Effect of the bovine TG5 gene polymorphism on milk- and meat-producing ability.

AIM: This study aimed to determine the effect of thyroglobulin (TG5) gene polymorphism on milk and meat productivity in the various cattle breeds currently bred in the Republic of Bashkortostan. MATERIALS AND METHODS: The test was performed on dairy cattle of Black-and-White, Bestuzhev, and Simmental breeds, and meat cattle of Hereford and limousine breeds. The purpose of the test was to search for associations between the polymorphic alleles of the thyroglobulin (TG5) gene and economically useful traits. RESULTS: All studied breeds showed a frequency predominance of the TG5C allele (from 0.56 to 0.71). A clear trend of an effect of the genotypes of the TG5 gene on milk-productivity indicators was revealed; cows with the TG5TT genotype have the highest milk yield and fat content in milk. The milk of cows of Bestuzhev and Simmental breeds that possessed this genotype was also characterized by higher protein content. CONCLUSION: We identified an effect of the polymorphism of the TG5 gene in the Hereford and limousine breeds on fat metabolism intensity indicators, such as fat output and fat content, in the longissimus muscle and in the general sample of ground beef.

The CCDC55 couples cannabinoid receptor CNR1 to a putative DISC1 schizophrenia pathway.

PURPOSE: Our previous study suggested that the coiled coil domain-containing 55 gene (CCDC55), also named as NSRP1 (nuclear speckle splicing regulatory protein 1 (NSRP1)), was encompassed in a haplotype block spanning over the serotonin transporter (5-HTT) gene in patients with schizophrenia (SCZ). However, the neurobiological function of CCDC55 gene remains unknown. This study aims to uncover the potential role of CCDC55 in SCZ-associated molecular pathways. EXPERIMENTAL DESIGN: Using molecular cloning, sequencing and immune blotting to identify basic properties, yeast two-hybrid screening and glutathione S-transferase (GST) pull-down assay to test protein-protein interaction, and confocal laser scanning microscopy (CSLM) to show intracellular interaction of proteins. PRINCIPAL FINDINGS: (i) CCDC55 is expressed as a nuclear protein in human neuronal cells; (ii) Protein-protein interaction analyses showed CCDC55 physically interacted with Ran binding protein 9 (RanBP9) and disrupted in schizophrenia 1 (DISC1); (iii) CCDC55 and RanBP9 co-localized in the nucleus of human neuronal cells; (iv) CCDC55 also interacted with the cannabinoid receptor 1 (CNR1), and with the brain cannabinoid receptor-interacting protein 1a (CNRIP1a); (v) CNR1 activation in differentiated human neuronal cells resulted in an altered RanBP9 localization. CONCLUSION: CCDC55 may be involved in a functional bridging between the CNR1 activation and the DISC1/RanBP9-associated pathways.

[20-year experience with laserofluorescent diagnostics in clinical microbiology].

Results of rapid laser-assisted identification of microorganisms for diagnostics of microbial processes based on auto-fluorescence effect in bacteria-containing materials are summarized. It is proposed to use the auto-fluorescence technique for express diagnostics of pyoinflammatory diseases, evaluation of microflora conditions (eubiosis, dysbiosis) and sensitivity to antibiotics, monitoring and prognostication, assessment of the quality of antibiotic therapy. Priority in the development of this medical technology for laserofluorescent diagnostics and its practical application is protected by 15 patents.

Initial isolation and analysis of the human Kv1.7 (KCNA7) gene, a member of the voltage-gated potassium channel gene family.

A novel human potassium channel gene was identified and isolated. The maximal open reading frame encodes a protein of 456 amino acids. The predicted product exhibits 91% amino acid identity to the murine voltage-gated potassium channel protein Kv1.7 (Kcna7), which plays an important role in the repolarization of cell membranes. Based on the high similarity, the human gene has been classified as the ortholog of the mouse Kcna7 and given the name Kv1.7 (KCNA7). A structural prediction identified a pore region characteristic of potassium channels and six membrane-spanning domains. Northern expression analysis revealed the gene is expressed preferentially in skeletal muscle, heart and kidney. However, it is expressed at lower level in other tissues, including liver. A single mRNA isoform was observed, with a size of approximately 4.5 kb. Using fluorescence in situ hybridization, the gene was mapped to chromosomal band 19q13.4 (269.13 cR(3000)). A genomic sequence was identified in the database from this region, and the KCNA7 gene structure determined. Computational analysis of the genomic sequence reveals the location of a putative promoter and a likely muscle-specific regulatory region. Initial comparison to the published murine Kcna7 cDNA suggested a different N-terminal sequence for the human protein, however, further analysis suggests that the original mouse sequence contained an error or an unusual polymorphism.

Isolation and chromosomal localization of a new human retinoblastoma binding protein 2 homologue 1a (RBBP2H1A).

Using a NotI linking clone NR-025 as a probe, we isolated a novel putative member of the RB binding protein family, namely a human retinoblastoma binding protein 2 homologue (RBBP2H1A). The maximal open reading frame encodes a protein of 1681 amino acids. Homology analysis indicated that the predicted product has an overall 56% amino acid identity to RBBP2, which plays an important role in RB tumor suppressor regulation. Many extended regions are 100% identical in amino acids sequences. The degree of nucleotide identity is lower. The structure prediction analysis identified three DNA-binding zinc finger domains and two bipartite nuclear localization signals. Northern expression analysis revealed expression in all tissues; however, the level of expression significantly varied between tissues. The highest level of expression was detected in testis and the lowest in skeletal muscle. The mRNA sizes corresponding to two major products are around 6kb and 7kb. Using fluorescence in situ hybridization, we mapped the gene to chromosomal band 1q32.1.

NotI clones in the analysis of the human genome.

Not I linking clones contain sequences flanking Not I recognition sites and were previously shown to be tightly associated with CpG islands and genes. To directly assess the value of Not I clones in genome research, high density grids with 50 000 Not I linking clones originating from six representative Not I linking libraries were constructed. Altogether, these libraries contained nearly 100 times the total number of Not I sites in the human genome. A total of 3437 sequences flanking Not I sites were generated. Analysis of 3265 unique sequences demonstrated that 51% of the clones displayed significant protein similarity to SWISSPROT and TREMBL database proteins based on MSPcrunch filtering with stringent parameters. Of the 3265 sequences, 1868 (57.2%) were new sequences, not present in the EMBL and EST databases (similarity < or =90%). Among these new sequences, 795 (24.3%) showed similarity to known proteins and 712 (21.8%) displayed an identity of >75% at the nucleotide level to sequences from EMBL or EST databases. The remaining 361 (11.1%) sequences were completely new, i.e. <75% identical. The work also showed tight, specific association of Not I sites with the first exon and suggest that the so-called 3' ESTs can actually be generated from 5'-ends of genes that contain Not I sites in their first exon.

DNA-encoding enzymatically active HIV-1 reverse transcriptase, but not the inactive mutant, confers resistance to experimental HIV-1 challenge.

The present study was undertaken to examine the immunogenicity of a single plasmid DNA representing the reverse transcriptase (RT) of HIV-1. Plasmids containing the enzymatically active RT as well as a mutated nonenzymatically active RT with nucleotide (nt)-binding motifs of YMDD and YMLL, respectively, were used to immunize mice. Both constructs induced similar good antibody and T cell responses, with a tendency towards antibody directed to peptides representing the active and mutated sites. Immunized mice were challenged with a murine pseudotype HIV-1/MuLV infected spleen cells. Seven out of 10 mice immunized with RT had no recoverable HIV-1, while 10 individuals immunized with the RT mutant and all the 18 controls had high levels of recoverable HIV-1. This indicates that mutation of RT reduces the desired immunogenicity.

Analysis of NotI linking clones isolated from human chromosome 3 specific libraries.

We have partially sequenced more than 1000 NotI linking clones isolated from human chromosome 3-specific libraries. Of these clones, 152 were unique chromosome 3-specific clones. The clones were precisely mapped using a combination of fluorescence in situ hybridization (FISH) and hybridization to somatic cell or radiation hybrids. Two- and three-color FISH was used to order the clones that mapped to the same chromosomal region, and in some cases, chromosome jumping was used to resolve ambiguous mapping. When this NotI restriction map was compared with the yeast artificial chromosome (YAC) based chromosome 3 map, significant differences in several chromosome 3 regions were observed. A search of the EMBL nucleotide database with these sequences revealed homologies (90-100%) to more than 100 different genes or expressed sequence tags (ESTs). Many of these homologies were used to map new genes to chromosome 3. These results suggest that sequencing NotI linking clones, and sequencing CpG islands in general, may complement the EST project and aid in the discovery of all human genes by sequencing random cDNAs. This method may also yield information that cannot be obtained by the EST project alone; namely, the identification of the 5' ends of genes, including potential promoter/enhancer regions and other regulatory sequences

Identification of new tumor suppressor genes based on in vivo functional inactivation of a candidate gene.

As a step towards developing a new functional test for the identification of tumor suppressor genes, human wild type and mutant RB genes were expressed in the mouse A9 fibrosarcoma cell line under the transcriptional regulation of the tetracycline repressor using two new vectors: pLNCtTA and pETI. Following passage of the transfectants in immunodeficient SCID mice, the wild type RB gene was deleted or functionally inactivated already after the first passage in all 20 tumors tested. In contrast, a non-functional mutant RB gene was maintained in all 10 tumors studied. These results suggest that tests for the identification of tumor suppressor genes may be based on their functional inactivation in vivo, rather than on growth suppression.

Somatic mutation and homozygous deletion of PTEN/MMAC1 gene of 10q23 in renal cell carcinoma.

We studied chromosome 10q loss of heterozygosity and PTEN/MMAC1 gene inactivation in renal cell carcinoma (RCC). Fifty-four cases of RCCs were analysed by three 10q RFLP markers. Forty one of them were heterozygous for at least one of the markers, of which fourteen showed LOH (34%). Six tumors which showed 10q deletion for RFLP markers and six randomly selected tumors without RFLP LOH were included in an extended study of 10q by eight microsatellite markers. Eight of these cases showed LOH with two smallest deleted regions (SRO) at 10q23 delineated by markers D10S541 and D10S579 while the other distal SRO is between markers D10S587 and D10S212 at 10q25-26. The five tumors with LOH covering 10q23 were selected for mutation analysis of PTEN/MMAC1 gene. One tumor without LOH of 10q23 was selected as a control. Using direct sequencing of nine exons, we found three different base pair changes in three tumors with LOH. Nine RCC cell lines were analysed for PTEN/MMAC1 gene inactivation. One homozygous deletion was detected in the cell line UOK147. No expression of PTEN/MMAC1 gene was detect by RT-PCR in the cell line UOK 147.

A cosmid and cDNA fine physical map of a human chromosome 13q14 region frequently lost in B-cell chronic lymphocytic leukemia and identification of a new putative tumor suppressor gene, Leu5.

B-cell chronic lymphocytic leukemia (B-CLL) is a human hematological neoplastic disease often associated with the loss of a chromosome 13 region between RB1 gene and locus D13S25. A new tumor suppressor gene (TSG) may be located in the region. A cosmid contig has been constructed between the loci D13S1168 (WI9598) and D13S25 (H2-42), which corresponds to the minimal region shared by B-CLL associated deletions. The contig includes more than 200 LANL and ICRF cosmid clones covering 620 kb. Three cDNAs likely corresponding to three different genes have been found in the minimally deleted region, sequenced and mapped against the contigged cosmids. cDNA clone 10k4 as well as a chimeric clone 13g3, codes for a zinc-finger domain of the RING type and shares homology to some known genes involved in tumorigenesis (RET finger protein, BRCA1) and embryogenesis (MID1). We have termed the gene corresponding to 10k4/13g3 clones LEU5. This is the first gene with homology to known TSGs which has been found in the region of B-CLL rearrangements.

A micro-dissection approach for isolation of NotI linking clones from regions frequently deleted in RCC and SCLC.

We demonstrate that micro-dissection can be used for isolating NotI linking clones from the human 3p21-pter region. This approach is an improvement to positional cloning techniques, since NotI linking clones are directly linked with genes.