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Summary The nucleotide sequence of the Bacillus licheniformis bacitracin-resistance locus was determined. The presence of three open reading frames, bcrA, bcrB and bcrC, was revealed. The BcrA protein shares a high degree of homology with the hydrophilic ATP-binding components of the ABC family of transport proteins. The bcrB and bcrC genes were found to encode hydro-phobic proteins, which may function as membrane components of the permease. Apart from Bacillus subtilis, these genes also confer resistance upon the Gram-negative Escherichia coli. The presumed function of the Bcr transporter is to remove the bacitracin molecule from its membrane target. In addition to the homology of the nucleotide-binding sites, BcrA protein and mammalian multidrug transporter or P-glycoprotein share collateral detergent sensitivity of resistant cells and possibly the mode of Bcr transport activity within the membrane. The advantage of the resistance phenotype of the Bcr transporter was used to construct deletions within the nucleotide-binding protein to determine the Importance of various regions in transport.

Aim: To analyze genetic alterations of p53 gene in Slovenian gastric cancer patients and to compare these alterations with clinicopathological parameters in order to assess the value of p53 as a prognostic factor. Methods: We analyzed the samples from 230 Slovenian patients with gastric cancer, collected between 1983 and 2001. p53 expression was evaluated immunohistochemically with DO-7 monoclonal antibody. In addition, loss of heterozigosity (LOH) and microsatellite instability (MSI) of p53 gene were evaluated, as well as its mutational status in the selected population of patients. Results: p53 expression was associated with poorer survival and it was an independent predictor in multivariate analysis, along with TNM (T – size of tumor, N – nodal involvement, M – distant metastasis) stage status. Loss of heterozigosity and microsatellite instability status did not influence survival, however we found association of loss of heterozigosity with Lauren’s (Mantel-Haenszel test, P = .004) and Ming’s (Mantel-Haenszel test, P

The Saami are regarded as extreme genetic outliers among European populations. In this study, a high-resolution phylogenetic analysis of Saami genetic heritage was undertaken in a comprehensive context, through use of maternally inherited mitochondrial DNA ( mtDNA) and paternally inherited Y-chromosomal variation. DNA variants present in the Saami were compared with those found in Europe and Siberia, through use of both new and previously published data from 445 Saami and 17,096 western Eurasian and Siberian mtDNA samples, as well as 127 Saami and 2,840 western Eurasian and Siberian Y-chromosome samples. It was shown that the “Saami motif” variant of mtDNA haplogroup U5b is present in a large area outside Scandinavia. A detailed phylogeographic analysis of one of the predominant Saami mtDNA haplogroups, U5b1b, which also includes the lineages of the “Saami motif,” was undertaken in 31 populations. The results indicate that the origin of U5b1b, as for the other predominant Saami haplogroup, V, is most likely in western, rather than eastern, Europe. Furthermore, an additional haplogroup (H1) spread among the Saami was virtually absent in 781 Samoyed and Ob-Ugric Siberians but was present in western and central European populations. The Y-chromosomal variety in the Saami is also consistent with their European ancestry. It suggests that the large genetic separation of the Saami from other Europeans is best explained by assuming that the Saami are descendants of a narrow, distinctive subset of Europeans. In particular, no evidence of a significant directional gene flow from extant aboriginal Siberian populations into the haploid gene pools of the Saami was found.

A gene-fusion expression strategy was applied for the heterologous expression of hTNF-α in A. niger AB1.13. The TNF-α gene was fused with the A. niger glucoamylase GII form as a carrier-gene, behind its transcription control and secretion signal. The protein was expressed in the cells in the form of a glucoamylase-fusion protein, but was not present in the culture medium. From the expression of two hTNF-α analogues, LK 811 (Cys95/148) and LK 802 (Cys95/148, His107/108) respectively, we concluded that oligomerisation was not the critical point for secretion of hTNF-α in A. niger, but more probably improper folding already at the stage of monomer formation, or even incorrect processing of the molecule during the secretion pathway. © Springer Verlag 2000 Chemicals/CAS: Glucan 1,4-alpha-Glucosidase, EC 3.2.1.3; Tumor Necrosis Factor-alpha

We report a large genomic deletion of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, viz.. a deletion that is frequently observed in Central and Eastern Europe. The mutation, termed CFTRdele2.3(21 kb), deletes 21,080 bp spanning introns 1-3 of the CFTR gene. Transcript analyses have revealed that this deletion results in the loss of exons 2 and 3 in epithelial CFTR mRNA, thereby producing a premature termination signal within exon 4. In order to develop a simple polymerase chain reaction assay for this allele, we defined the end-points of the deletion at the DNA sequence level. We next screened for this mutation in a representative set of European and European-derived populations. Some 197 CF patients, including seven homozygotes, bearing this mutation have been identified during the course of our study. Clinical evaluation of CFTRdele2.3(21 kb) homozygotes and a comparison of compound heterozygotes for Delta F508/CFTRdele2,3(21 kb) with pairwise-matched Delta F508 homozygotes indicate that this deletion represents a severe mutation associated with pancreatic insufficiency and early age at diagnosis. Current data show that the mutation is particularly common in Czech (6.4% of all CF chromosomes), Russian (5.2%), Belorussian (3.3%). Austrian (2.6%), German (1.5%), Polish (1.5%), Slovenian (1.5%), Ukrainian (1.2%), and Slovak patients (1.1%). It has also been found in Lithuania, Latvia, Macedonia and Greece and has sporadically been observed in Canada, USA, France, Spain, Turkey, and UK, but not in CF patients from Bulgaria, Croatia, Romania or Serbia. Haplotype analysis has identified the same extragenic CF-haplotype: XV-2c/KM. 19 “A” and the same infrequent intragenic microsatellite haplotype 16-33-13 (IVS8CA-IVS17bTA-IVSI7bCA) in all examined CFTRdele2,3(21 kb) chromosomes, suggesting a common origin for this deletion. We conclude that the 21-kb deletion is a frequent and severe CF mutation in populations of Eastern- and Western-Slavic descent.

A gene-fusion expression strategy was applied for heterologous expression of human lymphotoxin alpha (LTα) in the Aspergillus niger AB1.13 protease-deficient strain. The LTα gene was fused with the A. niger glucoamylase GII-form as a carrier-gene, behind its transcription control and secretion signals. Special attention was paid to the influence of different codon usage on secretion of protein. In the case of human tumor necrosis factor alpha (TNFα) a dramatic change of secretion has been observed when human cDNA sequence was used instead of synthetic E. coli biased codons. In the case of LTα such a change of codon usage brought improvement at the RNA level, however, no increase in the quantity of secreted protein was observed, due to the proteolitic activity of the host organism. The estimated yield of secretion of LTα from A. niger into the soya medium was 50 pg l-1 of culture. Chemicals/CAS: Glucan 1,4-alpha-Glucosidase, EC 3.2.1.3; Lymphotoxin-alpha; RNA, Messenger

Različite metode rekombinantne DNA tehnologije u dijagnostici cistične fibroze