The nucleotide sequence of the chromosomally encoded ribulose bisphosphate carboxylase/oxygenase (RuBPCase) large (rbcL) and small (rbcS) subunit genes of the hydrogen bacterium Alcaligenes eutrophus ATCC 17707 was determined. We found that the two coding regions are separated by a 47-base-pair intergenic region, and both genes are preceded by plausible ribosome-binding sites. Cotranscription of the rbcL and rbcS genes has been demonstrated previously. The rbcL and rbcS genes encode polypeptides of 487 and 135 amino acids, respectively. Both genes exhibited similar codon usage which was highly biased and different from that of other organisms. The N-terminal amino acid sequence of both subunit proteins was determined by Edman degradation. No processing of the rbcS protein was detected, while the rbcL protein underwent a posttranslational loss of formylmethionyl. The A. eutrophus rbcL and rbcS proteins exhibited 56.8 to 58.3% and 35.6 to 38.5% amino acid sequence homology, respectively, with the corresponding proteins from cyanobacteria, eucaryotic algae, and plants. The A. eutrophus and Rhodospirillum rubrum rbcL proteins were only about 32% homologous. The N-and C-terminal sequences of both the rbcL and the rbcS proteins were among the most divergent regions. Known or proposed active site residues in other rbcL proteins, including Lys, His, Arg, and Asp residues, were conserved in the A. eutrophus enzyme. The A. eutrophus rbcS protein, like those of cyanobacteria, lacks a 12-residue internal sequence that is found in plant RuBPCase. Comparison of hydropathy profiles and secondary structure predictions by the method described by Chou and Fasman (P. Y. Chou and G. D. Fasman, Adv. Enzymol. 47:45-148, 1978) revealed striking similarities between A. eutrophus RuBPCase and other hexadecameric enzymes. This suggests that folding of the polypeptide chains is similar. The observed sequence homologies were consistent with the notion that both the rbcL and rbcS genes of the chemoautotroph A. eutrophus and the thus far characterized rbc genes of photosynthetic organisms have a common origin. This suggests that both subunit genes have a very ancient origin. The role of quaternary structure as a determinant of the rate of accepted amino acid substitution was examined. It is proposed that the sequence of the dimeric R. rubrum RuBPCase may be less conserved because there are fewer structural constraints for this RuBPCase than there are for hexadecameric enzymes.

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...

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29 separated by an intervening stretch of DNA, variable in length depending on the particular species of origin (Curtis & Hazelkorn, 1983; Shinozaki & Sugiura, 1983). The gene from S,.rzc~c.lioc.oc~r~us PCC30 1 (Reichelt & Delaney, 1983) has been cloned into the ampr region of PLa2311 (Remaut ct al., 1981) with expression controlled by the strong temperature-sensitive leftward promoter (PI) of bacteriophage EL (Gatenby et al., 1985). The construct is expressed in E. coli host organisms and produces active Rubisco. At present the levels of enzyme are poor, with low specific activity, possibly due to an under-production of the S-subunit. However, the synthesis of an active, multisubunit enzyme in non-photosynthetic bacteria argues against the necessity of a complicated assembly mechanism for Rubisco from cyanobacteria. Better constructs are certain to be developed that can be manipulated by 'in vitro' techniques and produce large amounts of enzymes. These will then be exploited to determine the structural basis for the differences between Rubisco from diverse photosynthetic organisms and indicate ways of changing the partition coefficient of the bisphosphate substrate.