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Biomedical subjects

T L Azhikina

Publications and source records attributed to T L Azhikina.

16 recordsLinked to original sources
PubMed2005

Study of tissue-specific CpG methylation of DNA in extended genomic loci.

Modern approaches for studies on genome functioning include investigation of its epigenetic regulation. Methylation of cytosines in CpG dinucleotides is an inherited epigenetic modification that is responsible for both functional activity of certain genomic loci and total chromosomal stability. This review describes the main approaches for studies on DNA methylation. Under consideration are site-specific approaches based on bisulfite sequencing and methyl-sensitive PCR, whole-genome approaches aimed at searching for new methylation hot spots, and also mapping of unmethylated CpG sites in extended genomic loci.

Animals↗
PubMed2001

Full-sized HERV-K (HML-2) human endogenous retroviral LTR sequences on human chromosome 21: map locations and evolutionary history.

One of the evolutionary mechanisms for acquisition of novel functional sequences can be domestication of exogenous retroviruses that have been integrated into the germ line. The whole genome mapping of such elements in various species could reveal differences in positions of the retroviral integration and suggest possible roles of these differences in speciation. Here, we describe the number, locations and sequence features of the human endogenous retrovirus HERV-K (HML-2) long terminal repeat (LTR) sequences on human chromosome 21. We show that their distribution along the chromosome is not only non-random but also roughly correlated with the gene density. Amplification of orthologous LTR sites from a number of primate genomes produced patterns of presence and absence for each LTR sequence and allowed determination of the phylogenetic ages and evolutionary order of appearance of individual LTRs. The identity level and phylogenetic age of the LTRs did not correlate with their map locations. Thus, despite the non-random distribution of LTRs, they have apparently been inserted randomly into the chromosome relative to each other. As evidenced in previous studies of chromosomes 19 and 22, this is a characteristic of HERV-K integration.

Animals↗
PubMed1999

[Identification and mapping on human chromosome 19 of chromosome-specific repeated element preferentially binding with nuclear matrix].

From a library of sequences binding preferentially to nuclear matrix (matrix attachment regions, MARs), a fragment of about 300 bp in length (CEA (carcinoembryonic antigen)-MAR) was isolated and characterized. The CEA-MAR sequence was found in more than ten loci of chromosome 19 containing elements similar to genes of the CEA family. No sequences of this group were found on other human chromosomes. Two CEA-MAR-containing loci were sequenced, and sequences for another seven loci were found in GenBank. A comparative analysis of CEA-MARs and the flanking sequences is reported. Based on the sequence of the CEA-containing chromosome 19 loci, a hypothetical model of the domain structure of a 2-Mb chromosome region was constructed and the mutual arrangement of CEA-MARs and genes of CEA family was elucidated. The CEA-MARs were located 5-20 kb downstream of the CEA genes. These results suggest that the duplication unit of the CEA family may coincide with chromatin domains containing these genes.

Base Sequence↗
PubMed1997

[Structural characteristics of four long terminal repeats (LTR) of human endogenous retroviruses and features of their integration sites].

Four LTR-containing regions of human chromosome 19 were sequenced by the primer walking technique using strings of short oligonucleotides tightly bound to the template. A comparative and evolutionary analysis of sequences homologous to human endogenous retroviruses (HERV) was performed, and the prototypes of the LTRs were determined. Analysis of the chromosome 19 sequences adjacent to LTR revealed that LTRs of HERV-K share a common location with other retroposons.

Base Sequence↗
PubMed1996

Determination of the patterns of hexanucleotide distribution along DNA by electron microscopy.

Hexanucleotides containing modified bases (5-methylcytosine and 2-aminoadenine instead of cytosine and adenine) with increased capacities to bind complementary DNA sequences were used to map the distribution of their complementary sequences in a DNA target using electron microscopy. The method used hexamers to initiate DNA polymerase directed DNA synthesis at complementary sequences along a template. DNA synthesis was limited to about 200 residues by using a low concentration of deoxynucleotide precursors. During DNA synthesis a biotin ligand was incorporated to facilitate the subsequent binding of an electron-dense label (streptavidin-labeled colloidal gold particles) into newly synthesized DNA chains. The method can be implemented with commercially available products. The results demonstrate that the approach can be used to compare primary structural features of DNA fragments. The principles of the method can be adapted to a variety of single molecule detection methods such as electron, scanning tunneling, or atomic force microscopies.

Animals↗
PubMed1991

[Isolation of recombinant interleukin-3 produced by E. coli].

A synthetic gene coding for human interleukin-3 (hIL3) was cloned in the plasmid pTE2IL3, the gene expression being controlled by the phage fd PVIII promotor and the phage T7 gene 10 translational enhancer. Under constitutive biosynthesis conditions in E. coli, the accumulation of recombinant hIL3 (in the inclusion bodies) was up to 30-40% of the total cell protein. An effective procedure of the hIL3 isolation is suggested. The hIL3 was solubilized in 5 M guanidinium chloride, renaturated and purified to homogeneity by a single chromatographic step. The protein's yield was 34 mg/g wet cells. The isolated hIL3 showed a specific biological activity.

Chromatography, Gel↗
PubMed1989

Insertion of short hepatitis virus A amino acid sequences into poliovirus antigenic determinants results in viable progeny.

In an infectious poliovirus cDNA construct, the determinant encoding antigenic epitope N-Ag1 (in a loop located between two beta-strands in poly-peptide VP1) was altered by site-directed mutagenesis, to be partially similar with the determinants for presumptive epitopes in polypeptides VP1 or VP3 of hepatitis A virus (HAV). The modified constructs proved to be infectious. However, another construct, in which the same locus encoded a 'nonsense' and a relatively hydrophobic amino acid sequence, exhibited no infectivity. These data showed the feasibility of the insertion of foreign sequences in a specific antigenically active locus of the poliovirus icosahedron, and suggest some limitations with respect to the sequences to be 'transplanted'.

Amino Acid Sequence↗
PubMed1989

Effective method for obtaining long nucleotide chains on partially complementary templates. Processed bovine gamma-interferon gene obtained from human gamma-interferon gene.

The method of obtaining the bovine gamma-interferon gene by means of simultaneous multidirected mutagenesis of the human gamma-interferon gene is presented. The first strand of the bovine gamma-interferon gene was obtained by ligation of synthetic oligonucleotides, using the cDNA of human gamma-interferon, cloned in the single-stranded phage M13mp19 as a template. The second strand was synthesized using a large fragment of E. coli DNA-polymerase I. The double-stranded gene was then treated by restriction nucleases and cloned in a pUC-18 derived vector. The primary structure was confirmed by sequencing.

Animals↗
PubMed1989

[Isolation of the porcine alpha-interferon gene using simultaneous directed multipoint mutagenesis].

A method of obtaining the pig alpha-interferon gene by means of simultaneous multidirected mutagenesis of the human alpha 2-interferon gene is presented. Nucleotide homology between these genes is 80.4%. Fourteen synthetic oligonucleotides forming a pig alpha-interferon gene's strand were ligated on a single-stranded template, carrying cDNA of the human alpha 2-interferon gene. The obtained DNA fragment was cloned in the single-stranded or double-stranded form. It was found that the method does not affect the cloning efficiency. The primary structure of the gene was confirmed by sequencing.

Amino Acid Sequence↗
PubMedDate not supplied

[Deletion-insertion differences between the genomes of Mycobacterium tuberculosis highly virulent strain HN878 and less virulent strain CDC1551].

The modfied version of the method of subtracting hybridization for full-genome comparison of M. tuberculosis strain HN878, capable of inducing a nonpulmonary form of tuberculosis, with strain CDC1551 causing tuberculosis--with classical pulmonary symptoms. The clone library of differential fragments, responsible for differences between genome HN878 and genome CDC1551, was created. As the result of the structural analysis carried out in this study, the set of differential fragments was divided into. three main groups: new places of the integration of transposon IS6110; fragments resulting from the transformations of other repeating sequences of the genome; long unique nucleotide sequences, absent in genome CDC1551. Genome transformations may be a highly important factor of the modulation of the phenotypical properties of the pathogen, including those which jointly determined its virulence, and also served as valuable molecular genetic markers for diagnostic purposes.

Base Sequence↗