Distributions of two recently inserted long interspersed elements of the L1 repetitive family at the Alb and beta h3 loci in wild mice populations

The presence of the L1 sequences, L1Md4 next to the pseudogene beta h3 and I12 found in the twelfth intron of the albumin gene, in certain strains of laboratory mice but not of others has led to the suggestion that these sequences were recent insertions into the Mus mus domesticus genome. To be sure that they are really recent insertions and not relics of an ancestral chromosome, we investigated the presence or absence of these sequences in populations of wild mice belonging to the semispecies M. m. domesticus and M. m. musculus as well as in other species of the genus Mus and in related murids. The sequence I12 in the albumin gene was found in 34% of the chromosomes of the wild mice belonging to M. m. domesticus and to a lesser extent (6%) in M. m. musculus. Of 114 M. m. domesticus chromosomes, L1Md4 was found in only nine, seven of which came from the same locality. Its presence was associated with the haplotype Hbbp, which is relatively rare in European populations of M. musculus. Since there was no evidence for the presence of these two L1 sequences in more distantly related species, we conclude that they are recent insertions in the M. musculus genome.      

Analysis of the pigment content of an antenna pigment-protein complex from three strains of Rhodopseudomonas sphaeroides.

The pigment content of a B800-850 light-harvesting pigment-protein complex isolated from three different stains of Rhodopseudomonas sphaeroides has been determined. In each case the ratio of carotenoid to bacteriochlorophyll present is very nearly 1 : 3 an no specificity with regard to carotenoid type was observed. The fourth derivative of the infra-red absorption bands of the complex was determined and it is concluded that the minimal functional unit of B800-850 complex consists of 1 carotenoid molecule and three bacteriochlorophyll molecules. The data presented here, together with the previous study of Austin, (Austin, L.A. (1976) Ph.D. Thesis, University of California at Berkeley, Lawrence Berkeley Laboratory Report No. LBL 5512) suggest that the 800 nm absorption band represents one of these bacteriochlorophyll molecules while the remaining two bacteriochlorophylls are responsible for the 850 nm band. The absorption spectra and circular dichroism spectra of the complexes suggests that their structure has not been greatly altered during the purification.     

Protein and m-RNA expression of farnesyl-transferases,RhoA and RhoB in rat liver hepatocytes: action of perillyl alcohol and vitamin A <Emphasis Type="BoldItalic">in vivo</Emphasis>.

Summary We analysed the action, in rats in vivo, of the protein isoprenylation inhibitor perillyl alcohol (POH) and that of vitamin A, alone or in association, on m-RNA and protein expression of farnesyltransferases (FTases α and β subunits) and their protein substrates RhoA and RhoB, in isolated hepatocytes. Combined administration of POH and vitamin A induced a sharp decrease in FTase α protein after 96 h, suggesting an involvement not only of farnesyltransferases but also of geranylgeranyltransferases, which share the FTase α protein. FTase β protein did not decrease. POH plus vitamin A, in contrast with POH or vitamin A alone, induced a decrease in RhoB protein, probably because of different cleavages. No modification was observed in RhoA protein. Vitamin A alone increased RhoB m-RNA and protein expression. As one of the functions of RhoB is cell polarisation, these data support our previous hypothesis of a polarised transport of vitamin A from hepatocytes to hepatic stellate cells. As the behaviours of m-RNAs and proteins in this study were often different, cytoplasmic metabolic pathways must be considered for the parameters studied. The behaviour of Rho B, which is thought to have an antioncogene function, is discussed in view of its isoprenylated forms in the membranes. These preliminary findings stress the need, when studying the association of two isoprenoids in cancer therapy, to consider normal as well as tumour-bearing animals.     

The effect of atractylate and oligomycin on the behaviour of mitochondria towards adenine nucleotides

1. Investigation of a number of reactions involving both internal and externally added adenine nucleotides of isolated liver mitochondria has revealed that atractylate and oligomycin differ markedly in the site of their inhibitory action. 2. Both atractylate and oligomycin inhibited the respiratory-chain-level phosphorylation of added ADP. Neither compound inhibited the substrate-level phosphorylation of internal (endogenous) ADP or the respiration-dependent accumulation of bivalent metal ions (Ca²⁺, Sr²⁺ or Mn²⁺). 3. Atractylate, but not oligomycin, inhibited the substrate-level phosphorylation of externally added ADP, the ATP- and carnitine-dependent reduction of nicotinamide nucleotide by palmitate and the ATP-induced activation of succinate oxidation. 4. Oligomycin, but not atractylate, inhibited the respiratory-chain-linked phosphorylation of internal ADP, and the dephosphorylation of internal ATP that occurred on the addition of antimycin. 5. The enhancement of arsenate-stimulated respiration by ADP was prevented by atractylate added either before or after the ADP. Oligomycin abolished both the arsenate and ADP stimulation. 6. It is suggested that atractylate prevents the passage of adenine nucleotides across the mitochondrial membrane, whereas oligomycin interferes with the formation of a `high-energy' phosphorylated intermediate.     

Lotus corniculatus nodulation specificity is changed by the presence of a soybean lectin gene

Plant lectins have been implicated as playing an important role in mediating recognition and specificity in the Rhizobium-legume nitrogen-fixing symbiosis. To test this hypothesis, we introduced the soybean lectin gene Le1 either behind its own promoter or behind the cauliflower mosaic virus 35S promoter into Lotus corniculatus, which is nodulated by R. loti. We found that nodulelike outgrowths developed on transgenic L. corniculatus plant roots in response to Bradyrhizobium japonicum, which nodulates soybean and not Lotus spp. Soybean lectin was properly targeted to L. corniculatus root hairs, and although infection threads formed, they aborted in epidermal or hypodermal cells. Mutation of the lectin sugar binding site abolished infection thread formation and nodulation. Incubation of bradyrhizobia in the nodulation (nod) gene-inducing flavonoid genistein increased the number of nodulelike outgrowths on transgenic L. corniculatus roots. Studies of bacterial mutants, however, suggest that a component of the exopolysaccharide surface of B. japonicum, rather than Nod factor, is required for extension of host range to the transgenic L. corniculatus plants.     

Dissecting the pattern of proton release from partial process involved in ubihydroquinone oxidation in the Q-cycle

A key feature of the modified Q-cycle of the cytochrome bc₁ and related complexes is a bifurcation of QH₂ oxidation involving electron transfer to two different acceptor chains, each coupled to proton release. We have studied the kinetics of proton release in chromatophore vesicles from Rhodobacter sphaeroides, using the pH-sensitive dye neutral red to follow pH changes inside on activation of the photosynthetic chain, focusing on the bifurcated reaction, in which 4H⁺are released on complete turnover of the Q-cycle (2H⁺/ubiquinol (QH₂) oxidized). We identified different partial processes of the Q_o-site reaction, isolated through use of specific inhibitors, and correlated proton release with electron transfer processes by spectrophotometric measurement of cytochromes or electrochromic response. In the presence of myxothiazol or azoxystrobin, the proton release observed reflected oxidation of the Rieske iron?sulfur protein. In the absence of Q_o-site inhibitors, the pH change measured represented the convolution of this proton release with release of protons on turnover of the Q_o-site, involving formation of the ES-complex and oxidation of the semiquinone intermediate. Turnover also regenerated the reduced iron-sulfur protein, available for further oxidation on a second turnover. Proton release was well-matched with the rate limiting step on oxidation of QH₂ on both turnovers. However, a minor lag in proton release found at pH?7 but not at pH?8 might suggest that a process linked to rapid proton release on oxidation of the intermediate semiquinone involves a group with a pK in that range.     

Reaction center and UQH2:cytc 2 oxidoreductase act as independent enzymes inRps. sphaeroides

Turnover of the ubiquinol oxidizing site of the UQH₂:cyt c₂ oxidoreductase (b/c ₁ complex) ofRps. sphaeroides can be assayed by measuring the rate of reduction of cytb ₅₆₁ in the presence of antimycin (AA). Oxidation of ubiquinol is a second-order process, with a value ofk ₂ of about 3 × 10⁵ M^–1. The reaction shows saturation at high quinol concentrations, with an apparentK _m of about 6–8 mM (with respect to the concentration of quinol in the membrane). When the quinone pool is oxidized before illumination, reduction of the complex shows a substantial lag (about 1 ms) after a flash, indicating that the quinol produced as a result of the photochemical reactions is not immediately available to the complex. We have suggested that the lag may be due to several factors, including the leaving time of the quinol from the reaction center, the diffusion time to the complex, and the time for the head group to cross the membrane. We have suggested aminimal value for the diffusion coefficient of ubiquinone in the membrane (assuming that the lag is due entirely to diffusion) of about 10^–9 cm^–2 sec^–1. The lag is reduced to about 100 µsec when the pool is significantly reduced, showing that quinol from the pool is more rapidly available to the complex than that from the reaction center. With the pool oxidized, similar kinetics are seen when the reduction of cytb ₅₆₁ occurs through the AA-sensitive site (with reactions at the quinol oxidizing site blocked by myxothiazol). These results show that there is no preferential reaction pathway for transfer of reducing equivalents from reaction center tob/c ₁ complex. Oxidation of cytb ₅₆₁ through the AA-sensitive site can be assayed from the slow phase of the carotenoid electrochromic change, and by comparison with the kinetics of cytb ₅₆₁. As long as the quinone pool is significantly oxidized, the reaction is not rate-determining for the electrogenic process. On reduction of the pool below 1 quinone per complex, a slowing of the electrogenic process occurs, which could reflect a dependence on the concentration of quinone. If the process is second-order, the rate constant must be about 2–5 times greater than that for quinol oxidation, since the effect on rate is relatively small compared with the effect seen at the quinol oxidizing site when the quinol concentration is changed over theE _h range where the first few quinols are produced on reductive titration. When the quinone pool is extracted (experiments in collaboration with G. Venturoli and B. A. Melandri), the slowing of the electrochromic change on reduction of the pool is not enhanced; we assume that this is due to the fact that a minimum of one quinone per active complex is produced by turnover of the quinol oxidizing site. Two lines of research lead us to revise our previous estimate for the minimal value of the quinone diffusion coefficient. These relate to the relation between the diffusion coefficient and the rate constants for processes involving the quinones: (a) The estimated rate constant for reaction of quinone at the AA-site approaches the calculated diffusion limited rate constant, implying an improbably efficient reaction. (b) From a preliminary set of experiments, the activation energy determined by measuring the variation of the rate constant for quinol oxidation with temperature, is about 8 kcal mol^–1. Although we do not know the contribution of entropic terms to the pre-exponential factor, the result is consistent with a considerably larger value for the diffusion coefficient than that previously suggested.