Structure and active transport in the plasma membrane of the tubules of frog kidney.

The active transport of organic anions through the plasma membrane of the proximal tubules of frog kidney was studied. For this purpose a marker anion, fluorescein, was used, its flow into the tubules registered by the increase of fluorescense. The kinetics of transport was measured as function of time, concentration of substrate, concentration of a competing acid (p-aminohippuric acid) and temperature. The process is inhibited by strophantin, a specific poison for (Na++K+)-dependent ATPase. These data show that fluorescein transport is effected with the participation of a charged carrier, probably by the downfield mechanism postulated by Mitchell. To confirm this mechanism, a passive flow of K+ was created inwards across the membrane of the proximal tubules by means of valinomycin. It led to the discharge of the membrane and to the inhibition of fluorescein transport. Anions are transported downfield across the membrane, probably in a state of complexes with two Na+ ions. A magnetic field of 10000-28000 oersted inhibits the fluorescein transport strongly. This can be regarded as a proof of the liquid-crystalline structure of biological membranes and demonstrates the importance of this structure for active transport.

On the active transport of organic acids (fluorescein) in the choroid plexus of the rabbit.

The kinetics of active transport of an organic acid (fluorescein) through the membranes of the choroid plexus from the lateral ventricules of the brain of rabbit was studied both morphologically and functionally. It was shown that fluorescein is actively translocated through the apical and basal membrane of the epithelium and is accumulated in blood capillaries at a concentration exceeding one order of magnitude that in the incubation medium. The kinetic curves displaying saturation and the demonstration of inhibition by other acids shows that a specific carrier is involved in the transfer across the membrane. The active transport of fluorescein at 20 degrees C was found to be sodium independent. Total exclusion of sodium from the incubation medium does not change the Michaelis constant (Km) and maximal velocity (V). The active transport depends on the operation of (Na+ + K+)-ATPase as energy source but obviously no specific complexes with the participation of sodium are involved.

W-reactivation and W-mutagenesis of gamma-irradiated phage lambda.

UV irradiation of Escherichia coli wild-type cells manifested the phenomena of W-reactivation (WR) and W-mutagenesis (WM) of phage lambda irradiated by 60Co gamma-rays in broth. WR of gamma-irradiated phage was half as efficient as that of UV-irradiated phage, although the frequency of c mutations in conditions of WR was about the same in both phages. The xthA and recBrecC sbcB mutants were practically identical with wild-type cells in respect of WR and WM of UV- and gamma-irradiated phage. As in UV-irradiated phage, WR and WM of gamma-irradiated phage were absolutely dependent on the recA+ and lexA+ genes of the host cell. WR and WM required much smaller doses of UV radiation for induction in polA1 and uvrB mutants. The lig-ts mutant, temperature sensitive in polynucleotide ligase, was deficient in WR and WM of UV- and gamma-irradiated phage at the semi-permissive temperature of 37 degrees. The uvrE502 mutant and the allelic recL152 strain were absolutely deficient in WR and WM of gamma-irradiated phage. In UV-irradiated phage WR was reduced, but not eliminated, in the uvrE mutant, and WM was entirely suppressed. This is another example of uncoupling of WR and WM which shows that several repair systems are active in WR but only some of them are mutagenic.

Study of genetic effects of high energy radiations with different ionizing capacities on extracellular phages.

The inactivating and mutagenic action of high-energy radiations with different ionizing capacities (gamma-rays, protons, alpha-particles and accelerated ions of 12C and 20Ne) was studied by using coliphages lambda11 and SD as subjects. In particular the role of irradiation conditions (broth suspension, pure buffer, dry samples) and of the host functions recA, exrA and polA was investigated. The dose-response curve of induced mutagenesis was studied by measuring the yield of vir mutants in lambda11 and plaque mutants in SD. The following results were obtained. (1) The inactivation kinetics of phages under the action of gamma-rays and protons was first order to a survival of 10(-7). Heavy ions also showed exponential inactivation kinetics to a survival of 10(-4). At higher doses of 20Ne ion bombardment some deviation from one-hit kinetics was observed. For dry samples of phages the dimensions of targets for all types of radiation were approximately proportional to the molecular weights of phage DNA's. For densely ionizing radiation (heavy ions) the inactivating action was 3-5 times weaker than for gamma-rays and protons. (2) Mutagenesis was observed for all types of radiation, but heavy ions were 1-5-2 times less efficient than gamma-rays. For both phages studied the dose-response curve of mutagenesis was non-linear. The dependence on the dose was near to parabolic for lambda11. For SD a plateau or maximum of mutagenesis was observed for the relative number of mutants at a survival of about 10(-4). (3) Host-cell functions recA and exrA were practically indifferent for survival of gamma-irradiated phage lambda11, but indispensable for mutagenesis. Mutation recAI3 abolished induced vir mutations totally and exrA- reduced them significantly. The absence of the function polA had a considerable influence on phage survival, but no effect on vir mutation yield (if compared at the same survival level). (4) In conditions of indirect action of gamma-rays no vir mutations were induced. This is regarded as evidence that the single-strand breaks formed under indirect action conditions cannot serve as pre-mutational damage in DNA.

Double-stranded complex of polyguanylic and polycytidylic acids and its antiviral activity in tissue culture.

The antiviral activity and conditions of formation of the most active double-stranded complexes of synthetic homopolynucleotides, polyriboguanylic and polyribocytidylic acids, were studied on the model of primary trypsinized chick embryo cells and RNA-containing viruses. The (poly G).(poly C) complex was very active against the viruses tested; their replication in cell cultures was inhibited completely. The antiviral activity of the (poly G).(poly C) complex increased markedly in the presence of diethylaminoethyl- (DEAE-) dextran. After treatment with 1 mug/ml of (poly G). (poly C) for 1 hour in the presence of 100 mug/ml DEAE-dextran, the cell sheet remained protected for 5-7 days. Preparations of (poly G).(poly C) obtained under optimal conditions were as active as (poly I).(poly C) complexes and exceeded them markedly in the level of the therapeutic index which under the present experimental conditions was 5-10 times 10(3) for (poly G).(poly C). Highly purified homopolymers with sufficiently high molecular weight must be used for production of active and stable (poly G).(poly C) complexes.

Purification of influenza viruses on wide-pore glass columns.

Influenza viruses of different serotypes were purified by gel filtration on wide-pore glass. The virus thus purfied retained completely its haemagglutinating activity and infectivity, and by one purification cycle at least 99% of ballast proteins were removed.

[Quantitative relationships in the theory of active transport]. / O kolichestvennykh sootnosheniiakh v teorii aktivnogo transporta.

Quantitative relations for the active transport across a membrane are derived according to Mitchell's theory using an approach based on thermodynamic and kinetic principles. The main formula predicts that the flow rate depends on the "oxidative power" of the membrane in case of a big ionic flow. In case of a low current the rate is determined by the redox potential of the respiratory substrate. The relations derived in the paper are in qualitative agreement with Kaback's data on the active transport in bacterial vesicles.

[Changes in the induction and repair of double-stranded DNA breaks in pro- and eukaryote cells. I. Use of a Zimm elastoviscosimeter to study induction of double-stranded DNA breaks in gamma-irradiated Escherichia coli cells]. / Izuchenie induktsii i reparatsii dvunitevykh razryvov DNK v kletkakh pro- i éukariot. I. Primenenie élastoviskozimetra Zimma dlia izucheniia induktsii dvunitevykh razryvov DNK v gamma-obluchennykh kletkakh Escherichia coli.

The technique of elastoviscosimetry allows to study the induction of double-strand breaks in DNA of E. coli at low doses (on the order of D37). The dose dependence of retardation time to shows a characteristic maximum. It is shown that the ascending part of the curve is due to the phenomenon of relaxation of supercoiled DNA in the bacterial nucleoid. Relaxation is effected by different gamma-induced damages in DNA which are not double-strand breaks. The position of the maximum yields the average dose for the formation of the first double-strand break, which transforms the circular DNA into a linear chain. The descending part of the dose curve is explained by accumulation of additional double-strand breaks. The gamma-irradiation and lysis of cells was performed in different media. It was found that only in the case when the action of nucleases was substantially (but not completely) inhibited, the position of the maximum of the dose dependent of retardation time coincides satisfactorily with the value of D37 (14.5 +/- 2.3 and 12.5 +/- 3 krad correspondingly). If the medium does not contain inhibitors of nucleases then the position of the maximum corresponds to a 4.2 times lower dose of gamma-rays. This shows that double-strand breaks in gamma-irradiated DNA are generated mainly by enzymes participating in repair processes and that the first double-strand breaks seems to be the true reason of lethality because of inability to be repaired.

[Mechanism of action of D-amino acid oxidase. II. Evidence for the free radical mechanism of the reaction catalysed by the monomer form of the enzyme]. / Izuchenie mekhanizma deistviia oksidazy D-aminokislot. II. Dokazatel'stvo svobodno-radikal'nogo mekhanizma reaktsii monomernoi formy fermenta.

D-Amino acid oxidase was shown to dissociate into subunits in 2 M urea retaining the catalytic activity. This makes possible the direct observation of ESR spectra of the intermediate radical state of the enzyme when interacting with the substrate. We have shown that these radicals are really observable. Using the reversibility of the reaction and an equilibrium shift the amount of radicals can be increased up to 10% of all flavin groups present. The dependence of the radicals concentration on the amount of substrate and product can be predicted. The theory is confirmed by experimental data.

[The mechanism of action of d-amino acid oxidase. I. Evidence for a free radical mechanism of the reaction catalyzed b a dimeric form of the enzyme]. / Izuchenie mekhanizma deistviia oksidazy d-aminokislot. I. Dokazatel'stvo svebodno-radikal'nogo mekhanizma reaktsii, kataliziruemoi dimernoi formoi fermenta

d-Amino acid oxidase can oxidize the substrate to a ketoacid in the absence of oxygen. The stoichiometry of this reaction is precisely 1 molecule of keto acid for 1 molecule of enzyme, containing two flavin groups. Hence, the flavin must be in the semi-reduced free radical state. But these free radicals cannot be visualized by ESR spectroscopy because of closeness and strong interaction. After the acid denaturation of the protein the coenzyme is released as a semi-reduced free radical. An alternative method of registration is the transfer of the free radical state to an added excess of free flavin molecules. By both methods it is quantitatively determined that each flavin of the enzyme is reduced to a free radical. Therefore, we believe to have evidenced unambiguously that this enzymatic reaction proceeds via a free radical transition state.

Interaction of transfer RNA with 50S ribosomal subunits of Escherichia coli in absence of templates.

The equilibrium constant of a complex of tRNA with the 50S ribosomal subunit was measured in the absence of a template. It was shown that the stability of the complex increases with an increase in the concentration of Mg2+, it decreases with an increase in the concentration of univalent ions, and does not depend on the pH of the medium in the range of 7.0-8.2. Removal of the 3'-terminal nucleoside of tRNA weakens the association approximately 40-fold; the subsequent successive splitting off of another three nucleotides has little effect on the association constant. In 90% 2H2O the stability of the complex increases approximately four-fold, which points to the large contribution of the hydrogen bonds to the free energy of the interaction. The tetranucleotide TphiCG competes slightly with tRNA for sites on the 50S subparticles; this means that the TphiC segment of tRNA does not play an important role in the formation of the complex under investigation.

[Mechanism and kinetics of repair mutagenesis]. / Mekhanizm i kinetika reparatsionnogo mutageneza.

On the basis of experimental data a model of induced mutagenesis is proposed that takes into account the repair of DNA damage by the rec-system. The peculiar feature of the rec-system is the cleavage and resynthesis of long sequences near the recognized DNA-damage. Up to 100--2000 nucleotides are replaced in one act. Therefore a definite probability exists of finding a damaged point on the second strand serving as template. It is believed that at this point no requirements of complementary exist and that a random substitution can take place. This is the origin of point mutation. From the model a general formula for the dose-response curve of mutagenesis is deduced which also takes into account the possibility of simultaneously initiated repair on both complementary strands of DNA. The latter leads to a lethal event when the points are situated priximally. This formula fits the observations in different cases studied, for instance, in case of transition from uvr+ to uvr- strains. The absence of repair mutagenesis in case of transforming DNA damage is readily explained by one-strand integration.

[Escherichia coli K-12 mutants with enhanced resistance to ionizing radiation. IV. Recombination characteristics of Gamr mutants]. / Mutanty Escherichia coli K-12 s povyshennoi ustoichivost'iu k ioniziruiushchei radiatsii. Soobshchenie IV. Osobennosti rekombinatsii u mutantov Gamr.

In the radiation-resistant Gamr444 mutant the inheritance frequency of long F' episomes ORF1 (purE+ tsx+ procC+ lac+) and F'14 (ilv+--argE+) is lower, and the frequencies of chromosome mobilization and integrative suppression of temperature-sensitive dnaA46 mutation by the sex factor F are much higher than those in the wild-type strain AB1157 and another radiation-resistant mutant Gamr445. In this respect, the mutant Gamr444 is very similar to the recRC sbcB mutant (RecF-pathway of recombination).

[Escherichia coli K-12 mutants with enhanced resistance to ionizing radiation. III. The effect of rec and lexA mutations on radioresistance]. / Mutanty Escherichia coli K-12 s povyshennoi ustoichivost'iu k ioniziruiushchei radiatsii. Soobshchenie III. Vliianie mutatsii rec i lexA na radiorezistentnost'.

Lethal action of gamma-rays on derivatives of the wild-type strain AB1157 and of two radiation-resistant mutants (Gamr444 and Gamr445) containing additional mutations dnaA46, recB21, recF143, recA56, recA430, lexA3, lexA102 or lexA3 recAo98, was studied. When the mean number of genomes per cell was reduced by means of pre-incubation at 43 degrees C, radioresistance of the strains AB1157 dnaA46 and Gamr445 dnaA46 was not changed, and that of the strain Gamr444 dnaA46 was reduced to the level of the Gamr445 dnaA46 strain. Introduction of additional mutations recB21, recA56 or lexA3 (lexA102) into the genome of the strains Gamr444 or Gamr445 made them as radiosensitive as the corresponding variants of AB1157. Additional mutations recF143 or recA430 (lexB30) significantly decreased the radioresistance of Gamr444 and Gamr445 mutants, although did not level them to corresponding derivatives of AB1157. Operator-constitutive mutation recAo98 enhanced radioresistance of all lexA3 derivatives tested but not to the level of the corresponding lexA+ strains. The role of recombinational repair and the inducible SOS system in enhanced radioresistance of Gamr mutants is discussed. The data of post-irradiation DNA degradation in various derivatives of the strains AB1157 and Gamr suggest that Gamr mutants have a constitutive inhibitor of degradation which does coincide with RecA protein.

[Escherichia coli K-12 mutants with increased resistance to ionizing radiation. I. Isolation and study of cross resistance to different agents]. / Mutanty Escherichia coli K-12 s povyshennoi ustoichivost'iu k ioniziruiushchei radiatsii. Soobshchenie I. Vydelenie i izuchenie perekrestnoi ustoichivosti k razlichnym agentam.

The strains of Bacillus subtilis actively exchange genetic material during joint incubation on a properly composed selective solid medium at 37 degrees C. Predominantly, colonies of multiple recombinants are formed. If DNase is present in the medium, total number of recombinant colonies and the share of colonies of multiple recombinants decrease. At 46 degrees C the recombination process is inhibited. The results obtained are discussed using two hypotheses: 1) a donor cell transfers a significant part or even whole chromosome to a neighbouring recipient cell, 2) recombinations occur successively in mini-clones of intermediate recombinants. It is suggested that the phenomenon observed is close to the natural genetic transformation in Bac. subtilis.

[Escherichia coli K-12 mutants with enhanced resistance to ionizing radiation. II. Study of DNA damage and repair in gamma-irradiated cells]. / Mutanty Escherichia coli K-12 s povyshennoi ustoichivost'iu k ioniziruiushchei radiatsii. Soobshchenie II. Izuchenie povrezhdeniia i reparatsii DNK v gamma-obluchennykh kletkakh.

Formation and repair of single-stranded breaks (ssb) and double-stranded breaks (dsb) in DNA of gamma-irradiated cells of three Escherichia coli strains (the parental strain AB1157 and radiation-resistant mutants Gamr444 and Gamr445) were studied by centrifugation in alkaline or neutral sucrose gradients. The initial yield of ssb and the kinetics of their repair during post-irradiation incubation in a growth medium are similar for Gamr mutants and the wild-type strain. The yield of dsb in the chromosome of Gamr mutants is significantly lower than in chromosomal DNA of the parental strain, both immediately after gamma-irradiation and after three hours of post-irradiation incubation in a growth medium. The decreased yield of dsb in the Gamr mutants correlates with their relative radioresistance. It was found also that the level of DNA degradation is significantly lower in UV- or gamma-irradiated cells of Gamr mutants, as compared with the wild-type strain. It is suggested that enhanced radioresistance of the Gamr mutants is due to decreased formation of enzymatically induced dsb, as a consequence of moderated DNA degradation under the action of repair exonucleases and also to enhanced efficiency of dsb repair.

[Escherichia coli K-12 mutants with increased resistance to ionizing radiation. V. The effect of mutations on spontaneous and radiation mutagenesis]. / Mutanty Escherchia coli K-12 s povyshennoi ustoichivost'iu k ioniziruiushchei radiatsii. Soobshchenie V. Vliianie mutatsii na spontannyi i radiatsionnyi mutagenez.

The frequencies of spontaneous mutations (reversions his-4----His+ and forward mutations to rifampicin-, nalidixic acid- or valine-resistance) in radiation-resistant mutants Gamr444 and Gamr445 are much lower than in the wild-type strain AB1157. His+ revertants and rifampicin-resistant mutants Rifr are induced by low doses of gamma-rays more efficiently than in the wild-type. Low doses of UV light only enhanced mutagenic activity in Gamr strains for induction of His+ reversions but not for Rifr mutations. For the wild-type strain the frequencies of His+ and Rifr mutations increase proportionally to the square of dose both of UV light and gamma-rays. For the most radioresistant Gamr444 mutant the frequencies of UV- and gamma-rays-induced Rifr mutations and of gamma-rays-induced reversions increase linearly with the dose. Possible reasons for these anomalies of radiation-induced mutagenesis in Gamr mutants are discussed.

[Mutagenic action of alkylating agents on prophage lambda]. / Mutagennoe deistvie alkiliruiushchikh agentov na profag liambda.

The lethal and mutagenic effects of 7 alkylating agents: N-nitroso-N-methylurea (NMU), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), nitrogen mustard (HN2), mitomycin C (MC), bifunctional acridine mustard (AM)--and of cyanate (KNCO) on heat inducible lambda cI857 prophage were studied. After treatment of lysogenic cells with mutagens, prophage was heat-induced either immediately or after 90 min incubation in nutrient broth and c mutants forming clear plaques at 32 degrees C were scored. NMU (0.02 M) when immediately induced with heat, induces c mutants very efficiently (maximal yield 10%) not only in the wild-type cells but also in repair-deficient mutants recA13, lexA102, uvrA6 umuC36, recF143, xthA9, polA1, uvrD3 and uvrD502. These data show that NMU-induced mutations are fixed as replication errors due to mispairing modified bases. After delayed heat induction, the prophage survival enhances and the frequency of c mutations declines considerably in host cells of all repair genotypes tested. Carbamoylation is not involved in the mutagenic action of NMU, because KNCO (0.02 M) has a very slight lethal effect and does not induce mutations. MNNG (100 micrograms/ml) and EMS (0.1 M) also induce mutations by replicative mechanism, because maximal yield of c mutations does not depend on RecA+ and is about 15 and 2%, respectively. MMS is a mutagen of the repair type, since its mutagenic action is suppressed by recA mutation of the host. NH2 only inactivates prophage, but does not induce mutations. MC (50 micrograms/ml) and AM (150 micrograms/ml) induce mutations rather inefficiently (the maximal yield 0.1 and 0.3%, respectively) both in recA+ and recA- hosts. The mutagenic action of these agents is probably due to intercalation.

[Mutagenic action of nitrous acid on prophage lambda]. / Izuchenie mutagennogo deistviia azotistoi kisloty na profag liambda.

The lethal and mutagenic effects of nitrous acid (0,1 M NaNO2 in 0,1 M acetate buffer, pH 4.6) on prophage lambda cI857 ind- were studied in the wild-type cells of Escherichia coli and in 9 repair-deficient mutants: uvrA6, uvrA6 umuC36, uvrD3, uvrE502, polA1, recA13, lexA102, recF143 and xthA9. After treatment with HNO2, the prophage was heat-induced either immediately or after 90 min incubation in broth at 32 degrees C. The prophage survival after delayed induction was considerably higher than after immediate induction. The lethal action of HNO2 was highly expressed in uvrA- and uvrE- lysogens after delayed induction. The frequency of temperature-independent c mutants forming clear plaques at 32 degrees C reached 4% in the wild-type host after immediate induction, this value being 10-15% in uvrA, uvrA umuC, uvrD, uvrE, polA and xthA mutants, 0,8% in recF- lysogen and only 0,2-0,3% in recA and lexA mutants. Under these conditions, about 90% of c mutants are generated by recA+, lexA+-dependent repair mechanism (most probably, due to W-mutagenesis). After delayed induction, mutation frequency in the wild-type host declines considerably (down to 0,1%). Analogous phenomenon of mutation frequency decline was registered in uvrA, xthA, recF, polA, uvrE and uvrD lysogens. Under conditions of delayed induction, the frequency of HNO2-induced c mutations only slightly depends on the recA+ and lexA+ gene products and mutations are, apparently, fixed by replication.

[Mechanism of genetic recombination during bacterial recombination. VI. Single-stranded conjugation]. / Mekhanizm geneticheskoi rekom binatsii pri kon''iugatsii bakterii. Soobshchenie VI. Odnonitevaia kon''iugatsiia

The mutation BT43 in the dnaB gene inhibits conjugational DNA synthesis in the recipient cell at 42 degrees C. Since only one DNA strand is transferred from the donor to the recipient in these conditions, this single strand is integrated into the recipient chromosone. This is characterized by a high increase of recombination frequency per length unit, an effect well known in the case of transformation. This peculiar genetic process is proposed to be called "single stranded conjugation". It is more efficient in recipient cells recB-recC-sbcB-lacking two main degrading enzymes, exonucleases I and V. The proof of single strandedness was given by means of clonal analysis in a special experiment. The transfer of the selected marker into the thermosensitive recipient took place at 37 degrees C and the transfer of the non-selected marker -- at 42 degrees C. Thhe progeny of one merozygote must be mixed i.e. consist of cells with both alleles of the non-selected marker. This was confirmed by experimental data.