We have determined the complete primary structure (8031 base pairs) of an infectious clone of cauliflower mosaic virus strain CM1841. The sequence was obtained using the strategy of cloning shotgun restriction fragments in the sequencing vector M13mp7. Comparison of the CM1841 sequence with that published for another caMV strain (Strasbourg) reveals 4.4% changes, mostly nucleotide substitutions with a few small insertions and deletions. The six open reading frames in the sequence of the Strasbourg isolate are also present in CM1841.

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We have sequenced the genome of bean golden mosaic virus, which comprises two circular single-stranded DNA molecules (2646 and Geminiviruses are small viruses of plants characterized by morphologically unique geminate particles and single-stranded DNA (ss DNA) genomes (1, 2). Based on evidence from restriction and infectivity dilution analyses, we suggested (3) that the genome of one geminivirus, bean golden mosaic virus (BGMV), may be bipartite. This hypothesis has been amply validated by subsequent work with other whitefly-borne geminiviruses (4-8). Stanley (9) provided definitive evidence that cassava latent virus (CLV) has a bipartite genome by reproducing disease in Nicotiana benthamiana when leaves were inoculated with full-length clones of both DNAs and never when inoculated with only one DNA.The nucleotide sequence of CLV contains several interesting features (6). These include a common sequence of ==200 nucleotides found in both DNA components, the only extensive sequence homology between the two DNAs. Within the common region are direct repeats and an inverted repeat that could form a stem-loop structure. The CLV sequence contains 12 open reading frames (ORFs) that potentially encode proteins >10 kDa; ORFs were found in both the viral (+) strand and the complementary (-) strand. One of these, the ORF that may encode a 30.1-kDa protein, was postulated to be the coat protein gene based on its length and by correlation with coat protein amino acid composition.We now present the nucleotide sequence of the DNAs from BGMV. A comparison of BGMV and CLV sequences reveals features of the genome organization of these viruses that may play a role in temporal regulation of their expression and their different host ranges. MATERIALS AND METHODSDNA Preparation. ss DNA was purified from virus preparations (10) by treatment with 1% NaDodSO4 followed by phenol extraction and ethanol precipitation. Double-stranded DNA (ds DNA) was then prepared by extension of random primers (11) on the ss DNA template (12).Viral ds DNA was also obtained from extracts of infected plants. BGMV-infected bean leaves, harvested 7 days after inoculation and stored at -80'C, were crushed to a powder in liquid nitrogen in a mortar. The powdered tissue was thawed in 4 ml of 20 mM Tris'HCl, pH 7.6/10 mM EDTA/50 mM NaCl/0.4 ml of proteinase K solution (10 mg/ml). After addition of 0.4 ml of 20% sodium lauroyl sarcosine, the slurry was homogenized again. We then added 0.4 ml of boiled RNase (5 mg/ml), centrifuged the slurry for 5 min in a Beckman Microfuge B, and incubated the supernatant for 30 min at 370C. The DNA-containing solution was extracted twice with phenol/chloroform (1:1), once with chloroform, washed twice with ether, and precipitated by addition of 0.1 vol of 3 M NaOAc (pH 5.5) and 3 vol of ethanol. The precipitate was washed several times with ethanol, taken up in buffer without proteinase K, and fractionated on a 5-20% sucrose gradient by centrifugation for 16 hr at 25,000 rpm in a Beckman SW 41 rotor (4).Molecular Clonin...

SUMMARYAmino acid sequences of 16 geminivirus replication-associated proteins and 15 coat proteins were aligned and a new computer program was used to calculate the minimum mutation distances for all possible pairwise comparisons. These data were used to construct phylogenetic trees. Trees based on coat proteins had two main branches which were positively correlated with vector specificities of the viruses. Trees based on replication-associated proteins also had two main branches which were positively correlated with viral host specificities for either monocotyledonous or dicotyledonous plants. Therefore, evolutionary pressures on coat proteins and replication-associated proteins are probably highly influenced by vectors and hosts, respectively. Geminiviruses that infect dicotyledonous plants may be divided further by geographical origins into Old World and New World viruses. These results suggest the possible geographical origins of some geminiviruses, that new taxa should be erected, and have implications for distinguishing viruses and strains.

Thirty-four Eco RI* sites have been identified on the nucleotide sequence of CaMV, following cloning of Eco RI* fragments in M13mp2. From this sequencing data, we have deduced that Eco RI* recognizes sites that differ in a single position from the canonical Eco RI sequence, GAATTC. Any substitution can occur at any one of the six positions in the recognition site, with the exception of A leads to T or T leads to A changes within the central tetramer. The Eco RI* restriction patterns of phi x174 and pBR322 are consistent with these recognition criteria. Similarly, Bam HI* cleavage of phi x174 and SV40 (George et al., 1980) produces restriction patterns that are consistent with single-position degeneracy in the canonical Bam HI recognition site. Cohesive termini produced by Eco RI* cleavage were ligated into the Eco RI site of M13mp2, even when there was a base pair mismatch within the four nucleotide overlap. Mismatches were corrected asymmetrically during subsequent replication of M13 in E. coli.