Variability of the Expression of Reference Genes in Samples of Human Olfactory Epithelium.

We analyzed variability of the expression of three reference genes in biopsy samples of the olfactory epithelium obtained from healthy volunteers. The expression of B2M, HPRT1, and CASC3 genes was analyzed by real-time PCR. The pairs of genes B2M-HPRT1 and B2M-CASC3 were found to possess minimum individual variability of expression and can be reliable candidates for the reference genes in analysis of gene expression in neural cells.

[DNA structural features on the borders of ERBB2 amplicons in breast cancer].

In 25-30% of breast cancer tumor cases amplification of chromosome fragment around the ERBB2 underlies the increased expression of genes adjacent to ERBB2. Increased expression of genes within ERBB2-containing am- plicons may have an impact not only on the growth and development of the tumor, but on the sensitivity of the tumor to different types of anti-cancer therapies. The initial cause of amplification and the exact borders of ERBB2-amplified chromosome fragment are still not completely characterized. No specific DNA sequences were found on the junction regions at intrachromosomal DNA amplification. We hypothesized that amplification borders can be specified by DNA structural peculiarities rather than the particular DNA sequence. This study focused on the mapping of ERBB2 amplification borders in breast cancer and the search for unusual structural features of DNA at the borders of the identified amplicons. The copy number of 10 genes adjacent to ERBB2 were evaluated by real time PCR in 162 breast cancer samples. Several ERBB2-containing amplicons of various lengths were revealed. In the majority of the analyzed samples, the borders of these amplicons were located within ZNFNIA3 and RARA genes. A bioinformatics analysis of the nucleotide sequence peculiarities around ERBB2 gene revealed the presence of AT-rich DNA regions with high degree of flexibility. These regions were able to form stable secondary structures. Positions of these sites strongly coincide with the positions of the ERBB2-containing amplicon borders found in real time PCR experiments. On the base of results obtained one can suppose that the structural features of DNA are involved in the formation of ERBB2-containing amplicon borders in breast cancer cells and the data are of importance for understanding the mechanisms of oncogene amplification.

[Detection of nonsense and frame shift mutations in human BRCA1 gene using new plasmid vector pPhoA-frame].

The technique for the detection of frame shift and nonsense mutations in BRCA1 gene was suggested. The technique presumes the construction of recombinant plasmids where the tested DNA fragment placed in-frame with alkaline phosphatase gene of Escherichia coli (phoA). A plasmid pPhoA-frame was constructed for such analysis, the plasmid contains DNA fragment coding for alkaline phosphatase of E. coli. Synthetic DNA fragment with BglII, StuI, ApaI and SacII sites was inserted into the DNA fragment coding for alkaline phosphatase of E. coli between Ala218 and Gly219 codons to facilitate the cloning of BRCA1 gene fragments. Occurrence of the frame shift or nonsense mutation in the tested DNA fragment can be detected after transformation of E. coli by the recombinant plasmid containing the tested fragment. E. coli colonies with the newly constructed recombinant plasmids are plated out on the indicator agar. In the case of frame shift or nonsense mutation the colonies are not colored, DNA fragments without such mutations result in the formation of the blue colonies.

[Guest peptide insertions into the surface loops of alkaline phosphatase].

The paper presents the description of the experiments in line with the rational concept for the "safe" insertion of guest polypeptides into the alkaline phosphatase with the minimal influence of the inserts on the enzymatic activity of the protein. Several approaches are described in the paper for the surface loop length estimation and two loops were used as the sites for guest peptides introduction by gene engineering technique. The experiments clearly demonstrate that insertions of several peptides after Ala218 of alkaline phosphatase (the site was selected by loop length analysis) do not block the activity of the enzyme. According the experimental data, the selection of the loops for the guest peptides insertion can be defined by the mobility of backbone dihedral angles during molecular dynamics simulation of alkaline phosphatase. The paper demonstrates the possibility to use in practice the estimation of loop length based on the mobility of backbone dihedral angles during molecular dynamics simulation. Indeed, it looks that the proteins with new features can be constructed by the introduction of new polypeptides into the enzymatically active proteins.

[Prevalence of mutations BRCA1 5382insC, and CHEK2 1100delC in the population of Siberian region].

Frequencies of the 538insC mutation in the BRCA1 gene and the 1100delC mutation in the CHEK2 gene were compared in the group of breast cancer patients and the large-scale sample, consisting of 7920 DNA specimens from healthy residents of the city of Novosibirsk. Higher frequencies of these mutations in the patient group compared to the control sample (1.95 versus 0.25% for BRCA1 5382insC, and 1.78 versus 0.40% for CHEK2 1100delC) were observed, pointing to their association with susceptibility to breast cancer (OR = = 7.86, 95% CI 3.51-17.30 and OR =4.46, 95% C1 2.04-9.49, respectively).

[Comparison of different methods of molecular-genetic analysis of somatic mutations in K-ras gene in patients with colorectal cancer].

Two approaches to somatic point mutations in 12 and 13 codones of K-ras gene were analyzed: PCR/SSCP/ACRS/sequencing and allele-specific PCR in the real-life regimen (Russian set "KRAS-7M"). The comparison was carried out on 62 examples of genomic DNA extracted from frozen colon carcinomas, which underwent manual dissection. The results obtained in two attempts were consistent in 95,2% (N=59). Specificity and sensitivity of K-ras mutations detection using "KRAS-7M" set were 100 and 96,4% respectively, and 94,1 and 100% respectievly using PCR/SSCP/ACRS/automatic sequencing. False positive results were absent when detecting with "KRAS-7M" and accounted for 2 cases (5,9%) when using PCR/SSCP/ ACRS/automatic sequencing. The only false negative response (3,6%) was obtained analyzing mutations using "KRAS-7M".

[Detection of 5382insC mutation in human BRCA1 gene using fluorescent labeled oligonucleotides].

Analysis of genetic predisposition to cancer can provide valuable information for early cancer detection or even prevention. Insertion 5382insC in BRCA1 gene is the most frequent mutation among those associated with high risk of breast cancer in women of East European origin. The method of 5382insC detection using fluorescent labeled allele specific oligonucleotides in Duplex Scorpion format has been developed. The method can be used in real-time PCR conditions as well as in conditions of end-point fluorescence measurement followed regular PCR. The adequacy of the method was demonstrated in the study of 5382insC mutation frequency in breast cancer patients. 564 samples of genomic DNA from breast cancer patients were genotyped. Eleven patients (1.95%) were found to be heterozygous for BRCA1 5382insC mutation. 5382insC allele frequency in breast cancer patients group was 0.0098. The method can be used as in clinical practice to determine individuals of a high risk of breast cancer, as in wide-scale population studies.

Comparison of SYBR Green I and TaqMan real-time PCR formats for the analysis of her2 gene dose in human breast tumors.

We compared two technologies of real-time PCR (with the use of fluorescent SYBR Green I dye and specific TaqMan probe) for quantification of the dose of her2 gene in breast tumors. The maximum increase in the gene dose in TaqMan and SYBR Green I analyses was 10- and 5-fold, respectively. In was found that TaqMan and SYBR Green I technologies allow detection of the matrix in amounts corresponding to 1-100 and 2.5-40.0 ng genomic DNA, respectively. Tenfold increase in the gene dose leads to incorrect evaluation of multiplication ratio in the SYBR Green I analysis. These results suggest that TaqMan technology is more preferable for correct evaluation of her2 gene dose.

[Frequency of the 735G --> A mutation of the 5'-splice donor site of intron 14 of the dihydropyrimidine dehydrogenase gene (DPYD) in residents of Novosibirsk region (Russia) as revealed with fluorescent oligonucleotides].

A simple method was developed for end-point fluorescence detection of the 735G --> A mutation of the 5'-splice donor site of intron 14 of the dihidropyrimidine dehydrogenase gene (DPYD). The method was based on allele-specific PCR with duplex Scorpion primers. The genotyping results obtained by the fluorescent endpoint PCR technique completely coincided with the results obtained by allele-specific PCR with amplicon detection in agarose gel. Genotyping was performed in 291 DNA samples from residents of Novosibirsk region (Russia), and two heterozygotes (0.69%) were detected.

[Her-2 gene dosage analysis in human breast cancer by PCR with internal standard].

PCR assay with internal control was developed to quantify the her-2 gene dosage in human breast cancer tumor samples. Recombinant plasmid with a fragment of the her-2 gene containing the fragment of T7 phage DNA was designed especially to be used as an internal control in the PCR her-2 gene dosage assay. PCR conditions were optimized for the simultaneous usage of two templates--human genomic DNA and DNA of recombinant plasmid (internal control). The ability of the technology to discriminate a twofold increase of the her-2 gene dosage was demonstrated. Increased level of her-2 gene amplification was observed in 6 of 38 samples investigated. This new simple rapid assay can be an alternative to fluorescence in situ hybridization (FISH) and immunochemistry (ICH) tests for the detection of her-2 amplification in human tumors. This technique may be a useful tool for large randomized, prospective cooperative group trials and may support selection of optimal therapy for breast cancer patients in the future.

[Possibility of using fusion proteins for detection of nonsense mutations and frame-shift mutations in BRCA1 gene]. / Izuchenie vozmozhnosti ispol'zovaniia slitnykh belkov dlia detektsii nonsens-mutatsii i mutatsii sdviga ramki v gene BRCA1.

The aim of this study was to evaluate the possibility of detecting nonsense and frame-shift mutations in exon 11 of brca1 gene by constructing fusion open reading frame (ORF) "exon 11 ORF-alpha-peptide of beta-galactosidase". The ability/inability of this newly constructed ORF to cause alpha-complementation in E. coli delta M15gal cells transformed by the plasmid with the ORF may reflect the absence/presence of nonsense and frame-shift mutations in the studied fragment. A single ORF fragment of exon 11 of brca1 gene--LacZ' gene was designed in pGEN7Zf plasmid, the plasmid was shown to cause Lac+ phenotype in E. coli delta M15gal. Four frame-shift deletion mutations were introduced into exon 11 sequence in the plasmid. Surprisingly, the frame-shift deletion mutations did not influence the ability of plasmids to induce Lac+ phenotype in E. coli delta M15gal in 3 cases and only one deletion mutation resulted in inability of the plasmid to form Lac+ phenotype in E. coli delta M15gal. We suppose that the phenomenon can be explained by the alpha-peptide translation reinitiation from inframe ATG codons situated within the exon 11 sequence. Seven inframe ATG sequences were found in exon 11, at least two in-frame ATG-containing fragments were demonstrated to cause reinitiation. On the other hand, the only deletion mutation resulted in inability of the plasmid to form Lac+ phenotype in E. coli delta M15gal did not leave LacZ' in-frame ATG in econ 11 sequence. We conclude that it is possible to detect frame-shift mutations by in-frame cloning with the LacZ' reporter gene, but this possibility is strongly impeded by the reinitiation of alpha-peptide translation from the in-frame ATG codons within the studied sequence.

Phenobarbital-dependent protein binding to Barbie box-like sequences in the coding region of cytochrome P450BM-3 gene from Bacillus megaterium.

Phenobarbital-dependent protein binding was shown to occur to DNA fragments from the coding region of the cytochrome P450BM-3 gene from Bacillus megaterium. Incubation of the DNA fragments from the coding region of the gene with total cell extract from Bacillus megaterium revealed two DNA regions with protein-binding capacity: +237/+318 and +319/+425 considering 'O' as the start of cytochrome P450BM-3 translation. DNaseI footprint analysis of the fragment +319/+425 with the total cell extract showed that some protein(s) protected DNA stretches from the position +373 up to the position +389 on the transcribed strand and from the position +378 up to the position +398 on the non-transcribed strand. DNaseI footprint analysis of the fragment +237/+318 revealed the protection in the region +262/+277 on the non-transcribed strand. Three regions protected by cell extract protein(s) from DNaseI hydrolysis (+262/+277, +373/+389 and +378/+398) appeared to be strongly homologous to the Barbie box sequence. Barbie-box-like sequences were found in the majority of regulatory regions of phenobarbital-inducible genes whose regulatory sequences had been reported (Fulco et al., 1994). Our results suggest that a functional role of Barbie box sequence takes place not only in regulatory but also in the coding region of the gene. In line with that hypothesis we analyzed all cytochrome P450 genes in respect to the presence of Barbie box-like sequences in their coding parts. At least one cytochrome P450 gene (CYP6A1, phenobarbital-inducible gene from Musca domestica) was shown to contain Barbie box sequence in the coding part of the gene.

[Evaluation of allele frequency of the hypervariable locus D17S30 in a sample of populations of European origin from a number of Siberian regions]. / Otsenka chastot vstrechaemosti allelei gipervariabel'nogo lokusa D17S30 v vyborke naseleniia evropeiskogo proiskhozhdeniia iz riada regionov Sibiri.

Frequencies of alleles of the polymorphic locus D17S30 were estimated in a sample of 121 unrelated Siberian inhabitants of European origin. The main method of study was polymerase chain reaction (PCR). The consecutive use of two pairs of primers allowed increasing of the specificity and sensitivity of the amplification reaction. The results of this study may be of theoretical interest for studies in population genetics. They may also find practical application in forensic medical examination and in the diagnosis of some genetic abnormalities.

[The use of the polymerase chain reaction method for the analysis of polymorphic sections of the human genome in performing forensic medical expertise on material evidence]. / Primenenie metoda polimeraznoi tsepnoi reaktsii dlia analiza polimorfnykh uchastkov genoma cheloveka pri provedenii sudebno-meditsinskoi ékspertizy veshchestvennykh dokazatel'stv.

The method of expert evaluation is described which was carried out to define the appurtenance of seminal spots to one of two suspected subjects. Biological sample appurtenance was identified using genetic markers by analysis of allele status of loci containing variable numbers of tandem repeats. Two loci were analyzed, ApoB and IL-6. To determine the studied loci alleles polymerase chain reaction with the DNA isolated from material evidences used as the matrix was used as well as agarose gel electrophoresis. Analysis helped determine appurtenance of seminal spots to suspected subject V.

Protein-binding DNA regions inside and in the vicinity of the cytochrome P450bm-3 gene from Bacillus megaterium.

Protein-binding DNA-regions inside and in the vicinity of the phenobarbital-inducible P450bm-3 gene from Bacillus megaterium were characterized by gel-retardation technique and foot-printing analysis. Regions with induction-dependent protein binding capacity were shown to be situated in -279bp/-215bp, -215bp/+83bp and +236bp/+425bp fragments. Precise localization of DNA-protein contacts was established by foot-printing analysis for region -215bp/+83bp. The data obtained are discussed in line with previously reported information on the regulatory regions of various phenobarbital-regulated genes.

[Restriction fragment length polymorphism of the apolipoprotein A-I gene among inhabitants of Novosibirsk]. / Polimorfizm dlin restriktsionnykh fragmentov gena apolipoproteina A-I sredi zhitelei g. Novosibirska.

Restriction fragments' length polymorphism in the region of apolipoprotein A-I (apo A-I) gene was investigated in Novosibirsk (Siberia, USSR) population. Correlation between PstI apo A-I alleles (2,2 kb-P1; 3,3 kb-P2) and total cholesterol, triglycerides, high density polyproteins cholesterol, and apolipoprotein A-I level was analysed. A tendency to increase in cholesterol index of atherogenicity and to decrease in high density lipoproteins cholesterol as well as apolipoprotein A-I level was shown to occur for P1P2 genotype patients.

[Expression of synthetic human angiogenin gene in E. coli in cleaved beta-galactosidase-angiogenin protein]. / Ekspressiia sinteticheskogo gena angiogenina cheloveka i E. coli v sostave slitogo belka beta-galaktozidaza-angiogenin.

A synthetic gene coding for human angiogenin was cloned in pUR290 plasmid in frame with beta-galactosidase, both parts of the resultant fused protein being joined through an Asp-Pro sequence. The fused protein, synthesised in E. coli cells upon IPTG induction and isolated as inclusion bodies, possessed angiogenic activity on the chick chorioallantois membrane and was cleaved upon acid treatment to yield free angiogenin.