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.

Inhalt: (Insemination mit Gefriersperma beim Hund mit Hilfe einer neuen Besamungstechnik). In diesem Versuch sind 11 Hündinnen mit tiefgefrorenem Sperma besamt worden. Die spermienreiche Ejakulatsfraktion wurde unmittelbar nach der Samenentnahme mit einer Tris‐Fructose‐Zitronensüure‐Lösung, die 8 Vol. % Glyzerin und 20 Vol. % Eidotter enthielt, etwa 1: 4 verdünnt. Wührend einer 3stündigen Equilibrierungszeit wurde der Samen auf + 5 °C heruntergekühlt, and in PVC‐Röhrchen im Stickstoffdampf eingefroren. Die Inseminationsdosen, die ungefähr 150 × 106 Spermien enthielten und 3 Wochen bis 1 1/2 Jahre gelagert waren, wurden unmittelbar nach dem Auftauen für 6,5 Sek. im Wasserbad von + 75 °C, durch den Cervicalkanal intrauterin deponiert. Es wurde meistens zweimal mit etwa 48stündigen Intervallen inseminiert. Von den 11 Hündinnen konzipierten 10 und brachten von ein bis sieben Junge. Die höchste Zahl der lebendigen Welpen in einem Wurf war sechs. Contents: Insemination with frozen dog semen was performed in a small trial including 11 bitches. The semen was diluted about 1: 4 with Tris‐fructose‐citric acid extender containing 8 % (v/v) glycerol and 20 % (v/v) egg yolk, equilibrated for 3 hrs and frozen in P.V.C.‐straws by use of N2‐vapour. The insemination doses being stored in liquid N2 from 3 weeks to 1 1/2 year and containing about 150 × 106 spermätozoa, was deposited in the uterus via the cervical canal immediately after thawing at 75 °C for 6,5 sec. By this procedure conception was obtained in 10 of the 11 bitches. The litter size ranged from one to seven puppies, the highest number of living puppies being six.

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We have previously shown that functional ribulose bisphosphate carboxylase (RuBPCase, rbc) genes in Alcaligenes eutrophus ATCC 17707 are present both on the chromosome and on the indigenous plasmid pAE7. Here we demonstrate that the chromosomal rbc locus encodes both a large (rbcL)-and a small (rbcS)-subunit gene. A 2.3-kilobase DNA fragment containing both subunit genes was subcloned into the broad-host-range vector pRK310 to yield plasmid pAE312. This plasmid was transferred into Pseudomonas aeruginosa in which expression of both the rbcL and rbcS genes took place, as demonstrated by Western blot analysis. A high level of RuBPCase activity was observed for P. aeruginosa(pAE312), suggesting that assembly of the subunits took place. Plasmid pAE312 was mutagenized with TnS in Escherichia coli. Complementation of A. eutrophus RuBPCase structural gene mutants with pAE312 containing mapped TnS insertions allowed functional analysis of the rbc gene region. The polar effect of the TnS insertions suggested that the two subunit genes were cotranscribed in A. eutrophus, with rbcL located promoter proximal. Northern blot analysis of total RNA from P. aeruginosa(pAE312) confirmed cotranscription of the two subunit genes. DNA probes containing both the rbcL and rbcS genes, or fragments of each gene, all hybridized to a predominant transcript about 2.1-kilobases long. These observations indicate that the chromosomally encoded rbcL and rbcS genes of A. eutrophus constitute an operon.Most autotrophic organisms, from bacteria to plants, depend on the reductive pentose phosphate cycle for assimilation of CO2, with the initial step catalyzed by ribulose bisphosphate carboxylase/oxygenase (RuBPCase, rbc; EC 4.1.1.39) (39). The activity of RuBPCase is often rate limiting to primary productivity in C3 plants, in which its secondary oxygenase activity initiates photorespiratory carbon oxidation (41, 45). RuBPCase from eucaryotes and most procaryotes is a hexadecamer composed of eight large catalytic (L; Mr = 54,000) and eight small (S; Mr -14,000) subunits. Exceptions to this structure are found in some photosynthetic bacteria, with a dimeric enyzme (L2) in Rhodospirillum rubrum (55) and hexameric (L6) as well as hexadecameric enzymes in Rhodopseudomonas sphaeroides (25) and Rhodopseudomonas capsulata (26).In higher plants the large subunit is chloroplast encoded, while the small nucleus-encoded subunit is translated as a precursor which is transported into the chloroplast. This is in contrast to the cyanobacteria Anabaena strain 7120 (44) and Anacystis nidulans (50) and to cyanelles from the protozoan Cyanophora paradoxa (54), in which the two subunit genes are cotranscribed. RuBPCase large-and small-subunit genes from both eucaryotes and procaryotes, including Rhodospirillum rubrum (43)