Localization of the ribosomal RNA genes in Drosophila simulans

In situ hybridization of cloned rRNA genes from Drosophila melanogaster to D. simulans metaphase chromosomes shows that in the tested wild type strains both sex chromosomes contain a nucleolus organizer region. Silver grain counts support the published data that the X chromosomal rRNA gene number is significantly higher than the Y chromosomal.     

The construction and replication properties of hybrid plasmids composed of the r-determinant of R100.1 and the plasmids pCRI or pSC201

Summary We have cloned the entire r-determinant of the antibiotic resistance plasmid R100.1 on the plasmid vectors pCR1 and pSC201. We find that the hybrid plasmids segregate from cultures in which replication of the vector is blocked. This suggests that the r-det is not capable of autonomous replication.     

The mitochondrially encoded "phantom ATPase" of S. cerevisiae. II. A heterogeneous mitochondrial ATPase population in situ.

Summary The mitochondrial ATPase from a PHO 1 mutant (OLI 2, PHO 1, OLI 4 region on mit DNA of S. cerevisiae) was further examined. A new purification method using Lysolecithin instead of Triton allowed us to solubilize and separate a heterogeneous ATPase population from PHO 1-mitochondria: the major abnormal fraction had extremely low oligomycin-sensitivity (but normal specific immunological reactivity), while a minor normal fraction (representing about 20% of the initial mitochondrial ATPase activity) had high sensitivity and affinity for oligomycin.Moderate urea treatment of PHO 1-mitochondria leads to partial loss of ATPase activity and a concomitant increase of oligomycin-sensitivity, suggesting that a heterogeneous ATPase population exists in situ in the mitochondrial membrane: part of the major abnormal ATPase fraction is selectively inactivated by urea, producing a concomitant enrichment in the initially minor normal ATPase fraction.If the minor normal ATPase fraction is the only one capable of in vivo ATP synthesis, the deficient but oligomycin-sensitive cell growth and oxidative phosphorylation in vitro are readily explained.Further structural studies are under way to ascertain whether the minor normal ATPase fraction is strictly identical to the wild type, in which case PHO 1 is a regulatory gene, or not, in which case PHO 1 is a structural gene.     

Involvement of IS1 in the dissociation of the r-determinant and RTF components of the plasmid R100.1

Summary Detailed mapping localized the PHO 1 mutation between the OLI 2 and OLI 4 loci on mitochondrial DNA of Saccharomyces cerevisiae.In its mitochondrially integrated form, the PHO 1-ATPase³ was difficult to identify either immunologically or by specific inhibitors like oligomycin and DCCD. Solubilization by Triton X-100 allowed unambiguuous identification of this enzyme as an authentic mitochondrial ATPase. However, Triton extraction produced a 2 to 3 fold enhancement of the PHO 1-ATPase activity which also became drastically cold-sensitive. The wild type ATPase was neither activated nor made cold-labile by solubilization, and retained full sensitivity to oligomycin and DCCD.Sucrose gradient analysis of the Triton-extracted ATPase from wild type, PHO 1 mutant and rho ^- strains showed a density difference between the solubilized PHO 1-and wild type ATPase, and similarity between solubilized PHO 1-and rho ^- ATPase (F₁).Whole cells of the PHO 1 mutant present considerably increased respiration rates.Comparison of oligomycin-sensitivity in whole cells, coupled isolated mitochondria and membrane-bound ATPase indicates a contrast between oligomycin-resistance of the ATPase and oligomycin-sensitivity of in vivo or in vitro coupling systems, which might characterize the products of this region of mitochondrial DNA.     

Oxidation-reduction studies of the Mo-(2Fe-2S) protein from Desulfovibrio gigas.

Potentiometric titration followed by e.p.r. measurements were used to determine the midpoint reduction potentials of the redox centres of a molybdenum-containing iron-sulphur protein previously isolated from Desulfovibrio gigas, a sulphate-reducing bacterium (Moura, Xavier, Bruschi, Le Gall, Hall & Cammack (1976) Biochem. Biophys. Res. Commun. 728 782-789; Moura, Xavier, Bruschi, Le Gall & Cabral (1977) J. Less Common Metals 54, 555-562). The iron-sulphur centres could readily be distinguished into three types by means of g values, temperature effect, oxidation-reduction potential values and reduction rates. The type-I Fe-S centres are observed at 77 K. They show mid-point potential values of -260mV (Fe-S type IA) and -440 mV (Fe-S type IB). Centres of types IA and IB appear to have similar spectra at 77 K and 24 K. The Fe-S type-II centres are only observed below 65 K and have a midpoint potential of -28mV. Long equilibration times (30 min) with dye mediators under reducing conditions were necessary to observe the very slow equilibrating molybdenum signals. The potential values associated with this signal were estimated to be approx. -415 mV for Mo(VI)/Mo(V) and-530mV for Mo(V)/Mo(IV).     

NMR characterization of three forms of ferredoxin from Desulphovibrio gigas, a sulphate reducer

A NMR and magnetic susceptibility study of the oxidized and reduced states of three different oligomers (forms) of a [4Fe-4S] ferredoxin protein from Desulphovibrio gigas, FdI, FdI′, and FdII was carried out. FdI and FdI′ are different trimers and FdII a tetramer of the same basic subunit. A probable assignment of the contact shifted resonances is indicated. Since the temperature dependences of the contact shifted resonances associated with each [4Fe-4S] are not all similar a delocalized model for the spin densities on the 4Fe does not apply. The exchange rate between oxidized and reduced states is slow on the NMR time scale. The three oligomers are not magnetically equivalent. Using the “three state hypothesis” terminology it is shown that FdI_ox is predominantly in the C^2? state and changes upon reduction into the C^3? state, while FdII_ox is in the C^? state and changes into the C^2? state. FdI′ does not easily fit into this classification. This study shows a similarity of magnetic behaviour between FdI and bacterial ferredoxins (e.g. Bacillus polymyxa) and between FdII and HiPIP from Chromatium sp.. The influence of the quaternary structure on the stabilization of the different oxidation states of ferredoxins as well as on their redox potentials is discussed.     

Purification and characterization of cytochrome C3 (Mr 26,000) isolated from Desulfovibriodesulfuricans Norway strain

An acidic cytochrome c (P_i = 4.8) has been purified from $\text{Desulfovibrio}\text{desulfuricans}$ Norway. Its molecular weight was estimated to be 26,000 but a monomeric form of 13,500 molecular weight has been obtained. The comparison of its amino acid composition and N terminal sequence has characterized this cytochrome as a new cytochrome, different from cytochrome c₃ (Mr 13,000) and cytochrome c_553(550) studied in the same organism. Its optical spectrum was similar to cytochrome c₃ (Mr 13,000) accordingly it has 4 haems per subunit. The absence of absorption at 695 nm indicates that two histidine residues are implicated as fifth and sixth ligand for haem iron. This new cytochrome is homologous to the cytochrome C₃ (Mr 26,000) previously described for $\text{Desulfovibrio}\text{gigas}$ and $\text{Desulfovibrio}\text{vulgaris}$.     

Characterization of the periplasmic hydrogenase from Desulfovibrio gigas.

The hydrogenase of the sulfate-reducer $\text{Desulfovibrio gigas}$ has been purified to homogeneity. The pure enzyme shows a specific activity of 90 μmoles H₂ evolved/min./mg protein. Its molecular weight is 89,500 and its is composed of two different subunits (mol. wt. : 62,000 and 26,000) which are not covalently bound. The absorption spectrum of the enzyme is characteristic of an iron-sulfur protein. The millimolar extinction coefficients of the hydrogenase are 46.5 and 170 respectively at 400 and 280 nm. It contains about 12 iron atoms and 12 acid-labile sulfur groups per molecule and the quantitative extrusion of the Fe-S centers of the hydrogenase indicates the presence of 3 Fe₄S₄ clusters. This hydrogenase has 21 half-cystine residues per molecule and a preponderance of aromatic amino-acids.     

Purification,characterization and biological activity of three forms of ferredoxin from the sulfate-reducing bacterium Desulfovibrio gigas

Three forms of ferredoxin FdI, FdI′, and FdII have been isolated from Desulfovibrio gigas, a sulfate reducer. They are separated by a combination of DEAE-cellulose and gel filtration chromatographic procedures. FdI and FdI′ present a slight difference in isoelectric point which enables the separation of the two forms over DEAE-cellulose, while FdII is easily separated from the two other forms by gel filtration. The three forms have the same amino acid composition and are isolated in different aggregation states. Molecular weight determinations by gel filtration gave values of 18 000 for FdI and FdI′ and 24 000 for FdII, whereas a value of 6000 is determined when dissociation is accomplished with sodium dodecyl sulfate. The electronic spectra are different and their ultraviolet-visible absorbance rations are 0.77, 0.87 and 0.68 respectively for FdI, FdI′ and FdII. Despite these differences, the physiological activities of the three forms are similar as far as the reduction of sulfite by molecular hydrogen is concerned.