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Today, we have very powerful and effective machines and methods to sequence and analyze DNA sequences. Almost every week, new genomes are added to sequence databases. However, those data are useless without additional annotations. Genes need to be found and their functions defined. Experimental work is too slow to analyze each sequence of a potential gene but computational methods facilitate such analyses. Here, we review the methodology, potential problems and constraints in genes finding and their annotation. We describe some new approaches including comparative genomics.
EN
Identification of gene functions needs information from different molecular levels: transcriptome, proteome and metabolome. Chromatographic techniques combined with different types of detectors are methods of choice for secondary metabolites profiling. Mass spectrometry is one of the best methods for natural products identification due to its high selectivity and sensitivity. However, physico-chemical properties of secondary metabolites present in plant species have very strong influence on the applicability of chromatographic techniques for separation of different classes of organic compounds present in the samples extracted from plant tissue. There does not exist an analytical method capable for separation and identification of all metabolites present in plant tissue during a single analysis. This article describes chromatographic systems combined with different mass spectrometric techniques for identification of different classes of secondary metabolites present in plant material.
EN
The genetic construct WAP 6xHisHGH containing the gene encoding human growth hormone (hGH) and WAP promoter expressed in mammary gland of animals was prepared. The 5? end of the gene was modified by the addition of sequence encoding six histidine residues and the sequence recognized by thrombin. In this way, the growth hormone can be easily purified by affinity chromatography and cleaved with thrombin to an active form. In the next step, the genetic construct was introduced by microinjection into male pronuclei of fertilized oocytes. Transgene was detected in male rabbit of F0 generation (number 61). Twelve offspring of founder rabbit of generation F1 indicated transgene sequences. The presence of growth hormone was revealed in the samples of milk accumulated during the lactation of females of F1 generation. The genetic constructs containing chain 1 and chain 2 of Feld1, and the major allergen produced by cat (Fedlis domesticus) were prepared. Both genes were inactivated by introduction into the sequences a positive selectable marker aminoglycoside phosphotransferase (resistant to neomycin). Outside the region of homology to Feld1 chain 1 and chain 2 genes, the negative selectable marker ? thymidine kinase gene was introduced. The genetic constructs pNTKFd1 and pNTKFd2 can be used in further experiments involving the inactivation of Feld1 genes in cat cells. Both genes were modified by site-directed mutagenesis using megastarter with Stop codon for premature termination of translation. The presence of mutation was confirmed by sequencing. The genetic constructs with human hGH gene and cat Feld1 gene were introduced into the bovine and cat fetal fibroblasts respectively in co-transfection with plasmid pGT-N29 containing positive selectable marker by lipofection, precipitation and electroinjection methods. After the selection, surviving cells were subjected to further molecular analysis. The stabile incorporation of the genetic constructs WAP 6xHisHGH and WAPHGH into the genome were observed.
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