Construction and characterization of recombinant adenoviruses expressing human BRCA1 or murine Brca1 genes.

Recombinant adenoviruses expressing human BRCA1 (AdBRCA1), murine Brca1 (AdBrca1), three clinically relevant human mutant BRCA1 proteins (t340, C61G, and 1853Stop), or a murine Brca1 C-terminal deletion mutant were constructed and evaluated in vitro. These recombinants were capable of transducing high-level transgene expression to a wide variety of cell lines in vitro. Three independent methods were utilized to monitor cell growth following transduction with these recombinants. High-level expression of either the human or mouse wild-type BRCA1 protein was incompatible with maximal levels of cell growth. AdBRCA1 transduction inhibited the outgrowth of several human breast and ovarian cell lines in colony formation assays. Flow cytometric analysis revealed an accumulation of the transduced cells in the G0/G1 phase of the cell cycle. This BRCA1-mediated accumulation of cells in G0/G1 was accompanied by an increase in the cellular level of hypophosphorylated pRB. Ad mutant BRCA1 t340, C61G, and 1853Stop viruses were impaired, to varying degrees, in their ability to transduce a growth-arrested state to the target cells. Using these same three criteria, overexpression of murine Brca1 by AdBrca1 was also capable of transducing a growth-arrested state to human cells. Deletion of the C-terminus of Brca1 diminished this activity. This panel of adenoviruses may be useful reagents as part of an approach to understand the function of BRCA1/Brca1 in normal breast and ovary and help to define the tumor suppressor defect (s) conferred by clinical BRCA1 mutations in breast and ovarian cell tumorigenesis.

Changes in the size of pulse-labelled DNA fragments induced in human cells by inhibitors of uracil-DNA glycosylase and DNA methylation.

An inhibitor of uracil-DNA glycosylase, uracil, induces an increase in the size of pulse-labelled DNA fragments in human cells in vivo suggesting that dUMP incorporation into DNA and uracil-DNA glycosylase contribute to the small size of pulse-labelled DNA. It is also shown that inhibition of DNA methylation in vivo by ethionine and 5-azacytidine induces a decrease in the size of pulse-labelled DNA, and the effect is partially suppressed by uracil. In vitro experiments with purified uracil-DNA glycosylase from human placenta show that DNA hypermethylation inhibits the enzyme. The data make it possible to explain the antimutagenic effect of ethionine in mammalian cells [1] by stimulation of the repair of DNA containing incorporated uracil on the basis of the hypothesis that DNA-uracil repair stimulates mismatch correction leading to preferential excision of misincorporated nucleotides from daughter DNA strands.

[Uracil in DNA]. / Ob uratsile v DNK.

The data confirming the formation of dUMP residues in DNA to be a continuous process taking place in the living cells are reviewed. All living organisms produce specific enzymes repairing the lesion of this type. The possible ways for uracil incorporation into DNA are described. The main of them are as follows: cytosine deamination in DNA molecules and utilization of dUTP by DNA polymerases during replication. The spontaneous mutability, the decrease in chain length of the newly synthesized DNA and the increase in recombination frequencies are discussed as possible consequences of this phenomenon.

[UNG-dependent correction of molecular heteroduplexes of M13 phage DNA in Escherichia coli cells]. / UNG-zavisimaia korrektsiia molekuliarnykh geterodupleksov DNK faga M13 v kletkakh Escherichia coli.

Correction of heteroduplex DNA obtained by hybridization of uracil-containing single-stranded M13mp18 phage DNA and "mutant" synthetic oligonucleotide with deletion of cytosine in SalGI site was studied in ung+ and ung- E. coli strains. Uracil-containing DNA was prepared after growth of phage in an E. coli strain dut- ung-. The DNA was hybridized with "mutant" oligonucleotide then complementary DNA chain was synthesized by T4 DNA polymerase. Ung+ and ung- E. coli cells were transformed by DNA. In all experiments mutation frequency in ung+ was higher than in ung- cells (approximately 6-fold) and reached 11-50%. Absolute number of mutants was higher in ung+ cells. The results indicate that high level of mutagenesis depends on uracil repair system polarizing the correction of heteroduplex DNA.

[Uracil, contained in DNA, can direct the correction of mispaired nucleotides in human cells]. / Uratsil, soderzhashchiisia v DNK, mozhet napravliat' koretsiiu nesparennykh nukleotidov v kletkakh cheloveka.

The mismatch correction has been studied in human cells and presented in this paper. In the study the experimental model with half-containing hetroduplex (-1 residue in the polylinker region of lac gene) M13 DNA has been used. M 13 DNA was isolated from human cells 24 hours after transfection and transformed into ung+ and ung- cells of Escherichia coli. The percentage of lac- colonies (formed due to frameshift mutation in the lac gene) was analyzed. The increased percent of lac- mutations after human transfection indicated that DNA-uracil can polarize mismatch correction in human cells.

[Genetic transformation of somatic cells. XIV. Expression of gene coding for the human hepatitis B virus surface antigen in mammalian cells]. / Geneticheskaia transformatsiia somaticheskikh kletok. XIV. Ekspressiia gena, kodiruiushchego poverkhnostnyi antigen virusa gepatita B cheloveka, v kletkakh mlekopitaiushchikh.

A set of recombinant plasmids with a gene encoding surface antigen of hepatitis B virus (HBsAg) is constructed. The plasmids were transfected by DEAE-dextran method into different lines of cultured cells and transient expression of the HBsAg gene was studied. The results indicate that: transcriptional enhancer of hepatitis B virus situated downstream from HBsAg gene is active in green monkey kidney cells (CVI), promoter of 5 LTR of bovine leukemia virus is trans-activated in the goat or calf cells infected with BLV. The results are discussed in the light of hypothesis on the role of transcriptional enhancers in determination of tissue-specificity of hepatitis B virus.

[Uracil-DNA glycosidase studied with an immune serum]. / Izuchenie uratsil-DNK-glikozidazy s pomoshch(iu immunnoi syvorotki.

Immune antiserum to uracil-DNA glycosylase was obtained by immunizing rabbits with an enzyme isolated from the rat liver. Antiserum was found to suppress the activity of uracil-DNA glycosylase not only in the extracts of rat liver, but also in the extracts of brain, cardiac muscle, kidney, spleen, thymus of rats, and in those of human placenta too. This enables us to make a conclusion about the similarity in antigenic properties of the enzyme in cells of various types of differentiation. Indirect immunofluorescent test shows a slight staining of the periphery of the nucleus in normal liver hepatocytes and the intensive staining of the inner part of the nucleus in hepatocytes of regenerating liver. Therefore it is concluded that the enzymatic activity increases as cells proliferate. This may be the result of the appearance of uracil in DNA during replication.

[Activity of the DNA repair enzyme uracil-DNA-glycosylase in mammalian species differing in longevity and the level of liver cell ploidy]. / Atkivnost' fermenta reparatsii DNK uratsil-DNK-glikozilazy u vidov mlekopitaiushchikh, razlichaiushchikhsia po prodolzhitel'nosti zhizni i urovniu ploidnosti kletok pecheni.

The activity of uracil-DNA glycosylase (UDG) was studied for livers of 13 mammalian species belonging to four orders. DNA contents were also measured in isolated hepatocytes. The enzymatic activity was shown to increase with the increase in the mean ploidy of liver parenchymal cells. The activity of UDG was 20 times as high when the mean liver cell ploidy of different mammalian species doubled. A reverse dependence between the UDG activity and species life spans is also revealed.

[The levels of DNA repair in the cells of diploid and tetraploid cell cultures]. / Issledovanie urovnei reparatsii DNK v kletkakh diploidnykh i tetraploidnykh kletochnykh kul'tur.

Levels of unscheduled DNA synthesis after UV-irradiation were investigated in addition to the activities of DNA-repair enzyme uracil-DNA glycosilase in cells of both diploid (XC-D) and tetraploid (XC-T) cell cultures. It was shown that repair levels were increasing directly and proportionally to the cell ploidy.

[Insertion of the bacterial gene for dihydrofolate reductase into colony-forming cells of mouse bone marrow]. / Vvedenie bakterial'nogo gena digidrofolatreduktazy v kolonieobrazuiushchie kletki kostnogo mozga myshi.

Introduction of the plasmid containing the methotrexate-resistant (Mtx-r) bacterial gene of dihydrofolate reductase (DHFR) under the control of the early promoter of SV 40 into the donor bone cells of the mouse with subsequent transplantation of the cells into lethally irradiated mice results in the increase in the life span of mice under conditions of methotrexate selection. It is due to the stable transformation of the bone marrow colony-forming cells with the plasmic DNA and the synthesis of the bacterial Mtx-r DHFR in the spleen and bone marrow of the recipient mouse.

[Transformation of mice by a prokaryotic gene for dihydrofolate reductase]. / Transformatsiia myshei prokarioticheskim genom digidrofolatreduktazy.

In mice obtained after microinjection into the male pronucleus of fertilized eggs of the plasmid, containing the bacterial gene of dihydrofolate reductase (DHFR), under the control of the early promotor of the simian virus 40 (SV40), an integration of the foreign DNA into the mouse genome is found. About 30% of the treated animals contain the integrated plasmid DNA sequences, i.e. are transgenic. In 2 of 7 mice, containing the introduced plasmid in their genome, the methotrexate-resistant DHFR activity is found in the kidney and spleen, which may be due to the expression of gene DHFR. The plasmid DNA sequences and the ability to synthesise the methotrexate-resistant enzyme DHFR are transmitted to the next generation of mice.

[Uracil-DNA-glycosylase in the rat: correlation between the enzyme activity and rate of DNA synthesis in different tissues]. / Uratsil-DNK-glikozilaza krysy: korreliatsiia aktivnosti fermenta i sinteza DNK v razlichnykh tkaniakh.

Uracil-DNA-glycosylase which releases uracil residues from DNA has been purified from rat liver more than 300-fold. The enzyme has a molecular weight of about 28,0000, Km = 1.7 . 10(-9) M. The content of uracil-DNA-glycosylase in five different tissues of the rat is correlated with the rate of DNA synthesis in the tissues. The enzyme activity in rat liver increases more than 3 times after partial hepatectomy, showing a peak 28-32 hrs following the surgery. The results obtained suggest that uracil-DNA-glycosylase from mammalian cells releases uracil residues from DNA at time intervals close to those of replication.

Enzymes from Micrococcus luteus involved in the initial steps of excision repair of spontaneous DNA lesions: uracil-DNA-glycosidase and apurinic-endonucleases.

Uracil-DNA-glycosidase that releases free uracil from single-stranded or double-stranded deaminated DNA and poly d(A-U) has been partially purified from Micrococcus luteus. The enzyme has a molecular weight of about 16,000 and can be separated from uracil-endonuclease and endonucleases (AP-endonucleases) specific for apurinic and apyrimidinic sites. Uracil-DNA-glycosidase does not act on guanine residues opposite uracil in double-stranded DNA and on xanthine in deaminated DNA. The glycosidase generates apyrimidinic sites which can serve as substrate sites for different AP-endonucleases from M. luteus.

[Purification and various properties of uracil-DNA-glycosylase from human placenta]. / Ochistka i nekotorye svoistva uratsil-DNK-glikozilazy iz platsenty cheloveka.

Uracil-DNA-glycosylase was isolated from human placenta and purified 2100-fold. The apparent Km value for non-methylated DNA substrate of the enzyme is 3.10(-7) M. However, Km for uracil-DNA-glycosylase was 3 times as low when methylated DNA was used as a substrate. It was shown that the initial rate of uracil excision was greater for the non-methylated than for the hypermethylated DNA. The experimental results indicate that the postreplicative methylation of DNA can interfere with uracil excision.

BRCA1-associated growth arrest is RB-dependent.

BRCA1 is a susceptibility gene for breast and ovarian cancer with growth-inhibitory activity for which the mechanism of action remains unclear. When introduced into cells, BRCA1 inhibits growth of some but not all cell lines. In an attempt to uncover the mechanism of growth suppression by BRCA1, we examined a panel of cell lines for their ability to reduce colony outgrowth in response to BRCA1 overexpression. Of all variables tested, only those cells with wild-type pRb were sensitive to BRCA1-induced growth suppression. In cells with an intact rb gene, inactivation of pRb by HPV E7 abrogates the growth arrest imposed by BRCA1. In accordance with these observations, we found that BRCA1 could not suppress BrdUrd uptake in primary fibroblasts from rb-/- mice and exhibited an intermediate ability to inhibit DNA synthesis in rb+/- as compared with rb+/+ cells. We further found that the BRCA1 protein complexes with the hypophosphorylated form of pRb. This binding is localized to amino acids 304-394 of BRCA1 protein and requires the ABC domain of pRb. In-frame deletion of BRCA1 fragment involved in interaction with pRb completely abolished the growth-suppressive property of BRCA1. Although it has been reported that BRCA1 interacts with p53, we find the p53 status did not affect the ability of BRCA1 to suppress colony formation. Our data suggest that the growth suppressor function of BRCA1 depends, at least in part, on Rb.