RESUMO
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.
Assuntos
Carica/virologia , Genoma Viral , Potyvirus/genética , Potyvirus/isolamento & purificação , Sequência de Bases , DNA Complementar , Dados de Sequência Molecular , RNA Viral/genética , TailândiaRESUMO
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.
Assuntos
Arabidopsis/metabolismo , DNA Bacteriano/metabolismo , Glucuronidase/biossíntese , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases/biossíntese , Sequência de Aminoácidos , Fusão Gênica Artificial , Sequência de Bases , Clonagem Molecular , Sequência Conservada , DNA de Plantas/química , DNA de Plantas/metabolismo , DNA de Cadeia Simples/metabolismo , Escherichia coli , Dados de Sequência Molecular , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Rhizobium , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , TATA Box , TransfecçãoRESUMO
Vectors were constructed for the isolation of random transcriptional and translational beta-glucuronidase gene fusions in plants. This system is based on the random integration of the transferred DNA (T-DNA) into the plant nuclear genome. The Escherichia coli beta-glucuronidase coding sequence without promoter, and also devoid of its ATG initiation site in the translational gene fusion vector, was inserted in the T-DNA with its 5' end at a distance of 4 base pairs from the right T-DNA border sequence. Transgenic plants can be selected by using a chimeric (P35S-nptII-3' ocs) kanamycin-resistance gene present in the same T-DNA. Subsequent screening of these for beta-glucuronidase expression allows the identification of clones harboring a fusion of the beta-glucuronidase coding sequence with plant 5' regulatory sequences. After transformation of Arabidopsis thaliana C24 root explants, beta-glucuronidase expression was detected in 54% and 1.6% of the plants transformed with the transcriptional and translational fusion vectors, respectively. Several different patterns of tissue-specific beta-glucuronidase expression were identified. The plant upstream sequence of a beta-glucuronidase fusion that is specifically expressed in the phloem of all organs was cloned and sequenced. After introduction in A. thaliana C24 and Nicotiana tabacum SR1, this sequence mediates the same highly phloem-specific beta-glucuronidase expression pattern as in the original transgenic plant from which it was isolated. These data demonstrate that this system facilitates the isolation and analysis of plant DNA sequences mediating regulated gene expression.
Assuntos
Glucuronidase/genética , Plantas/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Bacteriano/genética , Regulação Enzimológica da Expressão Gênica , Vetores Genéticos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Regiões Promotoras Genéticas , Biossíntese de Proteínas , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
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.