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Structure
and
Properties
of
CaMV -
The GaMV particles are spherical, isometric, or icosahedral, about 50 mm in diameter, contain circular double stranded DNA of about 8 kb, and can be isolated from an inclusion body using urea and nonionic detergents. There are probably four struc1ural polypeptides but only two account for over 90% of the viral protein.
The major components have mol. wts of 37,000 and 64,000 and are present in a molar ratio of 5 to 1. From the proportion of these polypeptide species and the mol. wt of the virus particle, the most likely structure is an outer shell of 420 molecules of the 37,000 species surrounding a core of 60 molecules of the 64,000 species.
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The two remaining polypeptides have much higher mol. wts (96,000 and 88,000) and may be glycoproteins. Their function is not known.
The DNA molecule is about 8 kbp long and several varieties (totaling 50,000 bases) have been sequenced.
The DNA exists in linear, open, circular and twisted or knotted forms; however, none of the circular forms is covalently closed due to the presence of site specific single strand breaks.
These nuclease sensitive single strand breaks are not true gaps but short oligonucleotide overlapping regions, having sequence complementarity, and thus forming short triple stranded structures with a fixed 5'-end. There are three such sites, one (1) in the minus (coding or transcribed) strand yielding the large fragment and overlapped by eight residues.
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The other two (2 and 3) are in the plus (noncoding or nontranscribed) strand yielding the and t fragments having 18 and 15 residue overlaps respectively. One of the plus strand discontinuities is dispensable and. none is required for infection, as virus DNA previously cloned in bacteria and. lacking the "gaps" is as infectious as native DNA.
The whole base sequence has been determined and from the sequence one can identify eight open reading frames (ORFs). So far genetic evidence suggests that ORF VI codes for the matrix protein of the inclusion body. ORF IV codes for a coat protein. ORFs II and VII seem not to be necessary for a successful infection.
Sequence analyses of the DNA at these discontinuities in GaMV have shown that they in fact consist of short overlaps of 6-18 nucleotides so that there is a terminal redundancy in one strand of the DNA at the site of the discontinuity.
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Presumably as a result of these overlaps, the DNA can be isolated in a variety of twisted circular forms resembling supercoils and also as a linear form due to breakage. The function of the discontinuities in the DNA is not clear, although they are not required for infectivity.
Sequence data obtained from GaMV has revealed six major and two minor tightly packed, potential coding regions distributed between three reading frames.
Transcription of CaMV is found to be asymmetric, with only the strand producing stable transcripts. The mechanism of replication seems to be as follows: The infecting GaMV DNA enters the plant nucleus, where the single stranded overlaps are digested and the gaps ligated to give a supercoiled minichromosome.
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The function of this minichr0mosome is to act as a template for plant nuclear RNA polymerase II. The transcript thus formed is transported to the cytoplasm where it is either translated or replicated by reverse transcription. A site 600 b downstream of the promoter of the large transcript binds the proposed primer of reverse transcription, methionine tRNA.
The RNA transcript is then copied into the minus strand DNA. Synthesis of the plus strand DNA starts at two primer binding sites near "gaps" 2 and 3. From gap 2 synthesis proceeds to the 5'-end of the minus strand DNA, whereas synthesis from gap 3 continues to gap 2. This DNA molecule gets packed into virus particles or reel1ters the nucleus and undergoes another round of transcription and/or translation/replication.
Two major RNA transcripts are found inside infected cells. One covers the sequence of ORF VI. Another and larger one contains the base sequence of the whole genome. (The RNA polymerase goes all the way around the template strand and then continues for a short distance, repeating the first transcribed segment.) It is likely that the larger RNA acts as a polygenic messenger RNA.
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This suggests that any gene inserted into the genome will be transcribed. Genes inserted in the correct direction may well be translated; however, it is possible that the genes contain some special information for initiating translation besides the base triplet AUG. This special information may be something like the Shine Delgarno sequence, even though this sequence is not found in most eukaryotic mRNAs.
Two prominent polyadenylated transcripts are found in infected cells. The larger one (35S), appears to be a greater than full length transcript that originates at nucleotide position 7435 and terminates near position 7615 after 1.02 circuits of the molecules. A second transcript (19S) begins at nucleotide position 5765 and terminates at the same site as the full length transcript.
The nucleotide sequence shows typical eukaryotic promoters (including TATA boxes) just upstream from the initiation of these two transcripts, and the usual polyadenylation signal (AATAAA) near their common terminus. Several smaller, minor transcripts are found in infected cells but their origin is not clear
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