The microsatellites are specific for each individual genome or species. In order to evaluate the genetic diversity and the relationships within the genus Lycopersicon, microsatellites markers were used. The main objective of this study was to determine the usefulness of the locus LE21085 in the genetic differentiation among six morphologically different tomato varieties of Lycopersicon esculentum Mill. (var. grandifolium subsp. cultum; var. cerasiforme - red and yellow, var. pruniforme, var. pyriforme subsp. subspontaneum and var. racemigerum subsp. spontaneum). For the microsatellites locus LE21085 were detected two alleles in all estimated tomato varieties, that differed by one base pair (122 and 123 bp). The biggest allele frequency was found for the allelic variant of 122 bp, and its values were: 0.8462 for L. esculentum subsp. subspontaneum var. cerasiforme (red), 0.6923 for L. esculentum subsp. subspontaneum var. cerasiforme (yellow), 0.5769 for L. esculentum subsp. cultum var. grandifolium, 0.6923 for L. esculentum subsp. subspontaneum var. pruniforme, 0.6154 for L. esculentum subsp. subspontaneum var. pyriforme and 0.8077 for L. esculentum subsp. spontaneum var. racemigerum. The average of observed heterozygosity for the locus LE21085 (Ho = 0.5641) was higher than average expected heterozygosity (He = 0.4158). The average PIC value for the locus LE21085 was 0.3294 and it was classified as a modest informative markers. From the obtained results it can be concluded that locus LE21085 could be used in genetic differentiation of tomato varieties, but in combination with other polymorphic microsatellite loci.
Тhermophilic bacteria Bacillus caldolyticus isolated from the hot spring in Bansko, Republic of Macedonia, were used for the isolation of DNA polymerase. Bacterial cells were disrupted by sonication and the first step in the purification of DNA polymerase was 40% ammonium sulfate precipitation. This was followed by chromatographic procedures on Sephadex G-50, DE-52 and CM-52 cellulose. DNA polymerase activity was analyzed at each step of purification using the incorporation of 3 H dATP in activated calf thymus DNA. The purity of the DNA polymerase was analyzed on SDS PAGE. Optimal conditions of activity were determined for temperature, pH, dNTP and Mg ++ concentration. In addition, the effect of ethanol and EDTA as possible inhibitors of polymerase activity was also analyzed. The optimal temperature of DNA polymerase was 66 ºC; the optimal pH was 7.2, optimal MgCl 2 concentration was 2.5 mM, and the optimal substrate concentration was 2.5 × 10 -6 M dNTP. The inhibitory effect of EDTA and ethanol on DNA polymerase was above 10 mM and 10%, respectively. Keywords: Bacillus caldolyticus; DNA polymerase; purification; optimization; inhibitors ПРОЧИСТУВАЊЕ И ОПТИМИЗИРАЊЕ НА УСЛОВИТЕ ЗА ДНК-ПОЛИМЕРАЗА ИЗОЛИРАНА ОД ТЕРМОФИЛНАТА БАКТЕРИЈА BACILLUS CALDOLYTICUSТермофилната бактерија Bacillus caldolyticus изолирана од термалниот извор во Банско, Република Македонија, беше користена за изолација на ДНК-полимераза. Бактериските клетки се лизирани со ултразвук и првиот чекор во прочистувањето на ДНК-полимеразата беше исталожување со 40% амониум сулфат. Потоа следуваа хроматографски процедури на Sephadex G-50, DE-52 и CM-52 целулоза. Активноста на ДНК-полимеразата беше анализирана во секој степен на прочистувањето со инкорпорација на 3 H dATP во активирана ДНК од телешки тимус. Чистотата на ДНК-полимеразата беше анализирана со користење на SDS PAGE. Оптималните услови за активноста беа одредени за температура, pH, dNTP и Mg ++ . Дополнително беа анализирни ефектите на етанолот и EDTA како можни инхибитори на ДНК-полимеразната активност. Оптималната температура за оваа ДНК-полимераза е 66 ºC, оптималната pH е 7.2, оптималната концентрација на MgCl 2 е 2.5 mM, додека оптималната концентрација на dNTP е 2.5 × 10 -6 M. EDTA покажува инхибиторен ефект врз ДНК-полимеразата во концентрација над 10 mM, додека етанолот во концентрација над 10%.
as well as, to quantify the amount of those fractions. 12.5 % SDS-PAGE was used. The total amount of proteins in whey from cow white cheese was 0.73 % ± 0.15, while in cow kashkaval whey was 0.91 % ± 0.08. In whey from white cheese, the relative protein percentages were: lactoglobulin 67.29 % ± 4.99, lactalbumin 20.64 % ± 2.02 and other fractions related to bovine serum albumin with 12.07 % ± 3. 05. In whey from yellow cheese, the proteins percentages were: lactoglobulin 52.62 % ± 1.21, lactalbumin 17.62 % ± 1.26 and other fractions related to bovine serum albumin with 29.74 %, respectively. Predominantly, -lactoglobulin was present in the analysed samples. The valorisation of the waste whey obtained in the white cheese production, and development of new product also contributes in the environment protection.
The molecular techniques provide new possibilities to characterize advanced genetic materials for registration purposes and for the protection of breeders' rights. The microsatellites appear as suitable molecular markers due to their highly polymorphic character. Such microsatellites may generate polymorphism useful for the analysis of genetic diversity and relationships within the genus Lycopersicon. The focus of the following study was usefulness of the locus LEEF1Aa in the genetic differentiation among six morphologically different tomato varieties of Lycopersicon esculentum Mill. The fragment analyses were done using Applied Biosystems DNA analyzer (ABI 3130) and GeneMapper®Software program. The obtained data were analyzed using the specific program Power Marker Software. The number of detected alleles for the microsatellites locus LEEF1Aa was six in estimated tomato varieties (219-221-223-225-227-229bp). The allele with the length of 229 bp was noticed only in Lycopersicon esculentum subsp. cultum var. grandifolium, while the alleles (221, 225 and 227 bp) in 4 varieties, the allele of 219 bp in 3 varieties and the allele of 223 bp in 2 varieties. The average PIC value for the locus LEEF1Aa was 0.7552 and it belongs to the group of high informative markers. Obtained results showed that the locus LEEF1Aais good choice for genetic differentiation of tomato varieties in combination with other polymorphic microsatellite loci.
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