[Study of hybridization in four species of ground squirrels (spermophilus: Rodentia, Sciuridae) by molecular genetic methods]. / Izuchenie gibridizatsii chetyrekh vidov suslikov (Spermophilus: Rodentia, Sciuridae) molekuliarno-geneticheskimi metodami.

Four species of ground squirrel--yellow (Spermophilus fulvus), russet (S. major), small (S. pygmaeus), and spotted (S. suslicus)--occur in the Volga region. Between S. major and S. pigmaeus, S. major and S. fulvus, and S. major and S. suslicus, sporadic hybridization was reported. Using sequencing and restriction analysis, we have examined the mtDNA C region in 13 yellow, 60 russet, 61 small, 45 spotted ground squirrels, and 9 phenotypic hybrids between these species. It was shown that 43% of S. major individuals had "alien" mitotypes typical of S. fulvus and S. pygmaeus. Alien mitotypes occurred both within and outside sympatric zones. No alien mitotypes were found in 119 animals of the other three species, which suggests that only one parental species (S. major) predominantly participates in backcrosses. Phenotypic hybrids S. fulvus x S. major and S. major x S. pygmaeus) were reliably identified using RAPD-PCR of nuclear DNA. However, we could find no significant traces of hybridization in S. major with alien mitotypes. Analysis of p53 pseudogenes of S. major and S. fulvus that were for the first time described in the present study produced similar results: 59 out of 60 individuals of S. major (including S. major with S. fulvus mitotypes) had only the pseudogene variant specific for S. major. This situation is possible even at low hybridization frequencies (less than 1% according to field observations and 1.4 to 2.7% according to nuclear DNA analysis) if dispersal of S. major from the sympatric zones mainly involved animals that obtained alien mtDNA via backcrossing. The prevalence of animals with alien mitotypes in some S. major populations can be explained by the founder effect. Further studies based on large samples are required for clarifying the discrepancies between mitochondrial and nuclear DNA data.

[The hypereosinophilic variant of Ph'-positive chronic myeloleukemia]. / Giperéozinofil'nyi variant Ph"-polozhitel'nogo khronicheskogo mieloleikoza.

AIM: To confirm clonal nature of idiopathic hypereosinophilic syndrome (IHES), its relevance to Ph'-positive chronic myeloid leukemia. MATERIALS AND METHODS: 3 cases of idiopathic hypereosinophilic syndrome are reported with morphologic analysis of bone marrow cells and cytogenetic examinations. In one patient the presence of Ph'-chromosome was confirmed at fluorescent in situ hybridization (FISH) and molecular-genetic analysis (bcr/abl). Samples of bone marrow, spleen and liver were examined pathohistologically. RESULTS: The presence of chromosome anomaly t(9;22), i.e. Ph'-chromosome, associated with chronic myeloid leukemia (CML) was identified in all the 3 cases. There was also myeloid hyperplasia in the bone marrow (with primarily mature, eosinophilic granulocytes), spleen and liver, depression of megakaryocyto- and erythropoiesis. 2 patients had similar clinical symptoms which was not typical for CML in chronic phase: fever, elevated ESR, clear-cut anemia and thrombocytopenia. In the absence of hyperleukocytosis, blood and bone marrow eosinophils remained high (42.5, 21.5, 42.5% and 21.4, 7.1, 6.5%, respectively) due to "mature" forms. The number of blasts in the bone marrow was maximum 2.4%. CONCLUSION: The literature and the obtained data suggest closeness of idiopathic hypereosinophilic syndrome and Ph'-positive CML within myeloproliferative diseases.

[Polymorphism at codon 117 of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene]. / Polimorfizm v kodone 117 gena granulotsit-makrofag-koloniestimuliruiushchego faktora (GM-KSF).

A T-to-C substitution, replacing a hydrophobic isoleucine residue with a hydrophilic threonine residue in position 100 of a mature protein molecule, was found at codon 117 of the GM-CSF gene. The mutation frequencies were estimated in 51 DNA samples from healthy adult donors and also in 20 samples from patients with different neoplastic myeloid disorders. Almost equal substitution frequencies in patients and normal individuals were observed, suggesting that the defect was not associated with leukemia. Additionally the GM-CSF gene intron 1 sequence was refined.

[New polymorphic variants of the gene for human blood coagulation factor IX]. / Novye polimorfnye varianty gena faktora IX svertyvaemosti krovi cheloveka.

The polymorphism of Alu-repeats, which are located in the introns of the human factor IX gene (copies 1-3), was studied. To identify polymorphic variants, direct sequencing of PCR products that contained appropriate repeats was used. In each case, 20 unrelated X chromosomes were studied. A polymorphic Dra I site was found near the 3'-end of Alu copy 3 within the region of the polyA tract. A PCR-based testing system with internal control of restriction hydrolysis was suggested. Testing 81 unrelated X chromosomes revealed that the frequency of the polymorphic Dra I site is 0.23. Taq I polymorphism, which was revealed in Alu copy 4 of factor IX gene in our previous work, was found to be closely linked to Dra I polymorphism. Studies in linkage between different types of polymorphisms of the factor IX gene revealed the presence of a rare polymorphism in intron A that was located within the same minisatellite region as the known polymorphic insertion 50bp/Dde I. However, the size of the insertion in our case was 26 bp. Only one polymorphic variant was found among over 150 unrelated X chromosomes derived from humans from Moscow and its vicinity.

[A new polymorphism in the human factor IX gene, useful for determining carriers of hemophilia B]. / Novyi polimorfizm v gene faktora IX cheloveka, poleznyi dlia ustanovleniia nositel'stva gemofilii B.

A new Taq I polymorphism in Alu repeat 4 of the human factor IX gene is reported. This polymorphism is associated with a C-T transition at the 72-bp position of the Alu repeat consensus sequence. A simple PCR system for testing of this structural anomaly, with internal control of restriction hydrolysis, was developed. The frequency of the new polymorphic site and its linkage with other polymorphisms of the factor IX gene were also evaluated. The new polymorphism was used for establishing hemophilia B carriers.

[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.

[Nucleotide sequence of the E1B area and the adjacent 3'-terminal segment of the E1A oncogene from monkey adenovirus SA7 (C8)]. / Nukleotidnaia posledovatel'nost' oblasti E1B i primykaiushchego k nei 3'-kontsevogo uchastka oblasti E1A onkogena adenoviursa obez'ian SA7 (C8).

The SA7 (C8) simian adenovirus was sequenced from the 1478th to 3194th nucleotide. The region includes the 3'-terminal part of E1A and the major part of the E1B coding region. The sequence obtained was compared with the structure of SA7 (P) DNA previously determined in the region 1-2338, and many differences were found which are nucleotide substitutions, microdeletions and microinsertions. Among point substitutions the most frequent was the C-->T transition in CG pairs known as hot spots of mutations. Differences of our sequence from the previously published one was also revealed.

[Structure of integrated oncogens E1A and E1B in a malignant line of rat SH2 fibroblasts, transformed by monkey adenovirus SA7 (C8) DNA]. / Struktura integrirovannykh onkogenov D1A i E1B v zlokachestvennoi linii fibroblastov krysy SH2, transformirovannykh DNK adenovirusa obez'ian SA7 (C8).

In this investigation the primary structure of E1A and E1B regions of SA7 (C8) simian adenovirus integrated in malignant SH2 rat cell line was studied. Southern blotting revealed at least two copies of the SA7 oncogene integrated in the SH2 genome. PCR analysis of E1A and E1B regions showed heteroduplex structures, proving the different structure of the integrated copies. The heteroduplex molecules with different electrophoretic mobility were separated, and chains corresponding to different copies were sequenced according to the modified Sanger method. We found that two copies differ mainly in microsatellite regions, in E1A between positions 894-902 (GCG)3/(GCG)4, in E1B between positions 2037-2048 (GCA)3/(GCA)4. It is necessary to stress that all deviations found belong to the coding regions of the SA7 oncogene.

[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.

[Cloning the region of chromosome 22 participating in translocation t(9,22) in human chronic myeloid leukemia]. / Klonirovanie oblasti 22-i khromosomy, uchastvuiushchei v translokatsii t(9,22) pri khronicheskom mieloleikoze cheloveka.

Using oligonucleotide probes, sequences containing the Mbcr locus involved in chromosome translocation t(9:22) were cloned form the library of human genes in the Charon 4A vector. The recombinant clone lambda BCR 1.1 obtained contained Mbcr sequences, but the 3' region of the Mbcr locus in lambda BCR 1.1 clone was strongly altered. Subcloning of a fragment of the altered region and blot hybridization analysis using it as a DNA probe revealed recombination in the 3' region of the Mbcr locus in clone lambda BCR 1.1 which resulted in insertion of unknown sequences into the region. A modified system is suggested for chromosome 22 breakpoint identification using restriction analysis of genome DNA with four restriction endonucleases and one 5'-DNA probe.

[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.

[Increase of the negative charge of erythrocyte membrane proteins in hereditary neuromuscular diseases]. / Uvelichenie otritsatel'nogo zariada belkov membrany éritrotsitov pri nasledstvennykh nervno-myshechnykh zabolevaniiakh.

The parameters of erythrocyte ghost protein's fluorophores by nitrate's anions were studied in patients with various hereditary myodystrophy. In all the groups under examination the share of fluorophores accessible to a quencher was close to 1. In erythrocyte membranes of healthy donors the relevant constant quenchering was about 17.3 +/- 1.9 M-1 while those of patients were decreased by 3.1 (Duchenne's myodystrophy) and by about 2.0 (other forms of primary and secondary progressive muscular dystrophies). The most probable reason for the decreasing constant of quenchering is the increase of negative charges on the erythrocyte membrane proteins.

[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.