al., 2002). The infection cycle of plant viruses is not known to include an integration event, mainly because integration of retrovial DNA is facilitaded by a virally encoded integrase (Patience et al., 1997). Plant pararetroviruses generally lack the gene for this enzyme, and integration is not required for virus replication (Jakowitsch et al., 1999), even so there are is evidence of the integration of non-retroviral genomes into plant genomes (Tanne and Sela, 2005; Staginnus and Richert-Pöggeler, 2006).
With the absence of an integrase enzyme the integration of viral sequences into the plant genome must involve a recombination event. In the absence of viral sequence in the host genome, recombination must be non-homologous (Jakowitsch et al., 1999). If there are already viral sequences already present, the recombination could be homologous. It is still unknown if the complete viral DNA genome itself integrates or whether integration occurs through replication intermediates such as cDNAs. During replication, pararetrovirus genomes form multiple copies of nuclear mini-chromosomes and gapped dsDNA replication intermediates, and the ssDNA geminivirus genomes are replicated to high levels in the nucleus. Both modes of replication provide potential recombinogenic sequences for integration, via illegitimate recombination, in cells undergoing active genetic processes (Hull et al., 2000). Integrated sequences of banana streak virus and RTBV integrated sequences suggest that the cDNAs can integrate as well (Harper et al., 1999; Ndowora et al., 1999; Jakowitsch et al., 1999; Kunii et al., 2004). It is however possible that a retroelement integrase function can be used in trans, as has been suggested for SINE element integration from LINE element integrase (Eikbush, 1992). It might also be plausible that RNA virus segments are integrated after the viral segments get reverse-transcribed by reversetranscriptase used by other viruses in the cell.
It would seem likely that viral sequence integration can occur during every infection. However, as most viruses do not infect meristematic tissues, the integrations are not usually fixed and are lost on passage through seed. Furthermore cells and plants in which functional copies of the integrated virus can readily excise, be transcribed or otherwise result in disease might not be viable. Rapid rearrangement and/or deletion of viral sequences through recombination would effectively disrupt the process. This is a possible reason for all reported cases of integration involving rearrangement or deletions.
The possibility that viral DNA might insert regularly into plant genomes, has considerable implications for plant genome evolution. Little is yet known about the contribution of integrated viral sequences to plant genome organization, function and evolution. Similar to vertebrate endogenous retroviruses (Patience et al., 1997), integrated pararetroviral DNA could act as insertional mutagens; could contribute strong constitutive promoters to neighboring plant genes, altering gene expression patterns; or they could accumulate to generate new repetitive sequence families. As components of the genome, they can be altered, recombined and amplified, providing another source of variation.
Overall this analysis presents the first comprehensive assessment of viral integration and contribution to the rice nuclear genome.

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1 Introduction and literature review 
1.1 The eukaryotic genome
1.2 Origin of the chloroplast and mitochondrion
1.3 DNA transfer
1.3.1 DNA transfer between the organelles and nucleus
1.3.2 DNA transfer between viruses and plants
I. The banana streak virus (BSV)
II. Tobacco vein clearing virus (TVCV)
III. Petunia vein clearing virus (PVCV)
IV. Rice tungro bacilliform virus (RTBV)
1.3.3 DNA transfer between bacteria and plants
1.3.4 DNA transfer between fungi and plants
1.3.5 DNA transfer between plants and plant mitochondria
1.5 Models for gene transfer and integration
2 Materials and Methods 
2.1 Blast Analysis
2.1.1 Chloroplast and Mitochondrial Comparison
2.1.2 Viral Comparison
2.1.3 Bacterial Comparison
2.1.4 Fungal Comparison
3 Results 
3.1 Chloroplast and Mitochondrial comparison
3.1.1 Chloroplast homologies in the rice nuclear genome
3.1.2 Representation of the chloroplast genome in the rice nuclea genome
3.1.3 Chloroplast rRNA genes in the rice nuclear genome
3.1.4 Nature of the chloroplast insertions
3.1.5 Mitochondrial homologies in therice nuclear genome
3.1.6 Representation of the mitochondrial genome in the rice nuclear genome
3.1.7 Mitochondrial ATPase-β genes in the nucleus
3.1.8 Co-alignments between the chloroplast and mitochondria
3.1.9 Sice distribution of insertions
3.2 Viral Comparison
3.3 Bacterial Comparison
3.3.1 Bacillus sequence similarities in the rice nuclear genome
3.3.2 Xanthomonas sequence similarities in the rice nuclear genome
3.3.3 Pseudomonas sequence similarities in the rice nuclear genome
3.4 Fungal comparison
3.4.1 Magnaporthe sequence similarities in the rice nuclear genome
3.4.2 Dendogram type 1
3.4.3 Dendogram type 2
3.4.4 Dendogram type 3
4 Discussion 
4.1 Motivation for this study
4.3 Insertion dynamics
4.4 Models for DNA transfer
4.5 Benefits of insertion
4.6 Contribution of this study to science
4.7 Future perspectives
5 References

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Using whole genome comparison to detect sequence similarities between plants and microbes

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