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1.
Oncogene ; 36(13): 1770-1778, 2017 03 30.
Article in English | MEDLINE | ID: mdl-27669441

ABSTRACT

Cancer-associated fibroblasts (CAFs) comprise the majority of the tumor bulk of pancreatic ductal adenocarcinomas (PDACs). Current efforts to eradicate these tumors focus predominantly on targeting the proliferation of rapidly growing cancer epithelial cells. We know that this is largely ineffective with resistance arising in most tumors following exposure to chemotherapy. Despite the long-standing recognition of the prominence of CAFs in PDAC, the effect of chemotherapy on CAFs and how they may contribute to drug resistance in neighboring cancer cells is not well characterized. Here, we show that CAFs exposed to chemotherapy have an active role in regulating the survival and proliferation of cancer cells. We found that CAFs are intrinsically resistant to gemcitabine, the chemotherapeutic standard of care for PDAC. Further, CAFs exposed to gemcitabine significantly increase the release of extracellular vesicles called exosomes. These exosomes increased chemoresistance-inducing factor, Snail, in recipient epithelial cells and promote proliferation and drug resistance. Finally, treatment of gemcitabine-exposed CAFs with an inhibitor of exosome release, GW4869, significantly reduces survival in co-cultured epithelial cells, signifying an important role of CAF exosomes in chemotherapeutic drug resistance. Collectively, these findings show the potential for exosome inhibitors as treatment options alongside chemotherapy for overcoming PDAC chemoresistance.


Subject(s)
Cancer-Associated Fibroblasts/metabolism , Exosomes/metabolism , Pancreatic Neoplasms/metabolism , Actins/metabolism , Antimetabolites, Antineoplastic/pharmacology , Biomarkers , Cancer-Associated Fibroblasts/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Culture Media, Conditioned/pharmacology , Deoxycytidine/analogs & derivatives , Deoxycytidine/pharmacology , Drug Resistance, Neoplasm/drug effects , Humans , MicroRNAs/genetics , Models, Biological , Pancreatic Neoplasms/pathology , Signal Transduction/drug effects , Snail Family Transcription Factors/genetics , Vimentin/metabolism , Gemcitabine
2.
J Virol ; 77(5): 3247-56, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12584348

ABSTRACT

Point mutations were introduced into the major capsid protein (P3) of cloned infectious cDNA of the polerovirus beet western yellows virus (BWYV) by manipulation of cloned infectious cDNA. Seven mutations targeted sites on the S domain predicted to lie on the capsid surface. An eighth mutation eliminated two arginine residues in the R domain, which is thought to extend into the capsid interior. The effects of the mutations on virus capsid formation, virus accumulation in protoplasts and plants, and aphid transmission were tested. All of the mutants replicated in protoplasts. The S-domain mutant W166R failed to protect viral RNA from RNase attack, suggesting that this particular mutation interfered with stable capsid formation. The R-domain mutant R7A/R8A protected approximately 90% of the viral RNA strand from RNase, suggesting that lower positive-charge density in the mutant capsid interior interfered with stable packaging of the complete strand into virions. Neither of these mutants systemically infected plants. The six remaining mutants properly packaged viral RNA and could invade Nicotiana clevelandii systemically following agroinfection. Mutant Q121E/N122D was poorly transmitted by aphids, implicating one or both targeted residues in virus-vector interactions. Successful transmission of mutant D172N was accompanied either by reversion to the wild type or by appearance of a second-site mutation, N137D. This finding indicates that D172 is also important for transmission but that the D172N transmission defect can be compensated for by a "reverse" substitution at another site. The results have been used to evaluate possible structural models for the BWYV capsid.


Subject(s)
Aphids/virology , Capsid Proteins/genetics , Capsid/metabolism , Luteovirus/metabolism , Nicotiana/virology , Point Mutation , Amino Acid Sequence , Animals , Beta vulgaris/virology , Capsid Proteins/metabolism , Luteovirus/genetics , Luteovirus/physiology , Molecular Sequence Data , Plant Diseases/virology , RNA, Viral/chemistry , RNA, Viral/genetics , RNA, Viral/metabolism , Virus Assembly
3.
J Virol ; 76(13): 6815-24, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12050394

ABSTRACT

Higher plants employ a homology-dependent RNA-degradation system known as posttranscriptional gene silencing (PTGS) as a defense against virus infection. Several plant viruses are known to encode proteins that can suppress PTGS. Here we show that P0 of beet western yellows virus (BWYV) displays strong silencing suppressor activity in a transient expression assay based upon its ability to inhibit PTGS of green fluorescent protein (GFP) when expressed in agro-infiltrated leaves of Nicotiana benthamiana containing a GFP transgene. PTGS suppressor activity was also observed for the P0s of two other poleroviruses, cucurbit aphid-borne yellows virus and potato leafroll virus. P0 is encoded by the 5'-proximal gene in BWYV RNA but does not accumulate to detectable levels when expressed from the genome-length RNA during infection. The low accumulation of P0 and the resulting low PTGS suppressor activity are in part a consequence of the suboptimal translation initiation context of the P0 start codon in viral RNA, although other factors, probably related to the viral replication process, also play a role. A mutation to optimize the P0 translation initiation efficiency in BWYV RNA was not stable during virus multiplication in planta. Instead, the P0 initiation codon in the progeny was frequently replaced by a less efficient initiation codon such as ACG, GTG, or ATA, indicating that there is selection against overexpression of P0 from the viral genome.


Subject(s)
Gene Silencing , Luteovirus/physiology , RNA Processing, Post-Transcriptional/drug effects , Suppression, Genetic , Viral Proteins/pharmacology , Amino Acid Sequence , Green Fluorescent Proteins , Luminescent Proteins/genetics , Luteovirus/genetics , Luteovirus/metabolism , Molecular Sequence Data , Plant Leaves/genetics , Plant Leaves/virology , Plant Proteins/metabolism , Nicotiana/genetics , Nicotiana/virology , Transgenes , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/metabolism
4.
Plant J ; 29(5): 555-67, 2002 Mar.
Article in English | MEDLINE | ID: mdl-11874569

ABSTRACT

In plants, post-transcriptional gene silencing (PTGS) is part of a defence mechanism against virus infection. Several plant viruses have been shown to encode proteins which can counteract PTGS. In this paper it is demonstrated that P15 of peanut clump pecluvirus (PCV) has anti-PTGS activity. P15 is a small cysteine-rich protein with no sequence similarity to previously described PTGS-suppressor proteins which has several novel properties. It possesses four C-terminal proximal heptad repeats that can potentially mediate a coiled-coil interaction and is targeted to peroxisomes via a C-terminal SKL motif. The coiled-coil sequence is necessary for the anti-PTGS activity of P15, but the peroxisomal localization signal is not, although it is required for efficient intercellular movement of the virus.


Subject(s)
Gene Silencing , Nicotiana/genetics , Plant Viruses/genetics , Viral Proteins/physiology , Amino Acid Sequence , Arachis/virology , Cells, Cultured , Cloning, Molecular , Cysteine/genetics , Glyoxysomes/metabolism , Green Fluorescent Proteins , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Peroxisomes/metabolism , Plant Diseases/virology , Plant Viruses/growth & development , Plants, Genetically Modified , Sequence Homology, Amino Acid , Nicotiana/cytology , Nicotiana/virology , Viral Proteins/genetics
5.
Transgenic Res ; 10(4): 293-302, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11592709

ABSTRACT

Point mutations were introduced into the genes encoding the triple gene bock movement proteins P13 and P15 of beet necrotic yellow vein virus (BNYVV). Mutations which disabled viral cell-to-cell movement in Chenopodium quinoa were then tested for their ability to act as dominant negative inhibiters of movement of wild-type BNYVV when expressed from a co-inoculated BNYVV RNA 3-based replicon. For P13, three types of mutation inhibited the movement function: non-synomynous mutations in the N- and C-terminal hydrophobic domains, a mutation at the boundary between the N-terminal hydrophobic domain and the central hydrophilic domain (mutant P13-A12), and mutations in the conserved sequence motif in the central hydrophilic domain. However, only the 'boundary' mutant P13-A12 strongly inhibited movement of wild-type virus when expressed from the co-inoculated replicon. Similar experiments with P15 detected four movement-defective mutants which strongly inhibited cell-to-cell movement of wild-type BNYVV when the mutants were expressed from a co-inoculated replicon. Beta vulgaris transformed with two of these P15 mutants were highly resistant to fungus-mediated infection with BNYVV.


Subject(s)
Genes, Viral/genetics , Plant Leaves/genetics , Plant Viruses/genetics , Point Mutation , RNA Viruses/physiology , RNA, Viral/biosynthesis , Viral Proteins/genetics , Fungi/physiology , Genes, Dominant , Movement , Plants, Genetically Modified , RNA, Viral/genetics , Replicon/genetics , Transcription, Genetic
6.
Virology ; 264(1): 220-9, 1999 Nov 10.
Article in English | MEDLINE | ID: mdl-10544148

ABSTRACT

The subcellular localization of the first triple gene block protein (TGBp1) of peanut clump pecluvirus (PCV) was studied by subcellular fractionation and immunogold cytochemistry using TGBp1-specific antibodies raised against a fusion protein expressed in and purified from bacteria. In the inoculated and apical leaves of virus-infected Nicotiana benthamiana, TGBp1 localized to the cell wall and P30 fractions. Electron microscopy of immunogold-decorated ultrathin sections of the infected leaf tissue revealed TGBp1-specific labeling of the plasmodesmata joining mesophyll cells. In longitudinal sections of the plasmodesmata, the TGBp1-specific labeling was most commonly associated with the plasmodesmal collar region. In transgenic N. benthamiana, which constitutively expressed TGBp1, no TGBp1-specific immunogold labeling of plasmodesmata was observed, but plasmodesmata were gold decorated when the transgenic plants were infected with a TGBp1-defective PCV mutant, indicating that factors induced by the virus infection target and/or anchor the transgene TGBp1 to the plasmodesmata.


Subject(s)
Arachis/virology , Plant Viruses/physiology , Base Sequence , DNA Primers , Microscopy, Immunoelectron , Molecular Sequence Data , Open Reading Frames , Plant Leaves/virology , Plant Viruses/genetics , Plant Viruses/pathogenicity , Plants, Genetically Modified , Plants, Toxic , Polymerase Chain Reaction , Recombinant Proteins/biosynthesis , Subcellular Fractions/virology , Nicotiana/virology
7.
J Gen Virol ; 80 ( Pt 10): 2771-2778, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10573174

ABSTRACT

Luteoviruses such as beet western yellows polerovirus (BWYV) are confined to and multiply within the phloem compartment of their hosts. The readthrough domain (RTD) of the minor BWYV capsid protein P74 is required for efficient virus accumulation in Nicotiana clevelandii. Experiments were carried out to determine if the low virus titres observed following agro-inoculation of whole plants with certain RTD mutants are due to a defect in virus multiplication in the nucleate cells of the phloem compartment or to inefficient virus movement to new infection sites. Immuno-localization of wild-type and an RTD-null mutant virus in thin sections of petioles and in phloem cells of leaf lamina, as well as electron microscopy observations, were all consistent with the conclusion that the RTD is not essential for efficient virus multiplication in the nucleate phloem cells but intervenes in virus movement to increase the rate at which new infection foci are established and expand.


Subject(s)
Capsid/physiology , Luteovirus/physiology , Mutagenesis , Plant Leaves/virology , Plants, Toxic , Nicotiana/virology
8.
Mol Plant Microbe Interact ; 11(7): 618-25, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9650295

ABSTRACT

Cell-to-cell movement of beet necrotic yellow vein virus (BNYVV) requires three proteins encoded by a triple gene block (TGB) on viral RNA 2. A BNYVV RNA 3-derived replicon was used to express movement proteins to functionally substitute for the BNYVV TGB proteins was tested by coinoculation of TGB-defective BNYVV with the various replicons to Chenopodium quinoa. Trans-heterocomplementation was successful with the movement protein (P30) of tobacco mosaic virus but not with the tubule-forming movement proteins of alfalfa mosaic virus and grapevine fanleaf virus. Trans-complementation of BNYVV movement was also observed when all three TGB proteins of the distantly related peanut clump virus were supplied together but not when they were substituted for their BNYVV counterparts one by one. When P30 was used to drive BNYVV movement in trans, accumulation of the first TGB protein of BNYVV was adversely affected by null mutations in the second and third TGB proteins. Taken together, these results suggest that highly specific interactions among cognate TGB proteins are important for their function and/or stability in planta.


Subject(s)
Genes, Viral , Plant Viruses/physiology , RNA Viruses/physiology , RNA, Viral/biosynthesis , Movement , Plant Leaves , Plant Viruses/genetics , Plants, Edible/virology , Protoplasts/virology , RNA Viruses/genetics , RNA, Viral/genetics , Replicon , Transcription, Genetic
9.
J Gen Virol ; 79 ( Pt 2): 385-93, 1998 Feb.
Article in English | MEDLINE | ID: mdl-9472625

ABSTRACT

RNAs 1 and 2 of beet necrotic yellow vein virus (BNYVV) carry the functions enabling viral RNA replication, cell-to-cell movement, virus assembly and vascular movement of the virus in the systemic host Spinacea oleracea. In Beta macrocarpa, on the other hand, BNYVV RNA 3 is required for vascular movement. Replication-competent RNA 3 transcripts carrying various point mutations and deletions were coinnoculated with RNAs 1 and 2 to young leaves of B. macrocarpa and the ability of the virus to multiply on the inoculated leaves and to invade the plant systemically was examined. None of the RNA 3 mutants tested interfered with virus multiplication in the inoculated leaves. Point mutations designed to specifically block or truncate translation of the ORFs of the two known RNA 3 gene products, P25 and N, did not interfere with vascular movement. Vascular movement was not inhibited by deletions eliminating the short 5'-proximal ORF on RNA 3 (ORF A) or by point mutations blocking putative translation of the short 5'-proximal ORF (ORF S) on RNA 3sub, a subgenomic RNA derived from RNA 3. On the other hand, deletions in a 'core region' encompassing nucleotides 1033-1257 of RNA 3 completely blocked vascular movement of the virus while removal of sequences flanking the core region lowered its efficiency. The observations suggest that some feature of the RNA 3 sequence rather than an RNA-3 coded protein is important for vascular movement of BNYVV in B. macrocarpa.


Subject(s)
Chenopodiaceae/virology , Plant Viruses/physiology , RNA Viruses/physiology , RNA, Viral/physiology , Virus Replication , Chenopodiaceae/physiology , Mutagenesis, Site-Directed , Open Reading Frames , Plant Leaves , Plant Viruses/genetics , Polymerase Chain Reaction , RNA, Viral/biosynthesis , Sequence Deletion , Transcription, Genetic , Viral Proteins/biosynthesis
10.
Arch Virol ; 142(5): 1051-8, 1997.
Article in English | MEDLINE | ID: mdl-9191870

ABSTRACT

Beet necrotic yellow vein virus RNA 1 contains a single long ORF corresponding to the theoretical translation product of 237 kDa which contains the information necessary for replication of the viral genome. This ORF contains a putative papain-like proteinase domain which has been localized, on the basis of sequence alignments, between the helicase and polymerase domains. Here we show that the RNA 1 primary translation product can be cleaved autocatalytically in vitro into two species of 150 kDa and 66 kDa, the latter of which probably contains the entire polymerase domain. A 66 kDa protein was detected immunologically in infected C. quinoa protoplasts using an antiserum specific for the C-terminal region of the RNA 1 primary translation product, confirming that processing also occurs in vivo.


Subject(s)
Plant Viruses/genetics , RNA Viruses/genetics , RNA, Viral/genetics , Amino Acid Sequence , Molecular Sequence Data , Papain/pharmacology , Protein Biosynthesis
11.
J Gen Virol ; 77 ( Pt 5): 889-97, 1996 May.
Article in English | MEDLINE | ID: mdl-8609485

ABSTRACT

The triple gene block (TGB) of beet necrotic yellow vein virus RNA 2 is required for cell-to-cell movement of the virus RNA. The protein P42 encoded by the 5'-proximal gene of the TGB has consensus sequence motifs characteristic of an ATP/GTP-dependent helicase. P42 was over-expressed in Escherichia coli and shown to bind both single- and double-stranded RNA and DNA by Northwestern blotting. Site-directed mutagenesis located the nucleic acid-binding domain to the N-terminal 24 amino acids of the protein and a point mutation or deletions in the region of P42 containing the helicase consensus sequences did not affect nucleic acid-binding activity of the immobilized protein. Electrophoretic mobility-shift assays revealed that P42 also binds nucleic acids in solution and that deletion of the N-terminal region inhibits this binding. Mutations in both the N-terminal nucleic acid-binding domain and the helicase domain blocked infection of leaves, indicating that both regions of P42 are important for its activity in vivo.


Subject(s)
DNA/metabolism , Plant Viruses/chemistry , RNA Viruses/chemistry , RNA/metabolism , Viral Proteins/metabolism , Amino Acid Sequence , Binding Sites , Escherichia coli/genetics , Molecular Sequence Data , Molecular Weight , Plant Viruses/genetics , RNA Viruses/genetics , Recombinant Proteins/metabolism , Structure-Activity Relationship
12.
Arch Virol ; 140(6): 1109-18, 1995.
Article in English | MEDLINE | ID: mdl-7611881

ABSTRACT

The complete nucleotide sequence of the genomic RNA of beet mild yellowing virus, isolate 2ITB, is reported. The RNA consists of 5722 nucleotides and contains six long open reading frames which conform to the arrangement characteristic of Subgroup 2 luteoviruses. The three 3'-proximal open reading frames, which encode the viral coat protein, a putative movement protein and the Readthrough Domain, are highly homologous to the corresponding genes of beet western yellows luteovirus while the three 5'-proximal open reading frames are more closely related to the corresponding genes of cucurbit aphid borne yellows luteovirus. The sequence data thus indicate that beet mild yellowing virus should be considered a distinct virus rather than a strain of beet western yellows virus.


Subject(s)
Luteovirus/genetics , RNA, Viral/analysis , Vegetables/virology , Amino Acid Sequence , Base Sequence , DNA, Viral/analysis , Genome, Viral , Molecular Sequence Data , Nucleic Acid Conformation , RNA, Viral/chemistry
13.
Arch Virol ; 135(1-2): 143-51, 1994.
Article in English | MEDLINE | ID: mdl-8198439

ABSTRACT

Long internal deletions were introduced into cloned cDNA of beet necrotic yellow vein virus RNAs 1-4 and transcripts containing the deletions were tested for their ability to inhibit replication of viral RNA in Chenopodium quinoa protoplasts and plants. No inhibition was observed with the deletion mutants based on RNAs 1, 3 and 4 but the RNA 2 deletion mutants all provoked a dramatic inhibition of synthesis of viral RNAs 1 and 2.


Subject(s)
Defective Viruses/genetics , Plant Viruses/genetics , RNA Viruses/genetics , Blotting, Northern , Cloning, Molecular , DNA, Complementary/metabolism , Defective Viruses/chemistry , Genome, Viral , Plant Viruses/chemistry , Plants/metabolism , Plants/microbiology , RNA Viruses/chemistry , RNA, Viral/analysis , RNA, Viral/biosynthesis , RNA, Viral/genetics , Sequence Deletion , Transcription, Genetic
14.
Arch Virol ; 134(1-2): 195-203, 1994.
Article in English | MEDLINE | ID: mdl-8279955

ABSTRACT

RNA 2 of beet necrotic yellow vein virus carries the cistron for the 21 kd coat protein at its 5'-extremity. During translation, the coat protein cistron termination codon is suppressed about 10% of the time so that translation continues into the adjacent open reading frame to produce a 75 kd species, known as P75, which contains the coat protein sequence at its N-terminus. Immunoblotting experiments with a P75-specific antiserum showed that P75 is present in only trace amounts in purified virus preparations. Electron microscopic visualization of immunogold-labelled virions in crude tissue extracts has provided evidence for an association between P75 and at least a fraction of the BNYVV particles, with P75 being predominantly located near one end of the rod-shaped virions. This finding is discussed in the context of the current model for the role of P75 in virus assembly and vector transmission.


Subject(s)
Capsid/analysis , Plant Viruses/chemistry , RNA Viruses/chemistry , Immunohistochemistry , Microscopy, Immunoelectron , Myxomycetes , Plant Viruses/physiology , Plant Viruses/ultrastructure , RNA Viruses/physiology , RNA Viruses/ultrastructure , Virion/chemistry , Virion/ultrastructure , Virus Replication
15.
Proc Natl Acad Sci U S A ; 89(13): 5715-9, 1992 Jul 01.
Article in English | MEDLINE | ID: mdl-1631051

ABSTRACT

RNA 2 of the multipartite genome of beet necrotic yellow vein virus carries the cistron for 21-kDa viral coat protein at its 5' extremity. The amber termination codon of the coat protein cistron undergoes suppression approximately 10% of the time so that translation continues into an adjacent 54-kDa open reading frame, yielding a 75-kDa readthrough protein. The roles of coat protein and the readthrough protein in infection were investigated with biologically active transcripts of RNA 2. Much of the coat protein cistron of the RNA 2 transcript could be deleted without interfering with viral replication and local lesion formation on leaves, although formation of the rod-shaped virions did not occur. Mutants in which the amber coat protein termination codon was replaced with an ochre codon or a tyrosine codon were also viable. The ochre codon was suppressed both in vitro and in planta. The mutant containing the tyrosine substitution produced only the 75-kDa read-through protein and was deficient in viral assembly. Deletions in the 54-kDa readthrough domain were also viable in planta but had different effects on virus assembly. A deletion in the C-terminal portion of the readthrough domain did not interfere with RNA packaging but, unexpectedly, deletions in the N-terminal portion were assembly deficient, although 21-kDa coat protein was produced in planta. Thus, the 75-kDa protein can apparently intervene in virion assembly even though it has not been detected in purified virions.


Subject(s)
Plant Diseases/microbiology , Plant Viruses/genetics , RNA, Viral/genetics , Virus Replication , Base Sequence , Capsid/genetics , DNA/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Oligodeoxyribonucleotides/chemistry , Peptide Chain Termination, Translational , Plant Viruses/pathogenicity , Structure-Activity Relationship
16.
EMBO J ; 11(2): 479-88, 1992 Feb.
Article in English | MEDLINE | ID: mdl-1537331

ABSTRACT

RNA 3 of the beet necrotic yellow vein virus (BNYVV) quadripartite RNA genome is not essential for virus multiplication on leaves of Tetragonia expansa but has dramatic effects on symptom expression. Virus isolates containing RNA 3 produce bright yellow local lesions while isolates lacking RNA 3 produce much milder symptoms. Using directed mutagenesis of cDNA clones followed by in vitro synthesis of biologically active transcripts, a 25 kDa open reading frame (ORF) of RNA 3 was shown to be responsible for the yellow local lesion phenotype. In addition, two deletion mutants of RNA 3 were found to elicit the appearance of severe necrotic local lesions. Analysis of one of these mutants revealed that necrosis was due to the overexpression of a second short ORF, N, overlapping the 3'-terminal portion of the 25 kDa ORF. As shown by gene fusion studies, gene N is not detectably expressed from full-length RNA 3 but is translationally activated by deletion of upstream sequences. Introduction of gene N into the genome of the unrelated DNA virus, cauliflower mosaic virus, elicits a necrotic response instead of the typical mosaic symptoms, demonstrating that gene N can induce necrosis outside of the context of a BNYVV infection.


Subject(s)
Genome, Viral , Plant Viruses/genetics , Plants/microbiology , RNA, Viral/genetics , Viral Proteins/genetics , Amino Acid Sequence , Base Sequence , Chromosome Deletion , Molecular Sequence Data , Mutagenesis, Site-Directed , Open Reading Frames , Phenotype , Plant Viruses/pathogenicity , Plasmids , Recombinant Fusion Proteins/metabolism , Transcription, Genetic , Viral Proteins/metabolism
18.
EMBO J ; 2(2): 277-82, 1983.
Article in English | MEDLINE | ID: mdl-11894938

ABSTRACT

The double-stranded circular DNA encapsidated within cauliflower mosaic virus (CaMV) particles contains three single-stranded discontinuities, two in one strand and one in the other, so that, upon denaturation, three linear single-stranded DNAs are produced. Here we show that a fourth much smaller single-stranded DNA, termed alpha1, is also present in denatured CaMV DNA preparations. The 5' extremity of alpha1 is identical to that of the alpha-strand, the strand of DNA possessing only one interruption, while its 3' extremity lies just two nucleotides downstream from a major transcription initiation site. We also show that the interrupted strand at each discontinuity sometimes has a single ribonucleotide in place of a deoxyribonucleotide at its 5' extremity. Oligoribonucleotide chains of eight and 10 residues in length have also been detected at the 5' end of one of the discontinuities. These structures are thought to be the vestiges of primers which have not been completely excised prior to encapsidation of the DNA. The possibility that synthesis of the alpha-strand occurs by reverse transcription of viral RNA using initiator tRNA(met) as primer is discussed.


Subject(s)
Caulimovirus/genetics , DNA, Viral/analysis , Chromosome Mapping , DNA, Single-Stranded , Electrophoresis, Polyacrylamide Gel/methods , Nucleic Acid Denaturation
19.
Cell ; 30(3): 763-73, 1982 Oct.
Article in English | MEDLINE | ID: mdl-7139714

ABSTRACT

Four RNA transcripts encoded by cauliflower mosaic virus DNA have been detected in the polyadenylated RNA from virus-infected turnip leaves. Two of these transcripts, the major 35S and the 8S species, have the same 5' termini, at nucleotide 7435. A viral DNA fragment encompassing this region directs transcription initiation at this point in vitro. The 5' terminus of the 19S transcript is at nucleotide 5764, and a corresponding viral DNA fragment also directs transcription initiation in vitro. The major 35S RNA is a complete transcript of the circular viral genome, and is 3'-coterminal with 19S RNA at nucleotide 7615. The 8S RNA has its 3' extremity at delta 1, the single-stranded interruption in the transcribed strand of virion DNA. A minor 35S RNA has also been detected that has its 5' and 3' termini at delta 1.


Subject(s)
Mosaic Viruses/genetics , RNA, Viral/genetics , Transcription, Genetic , Base Sequence , Chromosome Mapping , DNA, Viral/genetics , Molecular Weight , Operon , RNA, Messenger/genetics , RNA, Viral/biosynthesis
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