Instead of binding calcium, one of the EF-hand structures in guanylyl cyclase activating protein-2 is required for targeting photoreceptor guanylyl cyclase.

Guanylyl cyclase activator proteins (GCAPs) are calcium-binding proteins closely related to recoverin, neurocalcin, and many other neuronal Ca(2+)-sensor proteins of the EF-hand superfamily. GCAP-1 and GCAP-2 interact with the intracellular portion of photoreceptor membrane guanylyl cyclase and stimulate its activity by promoting tight dimerization of the cyclase subunits. At low free Ca(2+) concentrations, the activator form of GCAP-2 associates into a dimer, which dissociates when GCAP-2 binds Ca(2+) and becomes inhibitor of the cyclase. GCAP-2 is known to have three active EF-hands and one additional EF-hand-like structure, EF-1, that deviates form the EF-hand consensus sequence. We have found that various point mutations within the EF-1 domain can specifically affect the ability of GCAP-2 to interact with the target cyclase but do not hamper the ability of GCAP-2 to undergo reversible Ca(2+)-sensitive dimerization. Point mutations within the EF-1 region can interfere with both the activation of the cyclase by the Ca(2+)-free form of GCAP-2 and the inhibition of retGC basal activity by the Ca(2+)-loaded GCAP-2. Our results strongly indicate that evolutionary conserved and GCAP-specific amino acid residues within the EF-1 can create a contact surface for binding GCAP-2 to the cyclase. Apparently, in the course of evolution GCAP-2 exchanged the ability of its first EF-hand motif to bind Ca(2+) for the ability to interact with the target enzyme.

Dimerization of guanylyl cyclase-activating protein and a mechanism of photoreceptor guanylyl cyclase activation.

Ca(2+)-binding guanylyl cyclase-activating proteins (GCAPs) stimulate photoreceptor membrane guanylyl cyclase (retGC) in the light when the free Ca(2+) concentrations in photoreceptors decrease from 600 to 50 nM. RetGC activated by GCAPs exhibits tight dimerization revealed by chemical cross-linking (Yu, H., Olshevskaya, E., Duda, T., Seno, K., Hayashi, F., Sharma, R. K., Dizhoor, A. M., and Yamazaki, A. (1999) J. Biol. Chem. 274, 15547-15555). We have found that the Ca(2+)-loaded GCAP-2 monomer undergoes reversible dimerization upon dissociation of Ca(2+). The ability of GCAP-2 and its several mutants to activate retGC in vitro correlates with their ability to dimerize at low free Ca(2+) concentrations. A constitutively active GCAP-2 mutant E80Q/E116Q/D158N that stimulates retGC regardless of the free Ca(2+) concentrations forms dimers both in the absence and in the presence of Ca(2+). Several GCAP-2/neurocalcin chimera proteins that cannot efficiently activate retGC in low Ca(2+) concentrations are also unable to dimerize in the absence of Ca(2+). Additional mutation that restores normal activity of the GCAP-2 chimera mutant also restores its ability to dimerize in the absence of Ca(2+). These results suggest that dimerization of GCAP-2 can be a part of the mechanism by which GCAP-2 regulates the photoreceptor guanylyl cyclase. The Ca(2+)-free GCAP-1 is also capable of dimerization in the absence of Ca(2+), but unlike GCAP-2, dimerization of GCAP-1 is resistant to the presence of Ca(2+).

[Polymorphism of B1-associated DNA fragments on various stages of ontogenesis of mice in tissues of different histogenesis]. / Polimorfizm B1-assotsiirovannykh fragmentov DNK na raznykh stadiiakh ontogeneza myshi v tkaniakh razlichnogo gistogeneza.

Pattern of B1-associated DNA fragments was studied by means of polymerase chain reaction (PCR) in the mouse tissues of different histogenesis of 15 and 20 day old embryos and of adult mice C57B1/6. As many as 20 DNA fragments were revealed on electrophoregrams differing in their molecular masses (m. m.) and amounts of amplified products. DNA fragments varied within a 100-10,000 bp range. The clusters of B1-associated DNA fragments, containing 100-200, 300-400 and 800-1000 bp, were most intensive in all studied electrophoregrams. The B1-associated DNA fragments from muscles of adult mice differed from those of other tissues by the presence of a DNA fragment with 800 bp. A comparative analysis of the spectra of B1-associated DNA fragments from hepatocytes of two inbred strains, C57B1/6 and C3HA, has shown their general similarities in m. m. values. But a significant distinction, that was found, involved the presence of a DNA fragment with m. m. approximately 6000 bp in the spectra of B1-associated DNA fragments from C3HA strain mice, that is absent in the spectra of respective fragments from hepatocytes of C57B1/6 strain mice. The obtained results allow to use the B1-PCR method for studying genome recombination during ontogenesis, intraspecies divergence and also at malignant cell transformation.

[Phenotypic variability in gag-myc transgenic mice]. / Fenotipicheskaia variabel'nost' u transgennykh myshei gag-myc.

The study of neoplastic growth capacity in gag-myc F3-F4 transgenic mice showed that they retain the capacity to develop hyperplasia and benign and malignant tumors same as F0-F2 transgenic mice do. However, the histological spectrum of tumors was much narrower, chiefly, due to the absence of rare patterns. The tumors in F3-F4 transgenic mice were mainly lymphomas and epithelial tumors of endocrine or exocrine glands. The incorporation of gag-myc in the genome of F3-F4 transgenic mice failed to tell on the variation of such quantitative polygenic characteristics as body mass and growth.

[Polymorphism of B1-associated DNA fragments from normal and transform ed murine hepatocytes]. / Polimorfizm B1-assotsiirovannykh fragmentov DNK v normal'nykh i transformirovannykh gepatotsitakh myshi.

Our study of B1-associated DNA fragment polymorphism in murine hepatocytes by means of polymerase chain reaction (PCR) allowed to reveal as many as 20 DNA fragments differing in their molecular masses (m. m.) and amount of amplified products varying within the range of 100-1000 bp. Within the same inbred strain of mice (C57B1/6), spectra of B1-associated DNA fragments were similar in different periods of ontogenesis (embryos of 15 and 20 days, adult mice), in different mice of the same or different litters. A comparative analysis of the spectra of B1-associated DNA fragments from hepatocytes of two inbred strains, C57B1/6 and C3HA, has shown in general their similarities in m. m. values. But a significant distinction, that was found, involved the presence of DNA fragments with m.m. approximately 600 bp in the spectra of B1-associated DNA fragments from C3HA strain mice, that is absent in the spectra of respective fragments from hepatocytes of C57B1/6 strain mice. The spectra of B1-associated DNA fragments from transformed hepatocytes of murine hepatoma MH-22a, in general, were the same as those from hepatocytes of C3HA strain mice. At the same time, a DNA fragment with m.m. of 450 bp, not detected in normal hepatocytes, was revealed in transformed ones. Nevertheless a DNA fragment with m.m. of 600 bp, characteristic of normal hepatocytes, was not observed in the transformed hepatocytes. The B1-PCR method can be used for studying genomic polymorphism both in different populations of mice, and during malignant growth.

Some characteristics of neoplastic cell transformation in transgenic mice.

The role of the expression of different cellular genes and viral oncogenes in malignant cell transformation is discussed. We pay special attention to the role of the genes for growth factors and their receptors and homeobox genes in oncogenesis. Based on both the literature and our own data, specific features of tumors developed in transgenic mice are discussed. All of these data are used to analyze current theories of multistep oncogenesis and the stochastic component in this process. We suggest that all known evidence about the mechanisms of oncogenesis be used in studying the problem at various structural and functional levels in an organism. The chapter shows that transgenic mice are a most suitable model for studying various aspects of malignant transformation from the molecular to the organismal and populational levels.

[The characteristics of homologous DNA recombination in somatic cells. III. Intrachromosomal recombinations of the aminoglycoside phosphoribosyltransferase gene in transgenic mice]. / Izuchenie osobennostei gomologichnoi rekombinatsii DNK v somaticheskikh kletkakh. III. Vnutrikhromosomnye rekombinatsii gena aminoglikozidfosforiboziltransferazy u transgennykh myshei.

Intrachromosomal homologous recombination has been revealed in the DNA from transgenic mice of three pedigrees. The recombination DNA of aminoglycosid phosphoribosiltransferase (neo) gene was observed in liver, kidney, spleen, heart, skeletal muscle, germinal glands and tail. It is concluded that the constructed model can be used for studying recombinations in cells of various organs and tissues both in the course of embryogenesis and during their malignant transformation and tumor progression.

[The use of B1-PCR method for studying the genomic polymorphism involved in malignant growth]. / Ispol'zovanie metoda v1-ptsr dlia izucheniia genomnogo polimorfizma pri zlokachestvennom roste.

The BI-PCT method showed the profile of BI-associated fragments of LNA in the cell line of the mouse hepatoma MH-22a to differ from that of the liver cells of C3HA mice, hepatoma cells incorporating the DNA fragments with 450 bp and those with 600bp disappearing. Application of the same method failed to reveal any differences in the profiles of BI-associated DNA fragments in the differentiated and non-differentiated cells of the embryonal carcinoma F9 induced by retinoic acid and cAMP dibutyryl treatment. It is suggested that the spectra of BI-associated DNA fragments might correlate with genetic stability in tumor cells.

[Oncogenesis in transgenic mice]. / Onkogenez u transgennykh myshei.

Oncogenesis in transgenic mice is at present a model, most adequately reflecting the natural conditions of tumor development. One of more important traits of this model is that it allows to study malignant growth simultaneously at all the structure-function levels in the context of the whole organism. This paper is a review of results of a series of experiments in which the localization of tumors was dependent or independent on the tissue specificity of a promoter, as well as development of multiple tumors with the use of viral regulatory sequences in genetic constructions. It has been shown that although a transgene is expressed in most of the tissues, tumors develop in some particular tissues only. These observations are interpreted by some authors in favour of the concept of multistep cancerogenesis. In this view, of primary importance are the results of studies on oncogenesis in transgenic mice, which contradict this concept and are regarded by their authors as an evidence of the possibility of a one-step transformation of normal cell into malignant one. The analysis of the obtained material enabled us to put forward an assumption that the key role in oncogenesis is played not only by certain genetic disturbances, but also by multi-level homeostatic mechanisms. Apparently, it is just the transgenic mice with cellular or viral oncogenes in their genome that represent a more adequate model for the detection of certain molecular-biological mechanisms underlying these disturbances. Also, of much importance is abundant material accumulated by now on oncogenesis of transgenic mice which shows a possibility of the effective use of various genetic constructions with prokaryotic and eukaryotic regulatory sequences, a possibility to induce not only tumors of some particular tissues, but also multiple hyperplastic and neoplastic changes in one and the same mouse. Development of tumors in such transgenic mice can be regarded as a model of different types of cancer disease.

[Leukemia in delta-GAG-MYC transgenic mice]. / Gemoblastozy u transgennykh myshei delta-GAG-MYC.

Transgenic mice carrying two delta-gag-myc genetic constructions were produced and kept under observation during their whole life. Nineteen out of 119 transgenic mice developed such hemopoietic diseases as lymphoid tissue hyperplasia, lymphoma, lymphosarcoma and myeloma. Lymphoid tissue hyperplasia and lymphoma generally involved multiple hyperplastic and neoplastic pathologies which were regarded, on the whole, as "malignant disease". In all cases, lymphosarcoma and myeloma were the only deadly pathologies. Lymphomas and myelomas were detected after 3-9 months, lymphosarcomas--18-29 months while lymphoid tissue hyperplasia occurred virtually throughout the entire life span--3-31 months. The study has shown that transgenic mice carrying delta-gag-myc gene in their genome can be used in the designing of special models for investigations of certain patterns of leukemia.

[The production and characteristics of transgenic mice expressing the surface protein gene of the human hepatitis B virus]. / Poluchenie i kharakteristika transgennykh myshei, ékspressiruiushchikh gen poverkhnostnogo belka virusa gepatita B cheloveka.

Transgenic mice were obtained that contained a gene of human hepatitis B surface antigen (HBsAg). The integrated HBsAg DNA sequences were inherited in the normal Mendelian fashion. 6 of 25 investigated transgenic mice expressed the HBsAg. The expression was detected in the serum and within the cytoplasm of hepatocytes. Specific tissue pathology was shown in these animals, including systemic hyperplasia of lymphoid tissue and degenerative changes in the liver and kidney parenchymatous cell elements.

[Viral myc oncogene in the genome of transgenic mice and their progeny]. / Virusnyi onkogen myc v genome trnasgennykh myshei i ikh potomstva.

The fused gag-v-myc oncogene was microinjected into fertilized mouse eggs, which were then implanted into foster mothers. Approximately 26% of the offsprings from injected eggs carried v-myc sequences. 26 of 32 progeny animals were found to be transgenic and some progeny containing the amplified oncogene (about 40 copies per genome). In one F0 and one F1 mice 1.5-2 months after birth the development of tumors was observed: rhabdomyosarcoma and sebaceous carcinoma. In both the cases the tumors were highly differentiated. Because spontaneous tumors of these types are seldom observed in common lines of mice it seems probable that the tumors observed in this study may be associated with the presence of oncogene v-myc.

[Tumors in transgenic mice]. / Opukholi u transgennykh myshei.

Transgenic mice are a suitable model for studying problems of modern biology on the molecular, cellular, and tissue levels. It is shown that some transgenic mice with integrated c-myc, v-myc, T-ag SV40, BPV have spontaneous tumours and may be used to study the role of oncogenes in the tumour development.