Characterization, cloning and sequence analysis of the CDC25 gene which controls the cyclic AMP level of Saccharomyces cerevisiae.

The cell division cycle of the yeast Saccharomyces cerevisiae is triggered at the stage called 'START'. Many results strongly suggest that adenylate cyclase is an essential element of the control of START. We report here results arguing for a positive control of the cAMP level by the CDC25 gene, another gene of START. Firstly, cdc25 cells can be rescued by extracellular cAMP. Secondly, the cellular cAMP content drops when thermosensitive cdc25 mutant cells are shifted to restrictive temperature. We report the molecular cloning of the CDC25 gene by complementation of cdc25 mutant cells. The identity of the cloned gene was confirmed by site-specific gene re-integration experiments and segregation analysis: the isolated fragment is shown to integrate into the cdc25 gene locus. When transferred in cdc25 mutant cells this DNA prevents the drop of the cAMP level at restrictive temperature. This gene is transcribed in a 5200-nucleotides mRNA. We have determined the nucleotide sequence of a 5548-bp DNA fragment which shows an uninterrupted open reading frame (ORF) coding for a 1587-amino acid polypeptide chain. Only the C-terminal part of the ORF appears to be essential for the complementation of the cdc25-5 allele, suggesting a multidomain protein.     

Adenovirus cyt+ locus, which controls cell transformation and tumorigenicity, is an allele of lp+ locus, which codes for a 19-kilodalton tumor antigen.

The early region E1b of adenovirus type 2 (Ad2) codes for two major tumor antigens of 53 and 19 kilodaltons (kd). The adenovirus lp+ locus maps within the 19-kd tumor antigen-coding region (G. Chinnadurai, Cell 33:759-766, 1983). We have now constructed a large-plaque deletion mutant (dl250) of Ad2 that has a specific lesion in the 19-kd tumor antigen-coding region. In contrast to most other Ad2 lp mutants (G. Chinnadurai, Cell 33:759-766, 1983), mutant dl250 is cytocidal (cyt) on infected KB cells, causing extensive cellular destruction. Cells infected with Ad2 wt or most of these other Ad2 lp mutants are rounded and aggregated without cell lysis (cyt+). The cyt phenotype of dl250 resembles the cyt mutants of highly oncogenic Ad12, isolated by Takemori et al. (Virology 36:575-586, 1968). By intertypic complementation analysis, we showed that the Ad12 cyt mutants indeed map within the 19-kd tumor antigen-coding region. The transforming potential of dl250 was assayed on an established rat embryo fibroblast cell line, CREF, and on primary rat embryo fibroblasts and baby rat kidney cells. On all these cells, dl250 induced transformation at greatly reduced frequency compared with wt. The cells transformed by this mutant are defective in anchorage-independent growth on soft agar. Our results suggest that the 19-kd tumor antigen (in conjunction with E1a tumor antigens) may play an important role in the maintenance of cell transformation. Since we have mapped the low-oncogenic or nononcogenic Ad12 cyt mutants within the 19-kd tumor antigen-coding region, our results further indicate that the 19-kd tumor antigen also directly or indirectly plays an important role in tumorigenesis of Ad12. Our results show that the cyt+ locus is an allele of the lp+ locus and that the cyt phenotype may be the result of mutations in specific domains of the 19-kd tumor antigen.     

Cloning of Pseudomonas aeruginosa algG, which controls alginate structure.

The biochemical mechanism by which alpha-L-guluronate (G) residues are incorporated into alginate by Pseudomonas aeruginosa is not understood. P. aeruginosa first synthesizes GDP-mannuronate, which is used to incorporate beta-D-mannuronate residues into the polymer. It is likely that the conversion of some beta-D-mannuronate residues to G occurs by the action of a C-5 epimerase at either the monomer (e.g., sugar-nucleotide) or the polymer level. This study describes the results of a molecular genetic approach to identify a gene involved in the formation or incorporation of G residues into alginate by P. aeruginosa. Mucoid P. aeruginosa FRD1 was chemically mutagenized, and mutants FRD462 and FRD465, which were incapable of incorporating G residues into alginate, were independently isolated. Assays using a G-specific alginate lyase from Klebsiella aerogenes and 1H-nuclear magnetic resonance analyses showed that G residues were absent in the alginates secreted by these mutants. 1H-nuclear magnetic resonance analyses also showed that alginate from wild-type P. aeruginosa contained no detectable blocks of G. The mutations responsible for defective incorporation of G residues into alginate in the mutants FRD462 and FRD465 were designated algG4 and algG7, respectively. Genetic mapping experiments revealed that algG was closely linked (greater than 90%) to argF, which lies at 34 min on the P. aeruginosa chromosome and is adjacent to a cluster of genes required for alginate biosynthesis. The clone pALG2, which contained 35 kilobases of P. aeruginosa DNA that included the algG and argF wild-type alleles, was identified from a P. aeruginosa gene bank by a screening method that involved gene replacement. A DNA fragment carrying algG was shown to complement algG4 and algG7 in trans. The algG gene was physically mapped on the alginate gene cluster by subcloning and Tn501 mutagenesis.     

Characterization of the multiple forms of cytochrome b559 in photosystem II

Cytochrome b559 is an essential component of the photosystem II (PSII) protein complex. Its function, which has long been an unsolved puzzle, is likely to be related to the unique ability of PSII to oxidize water. We have used EPR spectroscopy and spectrophotometric redox titrations to probe the structure of cytochrome b559 in PSII samples that have been treated to remove specific components of the complex. The results of these experiments indicate that the low-temperature photooxidation of cytochrome b559 does not require the presence of the 17-, 23-, or 33-kDa extrinsic polypeptides or the Mn complex (the active site in water oxidation). We observe a shift in the g value of the EPR signal of cytochrome b559 upon warming a low-temperature photooxidized sample, which presumably reflects a change in conformation to accommodate the oxidized state. At least three redox forms of cytochrome b559 are observed. Untreated PSII membranes contain one high-potential (375 mV) and one intermediate-potential (230 mV) cytochrome b559 per PSII. Thylakoid membranes also appear to contain one high-potential and one intermediate-potential cytochrome b559 per PSII, although this measurement is more difficult due to interference from other cytochromes. Removal of the 17- and 23-kDa extrinsic polypeptides from PSII membranes shifts the composition to one intermediate-potential (170 mV) and one low-potential (5 mV) cytochrome b559. This large decrease in potential is accompanied by a very small g-value change (0.04 at gz), indicating that it is the environment and not the ligand field of the heme which changes significantly upon the removal of the 17- and 23-kDa polypeptides.     

Equilibrium and kinetic studies of unfolding of homologous cytochromes c.

Extensive investigations of the unfolding equilibria and kinetics of oxidized and reduced cytochromes c are reported. It is found that all cytochromes c have similar unfolding free energies (deltaGD = 7 +/- 1 kcal/mol). Differences among species do not correlate in any way with the metabolic differences among species. The stabilization of cytochrome c on reduction is estimated at 1.1 kcal/mol. Stability differences among species are mirrored in their denaturation kinetics. For cytochrome c (III), the unfolding exhibits multiple phases. The rate constants for the two observable phases both change by a factor of 3 between horse cytochrome c (III) and cow cytochrome c (III). On reduction, all unfolding appears to occur in a single step. The rate of this unfolding still varies between species, however, the results can be accommodated to a sequential model, with some assumptions. The observations are consistent with chain reversal occurring at an early stage in the reaction and suggest that previously observed rapid processes may be ligand exchange processes.     

Probing the differences between rat liver outer mitochondrial membrane cytochrome b5 and microsomal cytochromes b5

Two distinct forms of cytochrome b5 exist in the rat hepatocyte. One is associated with the membrane of the endoplasmic reticulum (microsomal, or Mc, cyt b5) while the other is associated with the outer membrane of liver mitochondria (OM cyt b5). Rat OM cyt b5, the only OM cyt b5 identified so far, has a significantly more negative reduction potential and is substantially more stable toward chemical and thermal denaturation than Mc cytochromes b5. In addition, hemin is kinetically trapped in rat OM cyt b5 but not in the Mc proteins. As a result, no transfer of hemin from rat OM cyt b5 to apomyoglobin is observed at pH values as low as 5.2, nor can the thermodyamically favored ratio of hemin orientational isomers be achieved under physiologically relevant conditions. These differences are striking given the similarity of the respective protein folds. A combined theoretical and experimental study has been conducted in order to probe the structural basis behind the remarkably different properties of rat OM and Mc cytochromes b5. Molecular dynamics (MD) simulations starting from the crystal structure of bovine Mc cyt b5 revealed a conformational change that exposes several internal residues to the aqueous environment. The new conformation is equivalent to the "cleft-opened" intermediate observed in a previously reported MD simulation of bovine Mc cyt b5 [Storch, E. M., and Daggett, V. (1995) Biochemistry 34, 9682-9693]. The rat OM protein does not adopt a comparable conformation in MD simulations, thus restricting access of water to the protein interior. Subsequent comparisons of the protein sequences and structures suggested that an extended hydrophobic network encompassing the side chains of Ala-18, Ile-32, Leu-36, and Leu-47 might contribute to the inability of rat OM cyt b5 to adopt the cleft-opened conformation and, hence, stabilize its fold relative to the Mc isoforms. A corresponding network is not present in bovine Mc cyt b5 because positions 18, 32, and 47, are occupied by Ser, Leu, and Arg, respectively. To probe the roles played by Ala-18, Ile-32, and Leu-47 in endowing rat OM cyt b5 with its unusual structural properties, we have replaced them with the corresponding residues in bovine Mc cyt b5. Hence, the I32L (single), A18S/L47R (double), and A18S/L47R/I32L (triple) mutants of rat OM cyt b5 were prepared. The stability of these proteins was found to decrease in the following order: WT rat OM > rat OM I32L > rat OM A18S/L47R > rat OM A18S/L47R/I32L > bovine Mc cyt b5. The decrease in stability of the rat OM protein correlates with the extent to which the hydrophobic cluster involving the side chains of residues 18, 32, 36, and 47 has been disrupted. Complete disruption of the hydrophobic network in the triple mutant is confirmed in a 2.0 A resolution crystal structure of the protein. Disruption of the hydrophobic network also facilitates hemin loss at pH 5.2 for the double and triple mutants, with the less stable triple mutant exhibiting the greater rate of hemin transfer to apomyoglobin. Finally, 1H NMR spectroscopy and side-by-side comparisons of the crystal structures of bovine Mc, rat OM, and rat OM A18S/L47R/I32L cyt b5 allowed us to conclude that the nature of residue 32 plays a key role in controlling the relative stability of hemin orientational isomers A and B in rat OM cyt b5. A similar analysis led to the conclusion that Leu-70 and Ser-71 play a pivotal role in stabilizing isomer A relative to isomer B in Mc cytochromes b5.     

Characterization of plasmid transformation in Bacillus subtilis: kinetic properties and the effect of DNA conformation.

Summary Transformation of competent cells of Bacillus subtilis with antibiotic resistance plasmid DNA has shown that (a) competence for plasmid and chromosomal DNA develops with similar kinetics; (b) DNA linearized with a variety of restriction endonucleases does not transform; (c) CCC plasmid DNA is inactivated for transformation by a single nick; (d) T4 ligase restores transforming activity to both nicked and linearized DNA; (e) CCC relaxed DNA is fully active in transformation; (f) the DNA concentration-dependence of plasmid transformation is first order; and (g) plasmid transformation proceeds with a low efficiency, requiring the uptake of 10³ to 10⁴ DNA molecules per transformant.Based on this information, a model for the processing of chromosomal, plasmid and transfecting DNA is proposed.Abbreviations Cm Chloramphenicol - Em erythromycin - Km kanamycin - Sm streptomycin - CCC covalently closed circular - TBAB tryptose blood agar base - NCE nicking and closing enzymeIn partial fulfillment of the requirements for the doctoral degree in the Department of Microbiology at the New York University School of Medicine, for S.C.     

Derivation of the globins from type b cytochromes

Summary Similarities in the amino acid sequences of vertebrate and invertebrate globins, b₅ and b₂ cytochromes and chicken sulfite oxidase point to a common ancestry for all of these proteins. The distal heme ligand (histidine or its equivalent) is common to both sets of proteins, but the proximal histidine ligand of the cytochromes is replaced by another histidine residue in the globins. This explains why the heme is reversed between globins and b₅ cytochromes. It seems likely that the genes for primitive globins contained three exons, the first two of which were derived from a cytochromelike DNA sequence. A model is presented to show how globins may have evolved from a pre-existing type bcytochrome; the complexity of the required changes is an indication that all globins are monophyletic.     

A kinetic analysis of the estrogen receptor transformation.

The rate of the 4 to 5 S estrogen-binding protein (EBP) in vitro transformation was measured by sucrose gradient centrifugation analysis. The temperature-activated 4 to 5 S EBP transformation is found to be highly reproducible without loss of [3H]estradiol-binding activity in a buffer containing an excess of [3H]estradiol, 40 mM Tris, 1 mM dithiothreitol, and 1 M urea at pH 7.4. The presence of [3H]estradiol is necessary for the 4 to 5 EBP transformation. A kinetic analysis of the 4 to 5 EBP transformation shows that it is a bimolecular reaction, the dimerization of the 4 S EBP with a second (similar or dissimilar) monomer or subunit. In buffers containing 0.4 M KCl the apparent second order rate constant is 2.3 plus or minus 0-2 times 10-7 M minus 1 min minus 1 at 28 degrees. The reaction is independent of the initial receptor concentration, suggesting that the 4 S EBP is dissociated into monomeric units in buffers of high ionic strength. In buffers without KCl or with 0.1 M KCl the apparent second order rate constant of receptor transformation increases with decreasing receptor concentration. This suggests that the 4 S EBP is associated weakly with another macromolecule (or macromolecules) in buffers of low ionic strength. The rate of 4 to 5 S EBP transformation shows a 200-fold increase between 0 and 35 degrees. The Arrhenius energy of activation is 21.3 kcal mol minus 1 in buffer without KCl and 19.1 kcal mol minus 1 in buffer with 0.4 M KCl. Following the temperature-activated dimerization, the avidity of binding between the 4 S EBP and its complementary subunit is increased, 0.4 M KCl can no longer cause dissociation, and the 5 S EBP dimer appears. This kinetic analysis indicates that the avidity of binding between the subunits of the estrogen receptor is modulated by estradiol, temperature, and ionic strength. We propose that these interactions of the estrogen receptor's subunits reflect conformational changes involved in receptor activation.     

Identification of b,c, and d cytochromes in the membrane of Vitreoscilla

Cytochromes b, c, d, and o were identified by spectroscopic analysis of respiratory membrane fragments from Vitreoscilla sp., strain C1. Carbon monoxide difference spectra of the reduced membranes had absorption maxima at 416, 534, and 571 nm (ascribed to cytochrome o) and 632 nm (cytochrome d). Derivative spectra of the pyridine hemochromogen spectra of the membranes identified the presence of b- and c-type cytochromes in Vitreoscilla. The cyanide binding curve of the membranes was biphasic with dissociation constants of 2.14 mM and 10.7 mM which were assigned to cytochrome o and cytochrome d, respectively. Membranes bound carbon monoxide with dissociation constant 3.9 M, which was assigned to cytochrome o. Cytochrome c ₅₅₆ and a NADH-p-iodonitrotetrazolium violet reductase component were partially purified from Vitreoscilla membranes.Abbreviations INT p-iodonitrotetrazolium violet - RMF respiratory membrane fragments - K _d dissociation constant - CHAPS 3-[(3-cholamido propyl) dimethylammonio]-1-propanesulfonate - DOC sodium deoxycholate - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

The fine transformation between the oxidized and reduced forms of interleukin-1 receptor antagonist.

The active and pure recombinant forms of human interleukin-1 receptor antagonist (rHIL-1ra) were obtained from inclusion body expressed in Escherichia coli BL21 (DE3). However, the final purified protein was found to be in an unstable reduced form and easily converted to the other forms. Which form of the protein was involved and precisely how this occurred remained obscure. Therefore, we changed oxidizing and reducing conditions to delineate the subtle change between the reduced and oxidized states of rHIL-1ra based on high-pressure liquid chromatography (HPLC). The fine transformation between the two states was further verified through matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectroscopy. Meanwhile, which form might be beneficial to the protein was also investigated by testing its full bioactivity based upon methyl thiazolyl tetrazolium (MTT) method.     

Characterization of a protein fraction containing cytochromes b and c1 from mitochondria of Leishmania tarentolae

A soluble red band fraction was obtained from Leishmania tarentolae cells by sucrose gradient sedimentation of a Triton X-100 lysate. Spectral analysis indicated that cytochrome b was present in the red band: the reduced minus oxidized difference spectra revealed absorption maxima at 562,527, and 431 nm at room temperature and 562, 530, and 422 nm at 77K. In addition, a 28-kDa protein was identified in this fraction which retained heme-associated peroxidase activity even after denaturation on SDS-polyacrylamide gels. The amino acid composition of this protein showed a strong similarity to cytochrome c1 of both bovine and yeast.     

Electron conduction between b cytochromes of the mitochondrial respiratory chain in the presence of antimycin plus myxothiazol

The b haems of the bc1 complex of bovine heart mitochondria were poised with succinate and fumarate so that only the high-potential haem (b-562) was reduced, and then isolated from further redox exchange with the ubiquinone pool by adding antimycin and myxothiazol. A transmembrane electric potential difference was then developed, either by electron flow from [Ru(NH3)6]Cl2 to oxygen or by ATP hydrolysis. The small difference spectrum, caused by the electric field, indicated 32-55% oxidation of b-562 with concomitant reduction of b-566. No lag greater than 0.1 s was detectable between the initiation of respiration and the development of the difference spectrum, thus providing a direct demonstration of (fairly) rapid electron transfer between the b haems.     

Reactivity of cytochromes c and f with mutant forms of spinach plastocyanin.

The reduction of plastocyanin by cytochromes c and f has been investigated with mutants of spinach plastocyanin in which individual, highly conserved surface residues have been modified. These include Leu-12 and Phe-35 in the 'northern' hydrophobic patch and Tyr-83 and Asp-42 in the 'eastern' acidic patch. The differences observed all involved binding rather than the intrinsic rates of electron transfer. The Glu-12 and Ala-12 mutants showed small but significant decreases in binding constant with cytochrome c, even though the cytochrome is not expected to make contact with the northern face of plastocyanin. These results, and small changes in the EPR parameters, suggested that these mutations cause small conformational changes in surface residues on the eastern face of plastocyanin, transmitted through the copper centre. In the case of cytochrome f, the Glu-12 and Ala-12 mutants also bound less strongly, but Leu12Asn showed a marked increase in binding constant, suggesting that cytochrome f can hydrogen bond directly to Asn-12 in the reaction complex. A surprising result was that the kinetics of reduction of Asp42Asn were not significantly different from wild type, despite the loss of a negative charge.     

Effect of phospholipid on the redox potential and enzymic properties of cytochromes b.

The effects of phospholipid on the redox behavior of b cytochromes in succinate-cytochrome c reductase, the cytochrome b-c₁ complex, and an isolated cytochrome b preparation were investigated by the oxidative and reductive titrations. Three E_m values of cytochrome b were observed in the phospholipid-sufftcient and -depleted succinate-cytochrome c reductase. Their midpoint potentials at pH 7.4 are 75, 75, and ?100 mV for the sufficient and 10, ?30, and ?160 mV for the depleted reductase. The molar distribution of the b cytochromes of these E_m values correspond to 30, 30, and 40%, respectively. The E_m values of the isolated cytochrome b preparations were not affected by addition of phospholipids. The isolated b preparation contained two components of equal concentration with E_m values of ?85 and ?200 mV. No direct correlation between enzymic activity and the amount of high potential b cytochromes present in the systems was demonstrated. Very little difference was observed in redox behavior of b cytochromes between the aged inactive preparations of phospholipid-depleted reductase and that of freshly prepared reconstitutively active enzyme.