Biomedical subjects
Publications and source records attributed to R Komel.
The allele distribution of the systems DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385 and YCAII were investigated in a sample of 121 unrelated males from Slovenia
In order to apply a set of 14 short tandem repeat (STR) loci in parentage testing, we performed a population genetic study on a sample of 260 unrelated people from the Slovenian population. Genotypes for the 14 STRs were determined using three multiplex polymerase chain reactions (PCR) and automated fluorescent detection. The allele frequencies of the STR loci D5S818, D13S317, D7S820, D8S1179 and D18S51 showed no deviation from the Hardy-Weinberg equilibrium and agreed well with other Caucasian populations. We resolved a series of 181 parentage disputes of which 29 were exclusions. In all cases, evidence for exclusion was obtained by at least 4 informative STRs out of the 14 loci analysed. The 14 loci combined comprise a highly discriminating test suitable for paternity and identity testing in the Slovenian population, with an average estimated mutation rate of 1.2x10(-3), a combined calculated power of exclusion of 99.99974% and paternity index (PI) value of >10(6) in 72% of the inclusion cases and >10(5) in 91% of the inclusion cases.
It was found previously that Ecm11 interacts with Cdc6 in a two-hybrid screen. To verify that these two gene products can actually encounter in the living cell, ECM11 was expressed as a fusion with GFP. The fusion protein was located in the cell nucleus throughout all stages of mitosis. The protein was detected in discrete clusters in the nucleus. Clusters may be due to Ecm11 protein polymerisation and association with discrete structures in the nucleus. Two Ecm 11 molecules interact in the two-hybrid assay, suggesting the presence of functional dimmers. Ecm11 does not promote transcription of beta-galactosidase reporter gene, when fused with LexA DNA binding domain and fetch to the promoter of the reporter gene. According to our observation, we suppose that Ecm11 is a nuclear protein involved in the upholding chromatin structure.
To date, two genes encoding 5alpha-reductase isoenzymes are known (type I, type II), and one type I pseudogene. The divergent localization of these genes and the still not fully understood function of the encoded enzymes as well as the perplexing results we obtained after sequencing PCR-amplified SRD5A1 gene fragments (out of genomic DNA), made us assume that, in addition to the known SRD5A1 gene, one or more different human 5alpha-reductase type I coding genes may exist. Our research provide the first evidence for the existence of two new SRD5A1 related, previously unidentified sequences in the human genome. These sequences which were localized to chromosomes 6 and 8 are highly homologous (> 99%) to SRD5A1, and also do not contain any deletions or insertions that are otherwise a characteristic of the SRD5API pseudogene. Our results imply that these sequences may be either coding parts of yet unknown, active SRD5A1 genes, and/or of previously unidentified pseudogenes. These findings additionally support data of Chen et al. who confirmed the existence of various SRD5A1 proteins in cultured human skin cells.
We screened samples of tumour and peripheral normal tissue for differential expression of oncogenes by using an approach of detecting the differences in expression of a number of oncogenes simultaneously. Total RNA was isolated from 29 pairs of normal and tumour tissue samples from patients with gastric adenocarcinoma. Seven pairs of primers for oncogenes most probably associated with the process of carcinogenesis in stomach including cyclin E, c-erbB-3, HGR, c-met, TDGF/cripto, FGF-4, and EGF were used for the construction of fluorescent multiplex RT-PCR. Sense primers were 5' end-labelled with a fluorescent dye. 5-7 gastric oncogenes were simultaneously analysed for overexpression. Multiplex reverse transcription with a set of unlabeled primers was followed by a PCR reaction by adding the corresponding set of fluorescent labelled PCR-primers. Expression of oncogenes was compared to GAPDH internal standard. Multiplex fluorescent RT-PCR results were analysed by capillary electrophoresis on ABI-PRISM 310 Genetic Analyzer. Differential expression of oncogene mRNAs in tumour and normal tissue was assessed by comparison of oncogene/GAPDH ratios in tumours and their peripheral normal mucosa. Our results show, that in most patients, comparing to normal tissue, we could estimate overexpression of at least one oncogene in a sample.
Estrogen receptor alpha (ER alpha) encoding gene is one of the candidate genes to be involved in the development of osteoporosis. Until now correlation between three ER gene polymorphisms (identified with PvuII, XbaI and BstUI) and bone mineral density (BMD) have been investigated. The results of these studies are contradictory. Thus the aim of our work was to search for new, yet unknown, and probably more informative polymorphism(s) of the ER alpha gene. For detection of mutations the whole coding region of the ER alpha gene was screened systematically. In a group of 85 late postmenopausal women all of the eight exons were amplified by polymerase chain reaction (PCR) and fragments were further analyzed by single-stranded conformation polymorphism (SSCP) analysis. Mutations were confirmed by direct DNA sequencing. In the whole coding region of the ER alpha gene two silent mutations in codon 87 and 325, respectively, were found. The silent mutation in codon 85 of exon 1 (GCG-->GCC; A87A) was described previously, as BstUI polymorphism. On the other side, the silent mutation in codon 325 (CCC-->CCG; P325P), located in exon 4, has not been analyzed so far in correlation with BMD. According to the distribution of genotypes CC:CG:GG=49.4:41.2:9.4, we can affirm the existence of genetic polymorphism in codon 325 in our population of late postmenopausal women. The mean femoral neck BMD, but not the lumbar spine BMD, was significantly lower (P=0.029) in the homozygous GG-women with CCG/CCG codon 325 as compared to the homozygous CC-women with the normal codon CCC/CCC. Our results suggest that codon 325 sequence polymorphism of the ER alpha gene might be one of the factors associated with low femoral neck BMD.
Epidermolysis bullosa simplex is a hereditary skin blistering disorder caused by mutations in the KRT5 or KRT14 genes. More than 50 different mutations have been described so far. These, and reports of other keratin gene mutations, have highlighted the existence of mutation "hotspots" in keratin proteins at which sequence changes are most likely to be detrimental to protein function. Pathogenic mutations that occur outside these hotspots are usually associated with less severe disease phenotypes. We describe a novel K5 mutation (V186L) that produces a conservative amino acid change (valine to leucine) at position 18 of the 1A helix. The phenotype of this case is unexpectedly severe for the location of the mutation, which lies outside the consensus helix initiation motif mutation hotspot, and other mutations at this position have been associated in Weber--Cockayne (mild) epidermolysis bullosa simplex only. The mutation was confirmed by mismatch-allele-specific polymerase chain reaction and the entire KRT5 coding region was sequenced, but no other changes were identified. De novo K5/K14 (mutant and wild-type) filament assembly in cultured cells was studied to determine the effect of this mutation on filament polymerization and stability. A computer model of the 1A region of the K5/K14 coiled-coil was generated to visualize the structural impact of this mutation and to compare it with an analogous mutation causing mild disease. The results show a high level of concordance between genetic, cell culture and molecular modeling data, suggesting that even a conservative substitution can cause severe dysfunction in a structural protein, depending on the size and structure of the amino acid involved.
Mutation screening of the major autosomal dominant polycystic kidney disease (ADPKD) locus, PKD1, has proved difficult because of the large transcript and complex reiterated gene region. We have developed methods, employing long polymerase chain reaction (PCR) and specific reverse transcription-PCR, to amplify all of the PKD1 coding area. The gene was screened for mutations in 131 unrelated patients with ADPKD, using the protein-truncation test and direct sequencing. Mutations were identified in 57 families, and, including 24 previously characterized changes from this cohort, a detection rate of 52.3% was achieved in 155 families. Mutations were found in all areas of the gene, from exons 1 to 46, with no clear hotspot identified. There was no significant difference in mutation frequency between the single-copy and duplicated areas, but mutations were more than twice as frequent in the 3' half of the gene, compared with the 5' half. The majority of changes were predicted to truncate the protein through nonsense mutations (32%), insertions or deletions (29.6%), or splicing changes (6.2%), although the figures were biased by the methods employed, and, in sequenced areas, approximately 50% of all mutations were missense or in-frame. Studies elsewhere have suggested that gene conversion may be a significant cause of mutation at PKD1, but only 3 of 69 different mutations matched PKD1-like HG sequence. A relatively high rate of new PKD1 mutation was calculated, 1.8x10-5 mutations per generation, consistent with the many different mutations identified (69 in 81 pedigrees) and suggesting significant selection against mutant alleles. The mutation detection rate, in this study, of >50% is comparable to that achieved for other large multiexon genes and shows the feasibility of genetic diagnosis in this disorder.
The cDNA encoding for catalytic subunit of bovine enterokinase (EK(L)), to which the sequence for Kex2 protease cleavage site was inserted, was expressed in the protease deficient filamentous fungus Aspergillus niger AB1.13. Fungal transformants were obtained in which expression of the glucoamylase fusion gene resulted in secretion of the protein into growth medium. Fusion polypeptide was processed to mature EK(L) by endogenous Kex-2 like protease cleavage during secretory pathway. The highest quantity of EK(L), up to 5 mg l(-1), was obtained in soya milk medium. The secreted EK(L) was easily purified from other proteins found in A. niger culture supernatant, using ion exchange and affinity chromatography. The yield of the purified and highly active EK(L) was 1.9 mg l(-1) of culture.
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Osteoporosis is a common bone disease which affects one in three women after the 60th year of life and is a major cause of morbidity in older people. To identify patients with osteoporosis, measurement of bone mineral density (BMD) is recommended. The association of BMD with vitamin D receptor (VDR) genotype in Slovenian postmenopausal women was studied. We determined VDR genotype in 102 late postmenopausal women aged 47-77 years. BMD measurements were performed at the level of the lumbar spine (L2-L4) by dual X-ray absorptiometry. Our data show significantly lower BMD in BB women compared to those with bb genotype. The relative distribution of VDR genotypes and alleles in the Slovenian population was 18.6:57.8:23.6% for BB:Bb:bb, respectively. The results are consistent with those of a previous study which found an excellent correlation between BB VDR genotype and low BMD. The data were derived from a relatively small, but ethnically homogeneous population of the same socioeconomic status, with very similar dietary and physical activity habits. Dietary habits in particular seem to be important because of the relatively low calcium intake which may enhance the phenotypic expression of VDR gene polymorphisms.
Northern analysis of the mouse testis and liver RNA show two types of lanosterol 14alpha-demethylase (CYP51) transcripts, somatic and a testis-specific transcript. At least two CYP51 mRNAs are expressed in liver and testis, while the smallest transcript is expressed only in testis. The partial mouse Cyp51 cDNA (1052 bp) was obtained by RT-PCR cloning and was also found in expressed sequence tag (EST) clones. Bacterial artificial chromosome (BAC) clones containing mouse Cyp51 gene were also found. The coding part of the partial mouse CYP51 is 93.9% homologous to the rat and 87.9% to the human CYP51 cDNAs. There is less than 2% variation between the CYP51 EST clones to the RT-PCR cloned partial cDNA and BAC clones.
More than 800 mutations have been indentified in the CFTR gene. This vast mutation diversity makes the search for molecular defects in cystic fibrosis difficult. Out of 100 Slovenian CF families, we have screened 30, using DGGE and SSCP as mutation detection techniques, while the remaining 70 have been studied previously. Together our and the previous studies have been able to indentify 18 CF mutations which cover 77.6% of the CF alleles in those families. The relative frequency of deltaF508 is 62.7% which is significantly higher than the average reported for the Mediterranean South European region (51.6%). At the same time, significant differences in mutation frequencies were found for the G542X, R1162X, W1282X, N1303K and 3905insT mutations. Several, otherwise rare mutations have been detected, such as: I148T, Q552X, 457TAT-->G, R1006H, 2907delTT, 3667ins4, A559T and G576A. An interesting fact is that A559T was so far found mostly in CF patients of African-American origin. These results imply that a high heterogeneity of CF mutations occurs within the small population of Slovenia, consisting only of 2 million inhabitants. In view of the spectrum and frequencies of detected mutations, Slovenian population expresses characteristics of Mediterranean and central European countries, and at the same time shows also distinctive differences and unique region specific CF mutations (Q685X, D192G, S4X).
A sensitive nonradioactive northern blotting for the detection of acetylcholinesterase mRNA in mammalian tissues is described and compared to its radioactive version. Best results were obtained if digoxigenin labeled RNA probe was used for hybridization and CDP-Star, a chemiluminescent alkaline phosphatase substrate, for detection. The described nonradioactive technique for acetylcholinesterase mRNA determination is as sensitive as the radioactive one, but requires no protection against radiation and is less time consuming. Because of higher stability of the labeled probe, nonradioactive technique is also more convenient from the standpoint of experimental planning.
A gene-fusion expression strategy was applied for the heterologous expression of hTNF-alpha in A. niger AB1.13. The TNF-alpha 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-alpha 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-alpha 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.
CDC6 is an essential gene of yeast Saccharomyces cerevisiae. Although DNA sequence of the gene is available for a long time, biochemical function of Cdc6 protein in the cell cycle remains unclear. Using the interaction trap experiment we were looking for proteins interacting specifically with Cdc6. Four gene products interacting with Cdc6 were detected. By sequence analysis we found that ECM11 codes for the protein involved in the cell wall synthesis, YNL201 codes for the protein of unknown function, probably involved in the carbon metabolism, YOR279 codes for protein of completely unknown function with no significant similarity with any known protein, and the interaction with Ty1 retrotransposition element was also found. The strongest interaction with Cdc6 bait measured as beta-galactosidase activity was observed with ECM11 and YNL201; YOR279 interacts slightly weaker. The weakest beta-galactosidase activity was obtained by Ty1A element. The strongest suppression of cdc6-1 mutation was observed by Ty1A element, the slight one with ECM11 and YNL201 but no suppression of thermosensitive mutation was detected for YOR279.
A gene-fusion expression strategy was applied for heterologous expression of human lymphotoxin alpha (LTalpha) in the Aspergillus niger AB1.13 protease-deficient strain. The LTalpha 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 (TNFalpha) 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 LTalpha 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 LTalpha from A. niger into the soya medium was 50 pg l(-1) of culture.
The fungal biotransformation of steroids is of applied interest due to the economic importance of such stereo- and regiospecific reactions and also in the context of ergosterol pathway engineering to produce vitamin D and steroidal products. In Schizosaccharomyces pombe no steroid hydroxylation as is found in filamentous fungi was observed, but a cytosolic NAD(H)/NADP(H)-dependent hydroxysteroid dehydrogenase activity was identified. Progesterone was reduced at the delta 4 double bond (in vivo only) as well as at the C-3 and C-20 keto groups. Testosterone and 4-androstene-3,17-dione were interconverted and 5 alpha-pregnane-3,20-dione and 5 beta-pregnane-3,20-dione were reduced to 3-hydroxy products. The reactions were sometimes reversible and showed regio- and stereo specificity. In S. pombe more than one steroid dehydrogenase homologue is likely to occur, as has been observed in Saccharomyces cerevisiae. Our findings indicate that genes encoding soluble proteins should be examined as candidates for actual steroid dehydrogenase activity.