[The development of polymerase chain reaction diagnostic of colorectal cancer based on DNA integrity separated from feces of patient.]

The article presents procedure of diagnostic of colorectal cancer on the basis of analysis of integrity of fecal DNA. The samples of stool were picked out in 91 patients and out of them: 60 patients with colorectal cancer, 24 healthy volunteers (control group) and 7 patients with benign tumors. The evaluation of integrity of genome DNA using technique of polymerase chain reaction analysis specified involvement of two long fragments - sites of genes TP53 and MLH1. In stool of 47 patients with colorectal cancer the long fragments of DNA were detected while analysis of DNA from stool of individuals from control group detected only short fragments of DNA with size less than 200 np. The technique of non-invasive polymerase chain reaction diagnostic of colorectal cancer was developed. The sensitivity and specificity of this technique made up to 78% and 100% (p<0.0001). The sensitivity of technique has no significant differences for patients with different stages of disease that permits to apply it for early diagnostic of colorectal cancer.

[EFFECT OF ANTI-CANCER DRUG DOXORUBICINE ON CYTOMEGALOVIRUS INFECTED HUMAN FIBROBLASTS].

The anticancer antibiotic doxorubicine (DOX) is highly toxic and induces functional complications in vital organs. The effect of DOX on normal cells has not been examined in sufficient detail, and the search for compounds reducing DOX toxicity did not lead to success so far. It has been suggested that DOX induces death of cancer cells via p53-dependent apoptosis, however, the information regarding the role of p73 protein, a member of p53 tumor suppressor family, is scanty. Cytomegalovirus (CMV) induces an antiapoptosis program that allows its replication until death of the target cell. Our objectives were to examine the effect of DOX on normal cells (human fibroblasts), analyze the ability of CMV-induced antiapoptosis program to reduce DOX toxicity, and to evaluate the involvement of p73 protein and its isoforms in the regulation of death of CMV-infected and DOX-treated cells. Within a 24-h time period DOX caused death of about 70% human embryonic lung fibroblasts (HELF) in cell culture, this parameter decreased significantly in CMV-infected DOX-treated HELF cells. TUNEL has shown that the number of cells with DNA fragmentation decreases from 5.2% under the effect of DOX to 3.2% (P < 0.05) after combined CMV-DOX treatment. Analysis of mitotic figures revealed that DOX causes accumulation of mitotic cells, which was not observed in CMV-infected DOX-treated cells. PCR analysis of mRNA of two p73 protein isoforms (TAp73 and dNp73) has shown that in uninfected cells the expression of TAp73 isoform was low, while in CMV-infected cells level of TAp73 was significant and expression of dNp73 was demonstrated for the first time. Expression of TAp73 associated with lack of mitosis block. The activation of caspases 8, 9 and 3 in CMV-infected cells was registered but cell death was not, however, as massive as that caused by DOX. From these findings it can be concluded that CMV attenuates DOX-related damage to normal cells. It can be suggested that induction of TAp73 and dNp73 isoforms provides conditions for reduction of DOX effect which leads to DNA damage and death of normal cells.

[Role of constitutive and inducible repair in radiation resistance of Escherichia coli].

Radiation resistance of Escherichia coil cells depends on how efficiently DNA is recovered after damage, which is determined by the function of constitutive and inducible repair branches. The effects of additional mutations of the key genes of constitutive and inducible repair (recA, lexA, recB, polA, lig, gyr, recE, recO, recR, recJ, recQ, uvrD, helD, recN, and ruv) on radiation resistance were studied in E. coli K-12 strain AB 1157 and highly radiation-resistant isogenic strain Gam(r)444. An optimal balance ensuring a high gamma resistance of the Gam(r)444 radiation-resistant E. coli mutant was due to expression of the key SOS repair genes (recA, lexA, recN, and ruv) and activation of the presynaptic functions of the RecF homologous recombination pathway as a result of a possible mutation of the uvrD gene, which codes for repair helicase II.

[Expression of gene recA of Deinococcus radiodurans in Escherichia coli cells].

Plasmids pKS5 and pKSrec30 carrying normal and mutant alleles of Deinococcus radiodurans recA gene controlled by the lactose promoter slightly increase radioresistance of Escherichia coli cells with mutations at genes recA and ssb. The RecA protein of D. radiodurans is expressed in E. coli cells, and its synthesis can be supplementary induced. The radioprotective effect of the xenologic protein does not exceed 1.5 times and is essentially to the contribution of plasmid pUC 19-recA1.1 harboring the E. coli recA+ gene in the recovery of resistance of the deltarecA deletion mutant. These data suggest that the expression of D. radiodurans recA gene in E. coli cells does not complement mutations at gene recA in the chromosome possibly due to structural and functional peculiarities of the D. radiodurans RecA protein.

[Mutation in the cspH-cspG gene cluster enhances expression of heat-shock proteins and SOS repair system of Escherichia coli].

The recessive radioresistance allele gam12 cloned in plasmid pBC4042-gam12 slightly increases the radiation resistance of Escherichia coli wild-type cells. Meanwhile, irradiation by gamma-rays induces transition of r12 mutation to the homozygous state and causes a 3.37-fold increase in radiation resistance of these cells. The mutation gamr12 was located at 22.68 min of the chromosomal map in the region of cspH-cspG gene cluster of cold-shock proteins. Sequence analysis of gam12 allele revealed the nucleotide sequence of cold-shock gene cspG and insertions in the C-terminal part of the gene. Translation of mutant cspG gene can lead to synthesis of a truncated product that represents the N-terminal protein fragment with motifs governing binding with DNA and RNA. Analysis of the Escherichia coli genome revealed motifs recognized by proteins of the cspA family in genes of cold shock, heat shock, SOS regulon, and other systems. These data suggest the possibility of involvement of mutant RNA-chaperones of type CspA' and CspG' in the expression of key genes in systems of SOS repair and recombination or auxiliary stress systems, including heat-shock proteins, in radiation resistant mutants of E. coli.

[Operator-constitutive mutation in the recA gene enhances radiation resistance of Escherichia coli].

Plasmid pUC19-recAoc carrying a mutant allele of the recA gene, which plays the key role in the control of the SOS repair system and homologous recombinational repair, causes a 1.5-fold increase in radiation resistance of Escherichia coli DeltarecA cells, as compared to the wild-type recA+ cells. The protective effect of this plasmid is drastically reduced in mutant lexA3 recADelta21 deficient in the LexA protein and in induction of the SOS regulon. Plasmid pUC19-recAoc effectively suppresses UV sensitivity of the DeltarecA mutant. Mutation recAo20 allows constitutive high-level synthesis of the RecA protein. This mutation impairs the SOS box in the operator site of the recA gene and enhances heterology of the dimer LexA binding site. These data confirm that high level of the RecA protein synthesis per se is not sufficient for the expression of gamma-inducible functions and that the derepression of lexA-dependent genes, other than recA gene, is necessary for the complete induction of the SOS repair system.

[Effect of insertional mutation in the cspA gene encoding the major cold-shock protein on radiation resistance of Escherichia coli]. / Vliianie insertsionnoi mutatsii v gene cspA glavnogo belka kholodovogo shoka na radiorezistentnost' kletok Escherichia coli.

Plasmid pCspA::Km carrying a cloned mutant allele of the cspA gene for the major Escherichia coli cold-shock protein CspA with an insertion of the kanamycin resistance gene cassette from transposon Tn903 into the core region of the coding sequence causes a 2.3-fold increase in radioresistance of wild-type E. coli cells (cspA+). The radioprotective effect of this plasmid is abolished or drastically reduced in mutants recA13 and rpoH15 defective in RecA protein and in induction of the heat-shock protein regulon, respectively. Plasmid pCspA::Km causes a 1.3-fold elevation in the resistance to gamma-irradiation of E. coli mutants with an intermediate level of radioresistance (Gamr445 and KS0160) but slightly diminishes resistance of a highly radiation-resistant Gamr445 mutant. In the chromosome of E. coli with normal DNA repair systems, the cspA::Km mutation in the homozygous state enhances resistance to the lethal effect of gamma-rays and UV light 2.9 and 1.4 times, respectively. These data suggest that the system of cold-shock proteins can modulate resistance of E. coli cells to the lethal effect of gamma-rays and UV light.

[Effect of a null mutation in the priA gene on radioresistance of Escherichia coli]. / Vliianie nulevoi mutatsii v gene priA na radiorezistentnost' Escherichia coli.

According to Kogoma's model of DNA recombination by replication, the PriA protein is involved in the RecBCD pathway of double-strand break (DSB) repair, which is associated with extensive DNA degradation, at the stage of primosome assembly in D-loops (intermediates of strand exchange at the ends of DSB) for the subsequent switch to DSB-induced DNA resynthesis. Comparable data on possible involvement of the PriA protein in the repair of gamma-ray-induced lethal lesions in cells of the wild-type strain of Escherichia coli (strain AB1157) and in two radiation-resistant mutants Gamr445 and Gamr444 were obtained. In all the three strains examined, the null priA2::kan mutation in the structural priA gene was shown to markedly enhance the radiation sensitivity, causing a two- to threefold increase in the slopes of linear dose-survival curves. In the AB1157 strain, the inactivation of PriA is manifested most clearly in the range of low doses (up to 0.15 kGy) when the priA2::kan mutation had only a slight effect on the radiation resistance of Gamr mutants. It can be assumed that, in these mutants with a decreased level of postradiation DNA degradation, the PriA-dependent RecBCD pathway of DSB repair associated with extensive DNA resynthesis is not essential for the repair of lethal lesions at low doses. However, this pathway becomes crucial at higher doses (> 0.5 kGy) even for radiation-resistant strains, especially for the most resistant Gamr444 mutant.

[Constitutive inhibition of DNA degradation due to the enzyme RecBCD in the radiation-resistant Escherichia coli K-12 mutant Gam(r)444]. / Konstitutivnoe ingibirovanie degradatsii DNK, vyzvannoi fermentom RecBCD, u radioustoichivogo mutanta Gam(r)444 Escherichia coli K-12.

Exonucleolytic degradation of [3]H-labeled DNA was examined in partially purified fractions of lysates obtained from nonirradiated RecBCD enzyme-containing cells of Escherichia coli and in the radiation-resistant mutant Gamr444. The degradative activity was shown to be lowered in these cells to the same extent as in the recBC mutant. The efficiency of plating of the mutant phage T4 2-, DNA of which can be degraded by exonuclease V, was 400-fold higher on the strain Gamr444 than on the wild-type strain AB1157. This value was shown to be only twice as low as that on the recB mutant or on the strain AB1157 carrying plasmid pGam26 with a radiation-resistance allele gam26 cloned from mutant Gamr444. The data obtained confirmed the hypothesis that the Gamr444 mutant contains a constitutive inhibitor of exonucleolytic activity of the RecBCD enzyme in nonirradiated cells. This inhibitor was shown to be encoded by the gam26 allele that had previously been mapped at 56.8 min of the E. coli chromosome. A possible mechanism of the involvement of this inhibitor in enhanced radiation resistance of the mutant Gamr444 is considered.

[Natural plasmid pSD89 (Cmr) from Salmonella derby K89, which increases the radiation tolerance of Escherichia coli strain K-12]. / Prirodnaia plazmida pSD89 (Cmr) iz Salmonella derby K89, povyshaiushchaia radioustoichivost' shtammov Escherichia coli K-12.

Several plasmids with molecular mass of 1.3-9 MDa were found in a clinical isolate of Salmonella derby K89 by electrophoresis in agarose gel. One of these plasmids, designated pSD89 (Cmr), was derived from the K89 strain via transformation of the plasmidless recipient S. derby K82 to chloramphenicol resistance. The plasmid-carrying strain K89 and the K82 strain completely cured of plasmids were equally sensitive to the lethal action of UV light, whereas the plasmid-carrying strain was even more sensitive to ionizing radiation than the plasmidless variant. Nevertheless, transformants carrying only plasmid pSD89 (Cmr) were found to be more resistant to gamma-rays and UV light than the recipient. By using an intermediate host Escherichia coli Z80 (r-m+), plasmid pSD89 (Cmr) was introduced into different E. coli K-12 strains: polA-, recA-, uvrA-, umuC-, and the wild-type strain. A slight increase in radioresistance of E. coli wild-type cells and a significant complementation of a repair defect in recA and polA mutants, but not in uvrA and umuC, were observed.

[A mutant allele gam18, participating in the RecF repair path in Escherichia coli K-12]. / Mutantnyi allel' gam18, uchastvuiushchii v RecF-puti reparatsii u Escherichia coli K-12.

Plasmid pGam18 carrying one of the cloned mutant loci, responsible for enhanced radiation resistance in the strain Escherichia coli Gamr444, was shown to increase resistance to the lethal effect of gamma-rays with a dose modification factor DMF = 2. Enhanced resistance was observed in wild-type cells and in the mutant recBC sbcB, but not recFBC sbcA. This indicates the involvement of a product of the gam18 locus in the RecF pathway of recombinational repair. The protective effect of plasmid pGam18 against radiation was completely abolished by mutations in the most RecF pathway genes (recF, recJ, recR, recO, recQ, recN, and ruvB). However, three mutations in the uvrD gene, which encodes DNA helicase II and belongs to the RecF pathway, can be partially complemented by plasmid pGam18. These data suggest that the mutant allele gam18 affects the DNA helicase II activity at the presynaptic stage of the RecF pathway-mediated repair of DNA double-stranded breaks induced by gamma-irradiation.

[Growth of radiation resistance in bacteriophages as a result of intensifying expression of the stress system in host cells]. / Rost radioustoichivosti bakteriofagov kak rezul'tat usileniia ékspressii stressovykh sistem kletki-khoziaina.

By means of polyacrylamide gel electrophoresis (PAGE) of proteins from radiation-resistant Gamr mutants of Escherichia coli, it was shown that induction and elimination of RecA protein in these mutants are kinetically more rapid than in wild type cells, and heat-shock proteins (HSP) are hyperproduced even at a normal temperature (32 degrees C). gamma-and UV-irradiated bacteriophages were used to study the results of simultaneous enhanced expression of two stress repair systems. Radiation-resistant mutants are similar to wild type cells in their ability to reactivate phages lambda CI, phi 80 vir, and T 4D inactivated by gamma-rays and UV-light. W-reactivation of gamma-irradiated phages lambda and 80 vir is respectively 1.5 and 1.2 times higher in Gamr cells in which maximal w-reactivation was observed at wide range of doses (from 300 to 2000 Gy) whereas in wild type cells the peak of W-reactivation was registered at doses of 400 to 450 Gy. The phage lambda gamma-, irradiated upon adsorption on the cells of a radiation-resistant mutant, was two times more resistant to gamma-rays (DMF = 2 at LD10) than when irradiated upon adsorption on wild type cells. Postirradiation degradation of the phage lambda DNA, when irradiated within Gamr cells, was significantly lower than in wild type cells, and preirradiation of the cells decreased phage DNA degradation (12% in Gamr cells and 30% in wild-type cells). The role of an increased HSP level and expression of SOS-regulon in radiation resistance and possible interaction of stress systems in bacterial cells are discussed.

[Mutant alleles for radioresistance form the Escherichia coli strain Gam(r)444): cloning and preliminary characteristics]. / Mutantnye alleli radiorezisteentnosti iz shtamma Escherichia coli Gam(r)444: klonirovanie i predvaritel'naia kharakteristika.

Mutant alleles Gamr, which are able to increase the resistance to radiation of Escherichia coli wild-type cells, were cloned from the hyperradioresistant mutant Gamr444 on a plasmid mini-Mu vector MudII4042. The influence of recombinant plasmids on the sensitivity of wild-type and mutant (recA and htpR) cells to gamma-irradiation was studied. It was shown that the enhanced resistance of the Gamr444 strain to radiation was caused by mutations of two different classes, dominant and recessive. The cloned recessive mutation gamr12 increases resistance to radiation only after homogenization, that is, radiation-induced transfer from the plasmid to the chromosome, and it imposes constitutive expression of the heat-shock promoter htpG. Dominant mutant gamr alleles are active in the trans-position. A mutation-insertion into a chromosomal gene impaired by one of the dominant mutations, gamr18, was constructed. The insertion causes drastic cell radiosensitization on the recB sbcB background and probably disturbs the RecF pathway of recombination and repair. Dominant plasmids of the second type lead to the RecA-independent inhibition of DNA postirradiation degradation. The radioprotective action of recessive and dominant gamr mutations is additive.

[Mutants of Escherichia coli K-12 with increased resistance to ionizing radiation. VI. Increased radioresistance and heat shock proteins]. / Mutanty Escherichia coli K-12 s povyshennoi ustoichivost'iu k ioniziruiushchei radiatsii. Soobshchenie VI. Povyshennaia radiorezistentnost' i belki teplovogo shoka.

By means of one-dimensional electrophoresis, it is shown that in radiation-resistant Gamr444 and Gamr445 mutants of Escherichia coli K-12 high-molecular weight heat shock proteins are hyperproduced at 32-37 degrees C and are induced more intensively during heat shock (in comparison to the parental wild-type strain AB1157). When the missense htpR15 mutation of the positive regulatory htpR gene for heat shock proteins was introduced by transduction into the genome of the Gamr444 mutant, its enhanced radiation-resistance disappeared but could be restored upon introduction of pKV3 plasmid bearing the htpR+ gene. These data show that heat shock proteins are participating in the enhanced radioresistance of Gamr mutants.

[Thermoinduced radioresistance of Escherichia coli cells and heat shock proteins]. / Termoindutsirovannaia radiorezistentnost' kletok Escherichia coli i belki teplovogo toka.

Radioresistance of E. coli cells is slightly increased (dose modification factor (DMF) = 1.2) with temperature elevated from 4 degrees to 43 degrees C at the time of gamma-irradiation. However, an appreciable effect of the thermoinduced radioresistance (DMF = 1.7) was observed when the wild-type cells were exposed to gamma-radiation at 15-43 degrees C (but not at 4 degrees C) after 30-min preincubation at 43 degrees C. This effect was absent in htpR mutants, defective in induction of heat shock proteins, and coupled with the decreased post-irradiation DNA degradation in gamma-irradiated htpR+ cells. It is suggested that heat shock proteins are involved in the thermoinduced radioresistance.

[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.

[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 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. 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.