[Inheritable phenotypic normalization of rodent cells transformed by simian adenovirus SA7 E1 oncogenes by singled-stranded oligonucleotides complementary to a long region of integrated oncogenes]. / Nasleduemaia fenotipicheskaia normalizatsiia transformirovannykh E1 onkogenami adenovirusa obez'ian SA7 kletok gryzunov odnotiazhevymi oligonukleotidami, komplementarnymi protiazhennym uchastkam integrirovannykh onkogenov.

G11 mouse cells and SH2 rat cells transformed with simian adenovirus SA7 DNA showed inheritable oncogen-specific phenotypic normalization when treated with sense and antisense oligonucleotides complementary to long RNA sequences, plus or minus strands of the integrated adenovirus oncogenes E1A and E1B. Transitory treatment of the cells with the oligonucleotides in the absence of serum was shown to cause the appearance of normalized cell lines with fibroblastlike morphology, slower cell proliferation, and lack of ability to form colonies in soft agar. Proliferative activity and adhesion of the normalized cells that established cell lines were found to depend on the concentration of growth factors in the cultural medium. In some of the cell lines, an inhibition of transcription of the E1 oncogenes was observed. The normalization also produced cells that divided 2 - 5 times and died and cells that reverted to a transformed phenotype in 2 - 10 days. The latter appeared predominantly upon the action of the antisense oligonucleotides.

[Assessment of the frequency of finding polymorphic alleles of the human X-chromosome locus DXS52 in the Muscovite population]. / Otsenka chastoty vstrechaemosti polimorfnykh allelei lokusa DXS52 S-khromosomy cheloveka v Moskovskoi populiatsii.

The frequency of different polymorphic variants of the multiallelic locus DXS52 (St14) of the human X-chromosome, adjacent to the factor VIII gene, was evaluated by means of PCR for the heterogeneous population of Moscow and Moscow oblast'. It was shown that the heterozygosity index of this polymorphism in the studied population is much lower (0.71) than in Western Europe (0.80-0.85), which can apparently be explained by a higher frequency of the prevailing allele 1690 (0.52 compared to 0.36). Five new St14 alleles were detected during this study. The total informativity of the polymorphic markers St14 and HindIII (intron 19 of the factor VIII gene), which are most commonly used for hemophilia A detection, was evaluated. Among 83 investigated women, only 57 (69%) were heterozygous for at least one of the markers used, which is also much lower than in Western-European populations (90-95%).

[Spectrum of DNA haplotypes and beta-thalassemia mutations linked with them in the Azerbaijan Republic]. / Spektr DNK-gaplotipov i stseplennye s nimi beta-talassemicheskie mutatsii v Azerbaidzhanskoi respublike.

Haplotyping of the beta-globin gene cluster was performed on DNA samples from 110 Azerbaidzhanian beta-thalassemic patients and their families. During this study, we found 18 different haplotypes and determined the frequency of their occurrence. Nine of these haplotypes have never been observed earlier in the studied population. One of the haplotypes was found only in beta-thalassemia alleles. Several haplotypes were associated with beta-thalassemia mutations found earlier in Azerbaidzhan.

[Use of the polymerase chain reaction to detect beta-thalassemia mutations in heterozygous carriers from Azerbaijan while performing prenatal DNA-diagnosis]. / Ispol'zovanie polimeraznoi tsepnoi reaktsii dlia vyiavleniia beta-talassemicheskikh mutatsii u geterozygotnykh nositelei iz Azerbaidzhana pri provedenii prenatal'noi DNK-diagnostiki.

Prenatal DNA-diagnosis of beta-thalassemia in a family from Azerbaijan revealed two mutations new for this region--G-A transition at codon 15 and G-C transversion at position 5 of the intron 1. Prenatal diagnosis was carried out by direct sequencing of in vitro amplified (PCR) beta-globin gene fragments with a modified Sanger technique using thermostable DNA polymerase. The absence of parents mutations in the fetal DNA allowed us to conclude that the fetus is normal. The diagnosis was proved at hematological testing of the baby borne.

[A PCR-system of analyzing polymorphic markers in gamma-A and gamma-G globin genes and gene for human blood coagulation factor IX with an internal control of the completeness of restriction hydrolysis]. / PTsR-sistemy analiza polimorfnykh markerov v gamma-A i gamma-G globinovykh genakh i gene faktora koaguliatsii krovi IX cheloveka s vnutrennim kontrolem polnoty restriktsionnogo gidroliza.

New systems are proposed for the PCR analysis of HindIII polymorphic sites in the gamma A and gamma G globin genes and of TaqI polymorphic site in the human factor IX gene of blood population. DNA fragments amplified according to the systems described contain constant restriction site of the appropriate endonuclease, in addition to the polymorphic one, which significantly improves the reliability of the RELP analysis. The systems proposed are highly specific and may be used for DNA diagnosis of beta-thalassemia and haemophilia B.

[A case of prenatal diagnosis of beta-thalassemia by polymerase chain reaction]. / Sluchai prenatal'noi diagnostiki po povodu beta-talassemii na osnove tekhniki polimeraznoi tsepnoi reaktsii.

The prenatal diagnosis of beta-thalassemia in the Udin family, where the parents were the carriers of 2 bp deletion in the codon 8 (-AA) was undertaken using PCR. Five polymorphic restriction endonuclease sites in the beta-globin gene region were tested. They are: 2 HindIII sites in the gamma G and gamma A genes, 2 HincII sites located in the pseudogene and in its 3'-flanking region, and the AvaIII site in the second exon of the beta-globin gene. The heteroduplex analysis was also performed. Two HindIII polymorphic sites were informative and the HincII site in the pseudogene and the AvaII site in the beta-globin gene were partially informative. According to the results of the RFLP analysis, the embryo was heterozygous. The similar result was obtained by heteroduplex analysis.

[Molecular nature of beta-thalassemia in Tajikistan: a four base pair deletion in codons 41-42 of the beta-globin gene]. / Molekuliarnaia priroda beta-talassemii v Tadzhikistane: deletsiia chetyrekh par osnovanii v kodonakh 41-42 beta-globinobogo gena.

Thirty tajiks, whose relatives had beta-thalassemia traits (revealed in previous investigations by determination of the HbA-2 and HbF levels) were selected to screen beta-thalassemia mutations. DNA samples from each individual were subjected to the PCR (polymerase chain reaction) to amplify the 635 bp beta-globin gene fragment. One additional band was detected in three samples after the amplified fragment underwent electrophoresis in 2% agarose gel and the EtBr was stained, and two additional ones were revealed by 6% PAAGE and staining of the EtBr. All additional bands migrated more slowly than appropriate 635 bp fragment. It is supposed that additional bands are heteroduplexes formed from the wild type chains and mutated chains carrying a deletion or insertion. The 4 bp deletion of the 41-42 (-tctt) was detected after the direct sequencing of the amplified fragments. This mutation is common among Chinese but it was not revealed in the Middle Asia populations. The mutation can be easily screened using the PCR and electrophoresis in 2% agarose gel or PAAG of the amplified beta-globin gene fragments.

[Oncogene-directed mutagenesis in vivo. Polyalkylating derivatives of short single-stranded polynucleotides, complementary E1-adeno-oncogene, in the normalization of adenovirus-transformed rodent cell lines]. / Onkogen-naprovlennyi mutagenez in vivo. Polialkiliruiushchie proizvodnye korotkikh odnotiazhevykh polinukleotidov, komplementarnykh E1- adenoonkogenu, v normalizatsii transformirovannykh adenovirusom kloetochnykh linii gryzunov.

Polyalkylating derivatives of single-stranded polynucleotides (30-200-mers) complementary to the long E1 oncogene sequences of simian adenovirus SA7 cause inherited normalization of SH2 and G11 cells transformed with adenovirus SA7; certain deletions in the integrated proviral E1A oncogene were observed in several cases during this process. The transformed cells are indifferent to reagents noncomplementary to the E1 region. Thus polyalkylating derivatives of single-stranded 30-200-mers act as addressed mutagenes which react in a specific way with the integrated complementary DNA sequences of E1 oncogene in transformed rodent cells and realize oncogene-directed mutagenesis in vivo. During this treatment temporary normalized cells reverting to the initial transformed phenotype are also produced.

[Penetration of oligo/polynucleotides and their polyalkylating derivatives into rat cells transformed by simian adenovirus DNA]. / Proniknovenie oligo/polinukleotidov i ikh poliakriliruiushchikh proizvodnykh v kletki krysy, trnasformirovannye DNK adenovirusa obez'ian.

The transport regularity of the [32P]-oligo/polynucleotides and their polyalkylating derivatives into SH2 rat cells transformed with SA7 adenovirus DNA was investigated. Derivatives penetrate the SH2 cells and their distribution in the subcellular fractions are proportional to the concentration of reagent in the medium. The transport efficiency of the derivatives is inhibited sharply with cell concentration increase and practically does not depend on the action of cell metabolism inhibitors. The data obtained assumes the mechanism of the derivatives transport to be liquid endocytosis. Being distributed in the cell components the polyalkylating derivatives were accumulated by the cell nuclei up to 10(5)-10(7) molecules per nucleus. Transport efficiency is much greater in the anchored cells than in the suspended ones. Though essential dephosphorylation of the utilized substances is observed in the SH2 cells, part of them maintain native chain length and the 5'-phosphate group after 1 h incubation in nucleic acids obtained from the cell nuclei.

[Detection of carriers of hemophilia A by testing for HindIII polymorphism in the factor VIII gene by PCR]. / Vyiavlenie nositelei gemofilii A pri pomoshchi testirovaniia polimorfnogo saita HindIII v gene faktora VIII metodom PCR.

Representatives of 62 families from Moscow and Leningrad with haemophilia A observed in the pedigree were tested for HindIII polymorphism in the factor VIII gene. The proposed scheme of investigation was based on intron 19 of the FVIII gene amplification by the PCR technique followed by restriction analysis with the inner control of hydrolysis. 207 unrelated X-chromosomes were analysed, the frequency of the incidence of the polymorphic HindIII site in the given population found to be 0.29. The frequency of incidence of the HindIII heterozygotes calculated according to Hardy-Weinberg equation was 0.41. This value evidences for relatively high informativity of this polymorphism for carrier detection and prenatal diagnosis of haemophilia A. 23 families (37%) out of 62 examined in the study were informative for this criteria. The new scheme proved to be effective in testing HindIII polymorphism for haemophilia A carrier detection and prenatal diagnosis. The whole procedure takes one day, the radiolabelled probes are not used. The scheme described was inculcated in the All-Union Research Center for Haematology, Ministry of Health, USSR, Moscow, Research Institute for Obstetrics and Gynecology, Leningrad, Institute of Medical Genetics, Greifswald, DDR.

[DNA diagnosis of beta-thalassemia. Study of restriction fragment length polymorphisms in families with affected children]. / DNK-diagnostika beta-talassemii. Issledovanie PDRF v sem'iakh s porazhennym rebenkom.

A kit of DNA-probes directed at the cluster of human beta-globulin genes was used to study the incidence rate of 7 polymorphic restriction sites in beta-thalassemia patients and normal donors in the Azerbaijan SSR. Informative polymorphic sites Hind III were detected in GJ and AJ fetal globin genes, Hinc II in psi beta and Hinc III in 3' area of psi beta gene and Ava II in beta-globine gene differing in the incidence rate in the patients and donors. An analysis of haplotypes with respect to informative sites was made in two Azerbaijan families with an affected child. It has been found that the analysis with respect to one informative site is sufficient for prenatal diagnosis of the status of the following children.

[Detection of hemophilia A carriers by testing polymorphic Bcl I and HINDIII sites using the PCR method with internal splitting control]. / Vyiavlenie nositelei gemofilii A testirovaniem polimorfnykh Bcl I i Hind III saitov metodom PCR s vnutrennim kontrolem rasshchepleniia.

A new variant of the PCR test system is discussed which allows one to detect Bcl I and Hind III polymorphic sites of FVIII gene. It can be used for rapid and effective diagnosis of hemophilia A, especially, in combination with the blot-hybridization technique that detects other polymorphic variants of FVIII gene. The method proposed is highly accurate, reliable and simple. It allows one to analyze submicrogram quantities of DNA without using radiolabeled probes. The whole procedure takes several hours. The variant discussed can, possibly, be the part of the general scheme of hemophilia diagnosis completely based on the effective PCR test.

[Character of two mutations of the beta-globin gene in beta 0-thalassemia in Azerbaijan]. / Kharkter dvukh mutatsionnykh povrezhdenii beta-globinnovogo gena pri beta 0-talassemii v Azerbaidzhane.

Molecular nature of two beta 0-thalassaemia-causing mutations in beta-globin gene in Azerbaijanian population has been elucidated, viz., C-T transition in 39 codon (nonsense mutation) and previously unknown G deletion in 82/83 codons.

[Preparation and characteristics of a new transformed cell line G11 by transfection of NIH/3T3 mouse fibroblasts with DNA from the SA7 oncogene of simian adenovirus]. / Poluchenie i kharakteristika novoi transformirovannoi kletochnoi linii G11 pri transfektsii fibroblastov myshei NIH/3T3 DNK onkogena adenovirusa obez'ian SA7.

Murine fibroblasts NIH 3T3 were transfected with the plasmid pASP containing simian adenovirus oncogene insertion. Focus forming transformants were cloned with a final dilution technique and a new cell line G11 was created as a result. Transformed status of this cell line is evidenced by changes in morphology, specific cytochemical and adhesion properties, ability to grow in semisolid agar and FCS concentration growth independence. Presence of intact integrated E1a-region of adenovirus SA7 oncogene was shown by blot-hybridization technique. Transformed status of G11 cells can be explained by integration of SA7 oncogene, that is evidenced indirectly by the increased resistance to heat shock.

[Incorporation of yeast tRNA into mouse L 1210 lympholeukemic cells]. / Vkliuchenie tRNK drozhzhei kletkami limfoleikoza myshei L 1210.

[32P]tRNA from baker's yeast is incorporated without degradation into lympholeukotic cells of L1210 mice. The tRNA incorporation determined after tRNA hydrolysis on cell surface by RNAase increases linearly with a rise in the initial concentration from 0.5 to 500 micrograms per ml. According to gel electrophoresis of intracellular nucleic acids, after a 3 hour incubation the [32P]tRNA incorporated into the cells by 50% to form tRNA fragments without any conspicuous reutilization. The kinetic curve of tRNA incorporation during the first 60 min demonstrates a severalfold decrease in the initial maximal incorporation of [32P]tRNA into the cells (2 min), with a subsequent restoration of the incorporation within 2-3 hours.

[Blast cell differentiation of a continuous human leukemia line as affected by dimethyl sulfoxide]. / Differentsirovka blastnykh kletok perevivaemoi linii leikoza cheloveka pod deistviem dimetilsul'foksida.

The human blast cells line L-101 isolated from blood leukocytes of the patient with the cutaneous erythromyeloleukemia in the media with dimethyl-sulfoxide (DMSO) and maintained by permanent cultivation stimulate myeloid differentiation of this cells. The number of differentiated cells depends on DMSO concentration. Maximum of differentiated cells (up to 75%) have exposed on the 6th day of incubation with 0,75% DMSO. The cell line L-101 is a suitable model for investigation of myeloid differentiation.

[Isolation and characteristics of the DNA of donor and systemic lupus erythematosus patient blood plasma]. / Vydelenie i kharakteristika DNK plazmy krovi donorov i bol'nykh sistemnoi krasnoi volchanki.

DNA from blood plasma of healthy donors and patients with systemic lupus erythematosus (SLE) was isolated by various techniques. Adsorption chromatography of hydroxyapatite with subsequent deproteinization has shown that DNA of blood plasma from both healthy donors and patients with SLE is associated with serum protein and is low-molecular. This combination (DNA--protein) is resistant to the action of nuclease but susceptible to pronase. DNA that circulates in blood of SLE patients in conjunction with immunoglobulins is high-molecular as compared to DNA associated with other serum proteins.