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Transformation of cantaloupes with the coat protein (cp) gene of papaya ringspot virus type W (PRSV-W), Thai isolate, was used to introduce virus resistance. Binary vectors containing either the full length coat protein coding region under control of the 35S CaMV promoter(pSA1175), or the inverted-repeat of a coat protein coding region (pSA1304), were constructed and used for Agrobacterium-mediated transformation of cotyledonary explants of the cantaloupe cultivar Sun Lady. Four independent transgenic lines were obtained using pSA1304 and one using pSA1175. Integration of the PRSV-W cp gene into the genome of these transgenic lines was verified by PCR amplification, GUS assays and Southern blot hybridization. In vitro inoculation of these lines with PRSV-W revealed that whereas the line containing pSA1175 remained sensitive, the four lines containing pSA1304 were resistant. The presence of small RNA species, presumably siRNA, corresponding to regions of the viral cp gene in transgenic lines resistant to PRSV-W supports the involvement of post-transcriptional gene silencing in the establishment of resistance.

Modification of the histone proteins that form the core around which chromosomal DNA is looped has profound epigenetic effects on the accessibility of the associated DNA for transcription, replication and repair. The SET domain is now recognized as generally having methyltransferase activity targeted to specific lysine residues of histone H3 or H4. There is considerable sequence conservation within the SET domain and within its flanking regions. Previous reviews have shown that SET proteins from Arabidopsis and maize fall into five classes according to their sequence and domain architectures. These classes generally reflect specificity for a particular substrate. SET proteins from rice were found to fall into similar groupings, strengthening the merit of the approach taken. Two additional classes, VI and VII, were established that include proteins with truncated/interrupted SET domains. Diverse mechanisms are involved in shaping the function and regulation of SET proteins. These include protein-protein interactions through both intra- and inter-molecular associations that are important in plant developmental processes, such as flowering time control and embryogenesis. Alternative splicing that can result in the generation of two to several different transcript isoforms is now known to be widespread. An exciting and tantalizing question is whether, or how, this alternative splicing affects gene function. For example, it is conceivable that one isoform may debilitate methyltransferase function whereas the other may enhance it, providing an opportunity for differential regulation. The review concludes with the speculation that modulation of SET protein function is mediated by antisense or sense-antisense RNA.

The coat protein (CP) of Papaya ringspot virus (PRSV) was analyzed for presentation of the antigenic peptide of animal virus, Canine parvovirus (CPV), in Escherichia coli (E. coli). The 45 nucleotides fragment coding for the 15-aa peptide epitope of the CPV-VP2 protein was either inserted into the PRSV-cp gene at the 5', 3' ends, both 5' and 3' ends or substituted into the 3' end of the PRSV cp gene. Each of the chimeric PRSV cp genes was cloned into the pRSET B vector under the control of the T7 promoter and transformed into E. coli. The recombinant coat proteins expressed from different chimeric PRSV-cp genes were purified and intraperitoneally injected into mice. All of the recombinant coat proteins showed strong immunogenicity and stimulate mice immune response. The recombinant coat proteins containing the CPV epitope insertion at the C terminus and at both N and C termini elicited ten times higher specific antisera in immunized mice compared with the other two recombinant coat proteins which contain the CPV epitope insertion at the N terminus and substitution at the C terminus.

Miniature inverted repeat transposable elements (MITEs) are thought to be a driving force for genome evolution. Although numerous MITEs are found associated with genes, little is known about their function in gene regulation. Whereas the rice ubiquitin2 (rubq2) promoter in rice (Oryza sativa) line IR24 contains two nested MITEs (Kiddo and MDM1), that in line T309 has lost Kiddo, providing an opportunity to understand the role of MITEs in promoter function. No difference in endogenous rubq2 transcript levels between T309 and IR24 was evident using RT-PCR. However, promoter analysis using both transient and stably transformed calli revealed that Kiddo contributed some 20% of the total expression. Bisulfite genomic sequencing of the rubq2 promoters revealed specific DNA methylation at both symmetric and asymmetric cytosine residues on the MITE sequences, possibly induced by low levels of homologous transcripts. When methylation of the MITEs was blocked by 5-azacytidine treatment, a threefold increase in the endogenous rubq2 transcript level was detected in IR24 compared with that in T309. Together with the observed MITE methylation pattern, the detection of low levels of transcripts, but not small RNAs, corresponding to Kiddo and MDM1 suggested that RNA-dependent DNA methylation is induced by MITE transcripts. We conclude that, although Kiddo enhances transcription from the rubq2 promoter, this effect is mitigated by sequence-specific epigenetic modification.

The complete nucleotide sequence of a Thai isolate of Papaya ringspot virus (PRSV) type W (PRSV-W) was determined. The viral genome is 10,323 nucleotides (nts) long and contains an ORF encoding polyprotein 3,343 amino acids (aa) long, flanked with 5'- and 3'-non-coding regions (NCRs) of 85 and 206 nts, respectively. Out of the ten putative proteins P1 is the most variable (73.9% similarity) as compared to the PRSV type P (PRSV-P) sequences, while the CI protein is most conserved (99.1% similarity). The sequence similarity among the type W and P isolates also suggests that the P type arose from the W type. However, no significant difference between types P and W that would account for the host specificity was disclosed.

Three random translational beta-glucuronidase (gus) gene fusions were previously obtained in Arabidopsis thaliana, using Agrobacterium-mediated transfer of a gus coding sequence without promoter and ATG initiation site. These were analysed by IPCR amplification of the sequence upstream of gus and nucleotide sequence analysis. In one instance, the gus sequence was fused, in inverse orientation, to the nos promoter sequence of a truncated tandem T-DNA copy and translated from a spurious ATG in this sequence. In the second transgenic line, the gus gene was fused to A. thaliana DNA, 27 bp downstream an ATG. In this line, a large deletion occurred at the target site of the T-DNA. In the third line, gus is fused in frame to a plant DNA sequence after the eighth codon of an open reading frame encoding a protein of 619 amino acids. This protein has significant homology with animal and plant (receptor) serine/threonine protein kinases. The twelve subdomains essential for kinase activity are conserved. The presence of a potential signal peptide and a membrane-spanning domain suggests that it may be a receptor kinase. These data confirm that plant genes can be tagged as functional translational gene fusions.

Five recombinant E. coli clones exhibiting toxicity to Aedes aegypti larvae were obtained from a library of 800 clones containing XbaI DNA fragments of 110 kb plasmid from B. thuringiensis var. israelensis. All the five clones (pMU 14/258/303/388/679) had the same 3.8-kb insert and encoded a major protein of 130 kDa which was highly toxic to A. aegypti larvae. Three clones (pMU 258/303/388) transcribed the 130 kD a gene in the same direction as that of lac Z promoter of pUC12 vector whereas the transcription of the other two (pMU 14/679) was in the opposite direction. A 1.9-kb fragment of the 3.8 kb insert coded for a protein of 65 kDa. Partial DNA sequence of the 3.8 kb insert, corresponding to the 5'-terminal of the 130 kDa gene, revealed a continuous reading frame, a Shine-Dalgarno sequence and a tentative 5'-regulatory region. These results demonstrated that the 3.8 kb insert is a minimal DNA fragment containing a regulatory region plus the coding sequence of the 130 kDa protein that is highly toxic to mosquito larvae.