On the size and chemical nature of the polypeptide chain of bovine liver rhodanese.

Evidence from molecular weight studies and sequence analysis of bovine liver rhodanese indicates that the enzyme is a single polypeptide of molecular weight 35,200, and not a dimer of identical subunits half this size. The rhodanese molecule contains 317 amino acids including 5 methionines, 4 cysteines, and 5 tryptophans. As expected, six fragments were produced by cleavage with cyanogen bromide and these have been aligned in the enzyme with the aid of overlapping tryptic peptides isolated from a [¹⁴C] carboxymethylmethionyl rhodanese derivative. The cyanogen bromide fragments account for all of the amino acid residues of the parent rhodanese molecule. Methionine residues are located at positions 72, 112, 214, 217, and 235 in the polypeptide chain and the active site cysteine is at position 251, in the carboxyl-terminal segment of the molecule.     

A study of the single polypeptide nature of rhodanese. A comparison of different preparations.

The enzyme rhodanese (EC 2.8.1.1) appears as a single polypeptide chain protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of this species is approx. 33 000. This contrasts with previous reports that rhodanese behaves on gel filtration chromatography as a rapidly equilibrating monomer-dimer system composed of identical subunits with a molecular weight of 18 500. We have investigated this apparent discrepancy by isolating the enzyme by the two different preparative procedures used in the above investigations. The two crystalline samples were subjected to gel filtration chromatography under a wide variety of conditions and to sodium dodecyl sulfate disc gel electrophoresis. The two preparations yielded rhodanese which behaved identically and no evidence for the monomeric species was obtained under any experimental condition tested. Thin-layer gel chromatography of clarified liver homogenates gave no evidence of rhodanese species other than that present in the purified samples. The variation in molecular weights observed in gel filtration chromatography may be a reflection of the conformational mobility of the enzyme leading to solvent-dependent changes in Stokes radius. If rhodanese is dimeric, special interactions must stabilize it under the conditions tested here.     

Purification and characterization of phiX174 gene A protein. A multifunctional enzyme of duplex DNA replication.

Synthesis of phiX174 viral (+) strand circles in vitro requires gene A protein, rep protein, DNA binding protein, and DNA polymerase III holoenzyme (Eisenberg, S., Scott, J. F., and Kornberg, A., (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 3151-3155). We have used this reaction as an assay to isolate gene A protein in greater than 90% purity. Its molecular weight under denaturing conditions is 59,000. The protein tends to aggregate and lose activity at low ionic strength. Tritium-labeled gene A protein cleaves the phiX174 duplex replicative form and is bound to it in a 1:1 ratio as part of an active replication complex. The attachment, at the 5' phosphoryl end of the cleavage point, is apparently covalent. The complex was not dissociated by: (i) banding in CsCl, (ii) treatment with 0.2 M NaOH, or (iii) boiling in 1% sodium dodecyl sulfate and electrophoresis on a sodium dodecyl sulfate-acrylamide gel; only micrococcal nuclease digestion of the DNA released the protein.     

Identification of lysis protein E of bacteriophage phiX174.

The product of gene E, the lysis gene of phiX174, has been identified as a distinct band in a sodium dodecyl sulfate-gel electropherogram. The position of the band is consistent with the molecular weight of 10,589 calculated from the nucleotide sequence of the gene. The band is eliminated by a nonsense mutation in gene E. It is estimated that roughly 100 to 300 molecules of E protein are made in an infected cell; this appears to be less than one-tenth the amount of protein made by gene D, in which gene E is wholly contained.     

On the molecular length of the replicative form DNA of bacteriophage phiX174

This paper describes an electron microscopic study of the circular replicative form DNA of bacteriophage φX174. The study has been carried out using a preparative technique in which the DNA molecules are adsorbed from solution on to the cleavage surface of mica and visualized in the electron microscope as a metal-shadowed replica (Gordon &; Kleinschmidt, 1969,1970). Contour lengths of open circular molecules were measured in samples obtained from preparations in which the following experimental parameters were varied: the ionic strength of the solution from which the DNA was adsorbed on the mica and the way in which the molecules were dried before shadowing. At the 0.05 significance level, varying these parameters had no effect on the mean length and variances of samples of molecules obtained from five experiments; the samples were therefore regarded as being drawn from the same molecular population with a mean length and variance of, respectively, 1.83 μm and 0.0117 μm².It was argued that the DNA molecules adsorbed on the mica are “frozen” into the molecular conformation present in solution at the time of adsorption and that, therefore, the experimentally determined contour lengths represent authentic molecular lengths in solution. Based on current estimates of the replicative form DNA molecular weight, the mean contour length obtained was slightly but significantly larger than the length predicted for molecules in an exact B configuration. The variance was larger than could be attributed solely to experimental error, indicating that the molecular population in aqueous solution is heterogeneous in contour length. These experimental results were shown to be consistent with a model for DNA structure in aqueous solution in which individual molecules are dynamic variants of a perturbed B form structure (von Hippel &; Wong, 1971).     

Process of infection with bacteriophage phiX174. XXXVII. RNA metabolism in phiX174-infected cells.

The RNA produced in vivo from bacteriophage phiX174 DNA has been analyzed by polyacrylamide-agarose gel electrophoresis and sedimentation in dimethyl sulfoxide gradients, and the results of Hayashi and Hayashi (1970) have been confirmed and extended. An efficient procedure for recovery of RNA from gels, followed by a hybridization assay, has indicated the presence in infected cells of 18 distinct RNA species with sizes up to and greater than the unit (viral) length. The sizes of phiX mRNA's were similar irrespective of whether material was analyzed on gels or in dimethyl sulfoxide gradients. When virus-induced RNA was detected by a double-label method, seven additional low-molecular weight species were observed on gels and the resolution of dimethyl sulfoxide gradients was enhanced. The present results lend support to aspects of the model of Hayashi and Hayashi (1970) for the generation of these discrete mRNA species; an alternative model is also discussed.     

Evidence that DNA polymerase-alpha of calf thymus contains a subunit of molecular weight 155000.

Small samples of the 8-S species of enzymes (A1 and A2) occurring in the DNA polymerase-alpha fraction of calf thymus, have been extensively purified using non-denaturing (normal) polyacrylamide gel electrophoresis. When peak fractions of activity on normal gels were subjected to dodecylsulphate-polyacrylamide gel electrophoresis, a polypeptide at 155000 correlated with polymerase activity. Samples of the 7.3-S (C) enzyme prepared from A2 by treatment with 2.4 M urea or isolated directly without exposure to urea, also showed the presence of a 155000-Mr polypeptide. It is concluded that the 7.3-S (C) enzyme, of previously estimated molecular weight 155000-170000, is a single polypeptide and that the 8-S enzymes A1 and A2 contain an additional subunit of 50000-70000 molecular weight.     

Comparative reactivities of diolepoxide metabolites of carcinogenic hydrocarbons with phiX174 viral DNA.

The ØX174 DNA assay system developed earlier is utilized to determine the comparative reactivities with nucleic acid of the diolepoxide metabolites of a series of polycyclic aromatic hydrocarbons varying in carcinogenic potency. The infectious ØX174 viral DNA is exposed to the hydrocarbon derivative for 10 min., then infectivity of the treated DNA is assayed by incubation with $\text{E.}\text{coli}$ spheroplasts, counting plaque formation on agar plates. The bay region diolepoxides of benzo[a]-pyrene, chrysene, and dibenz[a,h]anthracene, implicated as the ultimately active carcinogenic metabolites of the parent hydrocarbons, exhibit potent viral inhibitory activity. On the other hand no correlation is evident between viral inhibitory activity and either the location of the diolepoxide function in a bay region or the theoretically calculated β-delocalization energies (ΔE_deloc.) of the carbonium ion arising from opening the epoxide ring. The significance of these findings with respect to theories of carcinogenesis is discussed.     

Subcellular localization of lethal lysis proteins of bacteriophages lambda and phiX174.

The gene products of the lethal lysis genes S and E of the bacteriophages lambda and phiX174, respectively, were shown to be associated primarily with inner membrane material by isopycnic sucrose gradient centrifugation of lysates of infected cells. A small amount of each polypeptide appeared to be in the outer membrane fraction.     

Coordinate repression of cholesterol biosynthesis and cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A synthase by glucocorticoids in HeLa cells.

The stability constants for the calcium and magnesium complexes of rhodanese are >10⁵m^?1 at both high and low substrate concentrations. The stoichiometry of alkaline earth metal ion binding totals close to 1 per 18,500 molecular weight. The usual assay reagents contain sufficient amounts of these metal ions to maintain added enzyme in its metal-complexed form. When reaction mixtures are treated with oxalate to remove calcium ions, inhibition of rhodanese activity is virtually complete under circumstances such that the contribution of magnesium ion is low.Zinc and a number of transition metal ions are inhibitors of rhodanese activity. Studies of the concentration dependence of these effects with zinc, copper, and nickel showed that: 1) Some cyanide complexes of these metals are competitive with the donor substrate, thiosulfate ion. The binding of the copper and zinc complexes is mutually competitive. 2) Another cyanide species of copper appears to combine with the free enzyme to form a functionally active complex. 3) The zinc cyanide species with a net positive charge is an inhibitor competitive with the acceptor substrate, cyanide ion.All of these observations are consistent with a model in which metal ions serve as the electrophilic site of rhodanese.     

Direction of Translation and Size of Bacteriophage phiX174 Cistrons

Translation of the bacteriophage X174 genome follows cistron order D-E-F-G-H-A-B-C. To establish this, the position of a nonsense mutation on the genetic map was compared with the physical size (molecular weight) of the appropriate protein fragment generated in nonpermissive cells. Distances on the X174 genetic map and distances on a physical map constructed from the molecular weights of X174 proteins and protein fragments are proportional over most of the genome with the exception of the high recombination region within cistron A.     

Heterologous transfection with bacteriophage phiX174 DNA. Unusual heterogeneous products.

Hydroxylamine-resistant infectious materials (HARIM) synthesized in natural non-host and progeny phage low productive bacterial spheroplasts upon transfection with bacteriophage phiX174 DNA were found to be unusually heterogeneous in their forms. Using Pseudomonas aeruginosa as a source of HARIM, it was shown that they have the following unusual features. (1) Almost all of the HARIM are denser than normal single-stranded (SS)- and double-stranded replicative form (RF)-DNAs of phiX174 found usually in the phage-infected host cells. (2) A great part of these heavy HARIM (approximately 84%) contain a variable length of single-stranded RNA associated with their infectious elements. (3) For most of the HARIM (approximately 80% of total molecules as the infectious elements of the heavy HARIM), the infectious elements are phiX-RFI-DNA. The wide-spread system for phiX-HARIM synthesis was shown to be present in many gram-negative bacterial cells.     

Synthesis of rhodanese in Hep 3B cells

Summary The biosynthesis of rhodanese was studied in human hepatoma cell lines by immunoblotting and pulselabeling experiments using polyclonal antibodies raised against the bovine liver enzyme. Rhodanese, partially purified from human liver, showed an apparent molecular weight of 33,000 daltons, coincident with that of rhodanese from Hep 3B cells. After pulse labeling of Hep 3B cells both at 37°C and 25°C, rhodanese in the cytosol fraction exhibited the same molecular weight as the enzyme isolated from the particulate fraction containing mitochondria. Moreover, newly synthesized rhodanese from total Hep 3B RNA translation products showed the same electrophoretic mobility as rhodanese from Hep 3B cells. These results suggest that rhodanese, unlike most mitochondrial proteins, is not synthesized as a higher molecular weight precursor.     

Cleavage map of bacteriophage phiX174 RF DNA by restriction enzymes.

phiX RF DNA was cleaved by restriction enzymes from Haemophilus influenzae Rf (Hinf I) and Haemophilus haemolyticus (Hha. I). Twenty one fragments of approximately 25 to 730 base pairs were produced by Hinf I and seventeen fragments of approximately 40 to 1560 base pairs by Hha I. The order of these fragments has been established by digestion on Haemophilus awgyptius (Hae III) and Arthrobacter luteus (Alu I) endonuclease fragments of phiX RF with Hinf I and Hha1. By this method of reciprocal digestion a detailed cleavage map of phiX RF DNA was constructed, which includes also the previously determined Hind II, Hae III and Alu I cleavage maps of phiX 174 RF DNA (1, 2). Moreover, 28 conditional lethal mutants of bacteriophage phiX174 were placed in this map using the genetic fragment assay (3).     

Origin and direction phiX174 double- and single-stranded DNA synthesis

The origin and direction of both φX174 double-stranded and single-stranded DNA synthesis has been determined by pulsing replicating viral DNA molecules with [³H]thymidine for periods of less than one round of DNA synthesis and examining distribution of activity in the Haemophilus influenzae restriction endonuclease (Hin) DNA fragments of these molecules. In early RFI and RFII DNA intermediates in double-stranded DNA replication, gradients of label were observed which started in the R3 fragment (cistron A) and increased towards the R4 fragment (cistron H). The origin of synthesis is near the R4/R3 junction of the R3 fragment. Thus, φX174 double-stranded DNA synthesis proceeds clockwise around the genetic map (5′ → 3′), in one direction only and starting in the region of cistron A, a conclusion consistent with the genetic experiments of Baas &; Jansz (1972). Similar experiments with the gapped late RFII DNA molecules that have just completed a round of single-stranded viral DNA synthesis demonstrated that φX174 single-stranded DNA synthesis also has a single origin of replication in the region of cistron A, and that the synthesis moves in the 5′ → 3′ direction, around the genetic map. The gap in both the early and the late RFII DNA molecules also appears to be in the R3 fragment containing cistron A.     

Denaturation maps of DNA: experimental and theoretical maps of phiX174 DNA.

A formaldehyde denaturation map of the replicative form of phiX174 DNA is obtained. The RFI DNA was converted into a linear state by restriction endonuclease pst I which introduces into this DNA a single double-stranded break. The map has four clear-cut peaks. Their positions excellently correlate with the peak positions on the map of equilibrium denaturation theoretically obtained earlier from the known nucleotide sequence of phiX174 DNA. The sequence is also used for a calculation of the maps of smoothed AT-content. The maxima on these maps correlate well with the peaks on the denaturation maps. To reveal the causes of a good correlation between the experimental formaldehyde and theoretical equilibrium denaturation maps, the theoretical formaldehyde denaturation maps are calculated for different conditions (temperature, formaldehyde concentration) using the detailed theory of DNA interaction with formaldehyde developed earlier.     

On the molecular weight of thiosulfate sulfurtransferase.

Bovine liver thiosulfate sulfurtransferase (rhodanese) (EC 2.8.1.1) HAS BEEN REPORTED TO EXIST IN SOLUTION IN A RAPID, PH-dependent equilibrium between monomeric and dimeric forms of molecular weights 18 500 and 37 000 (Volini, M., DeToma, F. and Westley, J. (1967), J. Biol. Chem. 242, 5220). We have reinvestigated the proposed dissociation using sodium dodecylsulfate-polyacrylamide gel electrophoresis. The smallest rhodanese species observed has a molecular weight around 35 000, which is not reduced by severe denaturing conditions, including alkylation in 8 M guanidine-HCl or dialysis against 2% sodium dodecylsulfate and 5% mercaptoethanol. After limited CNBr cleavage, intermediate products of greater than 18 500 molecular weight are formed. The apparent molecular weight of these intermediate fragments is not changed by addition of mercaptoethanol. The total apparent molecular weights of the CNBr fragments after exhaustive cleavage is approx. 45 000 plus or minus 15 000. These results are not consistent with a monomer molecular weight of approx. 18 500 for thiosulfate sulfurtransferase.     

Purification of Epstein-Barr virus DNA polymerase from P3HR-1 cells.

The Epstein-Barr virus DNA polymerase was purified from extracts of P3HR-1 cells treated with n-butyrate for induction of the viral cycle. Sequential chromatography on DNA cellulose, phosphocellulose, and blue Sepharose yielded an enzyme preparation purified more than 1,300-fold. The purified enzyme was distinct from cellular enzymes but resembled the viral DNA polymerase in cells infected with herpes simplex virus type 1 or 2. The active enzyme had an apparent molecular weight of 185,000 as estimated by gel filtration on Sephacryl S-300. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major polypeptide corresponding to a molecular weight of ca. 110,000. This polypeptide correlated with the catalytic function of the purified enzyme, whereas the other, less abundant polypeptides did not. By immunoblotting, the 110,000-molecular-weight polypeptide could be identified as a viral polypeptide. It could not be determined whether the native enzyme was composed of more than one polypeptide.     

Ultraviolet reactivation of the single stranded DNA phage phiX 174.

Summary When UV-irradiated X174 was grown in pre-irradiated host cells of various strains, ultraviolet reactivation (UVR) was observed only in recombination proficient strains such as E. coli C (uvrA ⁺ recA ⁺) and HF4704 (uvrA ^- recA ⁺), but not in the recombination deficient strain HF4712 (uvrA ⁺ recA ^-). By increasing the multiplicity of infection, no rise in the amount of such reactivation was observed. From the study of the neutral and alkaline sucrose gradient sedimentation patterns of DNA samples extracted from unirradiated cells infected with unirradiated phage, it appears that after the conversion of the viral single stranded (SS) DNA to the double stranded form (DS), nicks or scissions were produced on it within all three strains, which were ultimately sealed up in the recA ⁺ but persisted within the recA ^- host cells. When UV-irradiated phage infected unirradiated host cells, such nicking of the DS DNA appeared to be much more extensive in uvrA ⁺ recA ⁺, but slightly reduced in uvrA ⁺ recA ^- and severely suppressed in uvrA ^- recA ⁺ strains. When the host cells were also UV-irradiated, the conversion of the infecting viral SS DNA to DS DNA as well as its subsequent nicking were reduced in all the three strains to a much greater extent. Although nicking of the DS DNA molecule is an essential step even in the normal intracellular replication of X DNA, the production and the sealing up of such nicks appear not to have any positive correlation with UVR of these phages. A drastic reduction in nicking due te pre-irradiation of the host cells might, however, mean slowing down of the replication of the damaged parental RF molecules which would facilitate their repair perhaps through recombination with the homologous parts of the host genome.     

Physico-chemical and biological study of excision-repair of UV--irradiated phiX174 RF DNA in vitro.

We have studied excision-repair of UV-irradiated phiX174 RFI DNA in vitro with UV-specific endonuclease from Micrococcus luteus (UV-endo), DNA polymerase I from Escherichia coli and DNA ligase from phage T4 infected E. coli. Excision-repair was measured a) by physico-chemical methods, i.e. by determination of the conversion of RF I DNA into RF II DNA by UV-endo and by the subsequent conversion of RF II DNA ligase, b) by biological methods i. e. by measuring the ability of the reaction product to form phages upon incubation with spheroplasts from the appropriate strains of E. coli. Using the first method, we have shown, that more than 90% of the pyrimidine dimers can be repaired in vitro; with the latter method we have shown, that the molecules which are repaired as defined by method a) have regained full biological activity. Exonuclease III was found to be not essential for excision-repair in vitro and also did not stimulate repair. From this result we conclude that UV-endo generates 3'OH endgroups, in agreement with results obtained by Hamilton et al. (1974). The usefulness of the method presented in this paper with regard to the study of excision-repair is discussed.