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1.
Vavilovskii Zhurnal Genet Selektsii ; 27(7): 906-915, 2023 Dec.
Article in English | MEDLINE | ID: mdl-38213697

ABSTRACT

CHO cells are most commonly used for the synthesis of recombinant proteins in biopharmaceutical production. When stable producer cell lines are obtained, the locus of transgene integration into the genome has a great influence on the level of its expression. Therefore, the identification of genomic loci ensuring a high level of protein production is very important. Here, we used the TRIP assay to study the influence of the local chromatin environment on the activity of transgenes in CHO cells. For this purpose, reporter constructs encoding eGFP under the control of four promoters were stably integrated into the genome of CHO cells using the piggyBac transposon. Each individual transgene contained a unique tag, a DNA barcode, and the resulting polyclonal cell population was cultured for almost a month without any selection. Next, using the high-throughput sequencing, genomic localizations of barcodes, as well as their abundances in the population and transcriptional activities were identified. In total, ~640 transgenes more or less evenly distributed across all chromosomes of CHO cells were characterized. More than half of the transgenes were completely silent. The most active transgenes were identified to be inserted in gene promoters and 5' UTRs. Transgenes carrying Chinese hamster full-length promoter of the EF-1α gene showed the highest activity. Transgenes with a truncated version of the same promoter and with the mouse PGK gene promoter were on average 10 and 19 times less active, respectively. In total, combinations of genomic loci of CHO cells and transgene promoters that together provide different levels of transcriptional activity of the model reporter construct were described.

2.
Gene ; 520(2): 178-84, 2013 May 15.
Article in English | MEDLINE | ID: mdl-23481306

ABSTRACT

It is known that a lot of genes having a distinct expression pattern require the complex system of transcription regulation. The regulatory regions of such genes can include not only the 5'-flanking regions, but also other regions, particularly their intron sequences. The Drosophila melanogaster Trithorax-like (Trl) gene, encoding the GAGA protein, is one of the genes with complex expression pattern. GAGA is one of a few transcription factors that can regulate gene expression at multiple levels. The GAGA-mediated modulation of expression seems to be linked with modifications of the chromatin structure. Nowadays, the regulatory potential of the Trl 5'-flanking region that contains multiple GAGA binding sites has been analyzed, but the presence of the functionally significant elements in other Trl regions has not been examined. We found DNase I hypersensitive sites, evolutionary-conserved sequences and numerous GAGA binding sites in the second intron of the Trl gene. Interestingly, these sequences localize in two main regions of the intron in immediate proximity to preferred regions of transposon insertions. Additionally, we revealed that deletion of the intron fragment in the Trl(1-72) mutants caused an alteration of the Trl expression pattern. These results allow us to conclude that the second intron of the Trl gene contains functionally significant elements.


Subject(s)
DNA-Binding Proteins/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Introns/genetics , Regulatory Sequences, Nucleic Acid/genetics , Transcription Factors/genetics , Animals , Base Sequence , Binding Sites/genetics , Chromosome Mapping , Cloning, Molecular , DNA-Binding Proteins/metabolism , Drosophila Proteins/metabolism , Gene Expression Regulation , Regulatory Sequences, Nucleic Acid/physiology , Transcription Factors/metabolism
3.
Ontogenez ; 43(3): 163-74, 2012.
Article in Russian | MEDLINE | ID: mdl-22834131

ABSTRACT

The development of dorsal appendages of the chorion (specialized structures in the D. melanogaster egg which look like elastic tubes and ensure the breathing of the developing embryo) is an attractive model for the study of genetic mechanisms of the development of organs and tissues, whose generation is based on transformation of the epithelial tissue in the tubular structures. In the present review, we present information on genes and proteins that control the development of dorsal appendages of the chorion. We demonstrated that three signal pathways (EGFR, DPP, and NOTCH), which are combined together in a single gene network through a number of components, play a major role in the development of dorsal appendages of the chorion.


Subject(s)
Chorion/embryology , Drosophila Proteins/genetics , Drosophila melanogaster/embryology , Drosophila melanogaster/genetics , Animals , Drosophila Proteins/metabolism , Embryo, Nonmammalian , Epithelium , ErbB Receptors/genetics , ErbB Receptors/metabolism , Gene Expression Regulation, Developmental , Receptors, Invertebrate Peptide/genetics , Receptors, Invertebrate Peptide/metabolism , Receptors, Notch/genetics , Receptors, Notch/metabolism , Signal Transduction/genetics
4.
Zh Obshch Biol ; 73(3): 198-209, 2012.
Article in Russian | MEDLINE | ID: mdl-22834166

ABSTRACT

Insects is a taxon surprisingly rich with species and varieties, and its representatives are considered as the most fitted and "evolutionary successful" living things. Insects are distinguished by diversity and abundance of adaptations to environmental conditions, representatives of this class inhabit different ecological niches, they can be found practically in every corner of the Earth and, in particular, in close adjacency to man. Among them are those who man benefits from and those who man struggles against. This determines man's interest in studying peculiarities of their development as well as adaptations formed by them in the course of evolution to become more viable. In the paper, data are presented on morphological structure of respiratory systems in insect egg envelopes that ensure respiration process of developing embryo. Variability of these systems and their dependence on environmental conditions are demonstrated for different insect species. The information about genes controlling development of respiratory systems in fruit fly eggs is brought together, and occurrence of evolutionary conservative genes participating in development of such systems in other insect species is ascertained.


Subject(s)
Cell Respiration/physiology , Egg Proteins/metabolism , Insecta/growth & development , Ovum/metabolism , Animals , Chorion/metabolism , Chorion/ultrastructure , Drosophila Proteins/metabolism , Drosophila melanogaster/embryology , Drosophila melanogaster/genetics , Drosophila melanogaster/growth & development , ErbB Receptors/metabolism , Insecta/embryology , Insecta/genetics , Receptors, Invertebrate Peptide/metabolism , Receptors, Notch/metabolism
5.
Comput Biol Chem ; 35(6): 363-70, 2011 Dec 14.
Article in English | MEDLINE | ID: mdl-22099633

ABSTRACT

The transcription factor GAGA, encoded by the gene Trl, controls expression of many Drosophila melanogaster genes. We have compiled the presently largest sample (120 sites) of published nucleotide sequences with experimentally confirmed binding to GAGA protein. Analysis of the sample has demonstrated that despite an apparent structural diversity of the GAGA sites, they fall into four distinct groups, namely, (1) the sites containing two GAG trinucleotides with no more than one nucleotide substitution in each and separated by spacers with a length of 1 or 3 nucleotides (GAGnGAG and GAGnnnGAG); (2) the sites containing a single GAGAG motif; (3) (GA)(3-9) microsatellite repeats; and (4) the sites corresponding to three and more direct repeats of GAG trinucleotide homolog and its inverted repeats separated by spacers of various lengths. Using the software package SITECON, the methods were elaborated for recognizing the sites of GAGnGAG (method 1) and GAGnnnGAG (method 2) types in DNA sequences. Experimental verification confirmed the ability to interact with the GAGA factor for 72% of the sites predicted using method 1 and 94.5% of the sites predicted by method 2. Application of the experimentally verified methods to analyzing the localization of potential GAGA binding sites in the target genes of this transcription factor has demonstrated that the 5'-untranslated regions (5'UTRs) and first introns are enriched for these sites (two-threefold relative to the average occurrence frequency in the D. melanogaster genome) as compared with a moderate enrichment (not exceeding 1.5-fold) of promoter regions (-4000/+200 bp or -1000/+100 bp).


Subject(s)
DNA-Binding Proteins/metabolism , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Genes, Insect/genetics , Response Elements/genetics , Transcription Factors/metabolism , 5' Untranslated Regions/genetics , Animals , Base Sequence , Binding Sites , DNA/genetics , Drosophila Proteins/metabolism , Introns/genetics , Protein Conformation , Recombinant Fusion Proteins/genetics
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