Theses - Department of Molecular Biology and Genetics

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Open Access
Establishment of a rapid mutation detection technique for screening of BRCA1 and BRCA2 genes
(Bilkent University, 1997) Öktem, Emre; Özçelik, Tayfun
People bearing germline mutations in either BRCAl or BRCA2 genes are more prone to breast cancer than other people. These two recently identified genes account for nearly 90% of the hereditary cancer cases. This number corresponds approximately to 100,000 women each year in Turkey. The individuals who carry these mutations are at high risk and characterizing these mutations will be helpful for providing them genetic counseling. This includes the estimation of risk for both the individual and his/her progeny. In addition, discovering the mutations is an important step in finding out the iunctions of these genes, which will give insights about how breast cancer is occurring In this study, we have established an easy and rapid mutation detection strategy; heteroduplex analysis, and tested its efficiency in 15 hereditary breast cancer patients. These patients have been screened for the entire exon 11 of BRCAl, which is 50% o f the entire coding region. In addition, half of the exon 11 of BRCA2, which is roughly one fourth of the coding region was examined in 10 patients. As yet, no mutation in the Turkish patients have been encountered These results exclude exon 11 of the BRCAl gene and part of exon 11 of the BRCA2 gene for nucleotide deletions and insertions as the cause of hereditary breast cancer in Turkey. In order to verily the efficiency of the technique, we have also screened four French hereditary breast cancer patients with previously characterized BRCAl mutations in exon 11, and confirmed the presence o f the mutations in the majority o f the cases With the technique, firmly established in our laboratory, we plan to analyze the remaining coding regions of the BRCAl and BRCA2 genes in our patient samples and in additional patients.
Open Access
Development of a non-immunological system for the study of the cellular localization of BRCA1 gene product in living cells
(Bilkent University, 1997) Çağatay, Tolga; Yuluğ, Işık G.
BRCAl, is a familial breast and ovarian cancer susceptibility gene that has been cloned and shown to be either lost or mutated in families with breast and ovarian cancer. BRCAl, has been postulated to encode a tumor suppressor, a protein that acts as a negative regulator of tumor growth. To explore the biolo^cal function of BRCAl, several studies have been performed for the identification of cellular localization of BRCAl gene product. Results obtained from these immunofluorescent/ immunohistochemical studies generated two opposing views, cytoplasmic localization versus nuclear localization. Here, we describe a non-immunological system employing the Eukaiyotic Green fluorescent Protein (EGFP) tag for the study of the cellular localization of BRCAl gene product in living cells. Proteins carrying the green fluorescent protein (GFP) of Aequorea victoria provide a powerful system to analyze protein expression and targeting in living cells. Fusion proteins containing the GFP tag are therefore valuable tools to analyze nuclear trafficking in living cells. Here, we reporte the use of a mutant GFP, namely Eukaryotic Green Fluorescent Protein (EGFP), as a marker for the protein import into mammalian nuclei. We have analyzed the behavior of a protein domain of the BRCAl, that contains five putative nuclear localization signals (NLSs), in vivo using a chimera constructed from this polypeptide and the EGFP. This in vivo studies showed that EGFP was distributed uniformly throughout the cytoplasm and the nucleus. When EGFP was fused to NLSs containing domain of the BRCAl protein, fluorescent was predominantly detected in the nucleus, showing that these potential NLSs consensus sequences may destínate the full-lengh BRCAl producy into the nucleus of mammalian cell. This study has also shown that EGFP can be used as a potential fluorescent tag for visualization of gene expression and cellular protein localization in living cells.
Open Access
Production of recombinant human BRCA1 encoded proteins in E coli
(Bilkent University, 1997) Özçelik, Berna S.; Öztürk, Mehmet
Among the risk factors that cause breast cancer, heredity emerges as the major determinant. BRCAl is the first gene which was found to be associated with inherited early-onset breast cancer. BRCAl is spread over a 100 kb region on human chromosome 17q21 and consists of 22 coding exons which are transcribed into a 7.8 kb long mRNA. This mRNA is abundant in breast and ovarian tissues and encodes a polypeptide of 1863 amino acids. The exact cellular function of BRCAl remains to be elucidated. As there are many gaps of knowledge and many conflicts about the cellular functions of BRCAl, new points of views and technical approaches should be generated. As such studies require the presence of purified protein products, we aimed to express and purify BRCAl encoded proteins. In this study we cloned the BRCAl gene in four overlapping fragments into the pCR-Script Amp, sk (+) cloning vector and subcloned the carboxyl terminal into the pQE expression vector. The 73 kD gene product was purified by affinity chromatography.
Open Access
Establishment of an experimental system to study p53 effects in Saccharomyces cerevisiae
(Bilkent University, 1997) Dinçer, Tuba; Öztürk, Mehmet
The aim of this vv^ork was to establish an experimental model to study human wild type and mutant p53 protein effects in yeast Saccharomyces cerevisiae cells. Wild type p53 was previously shown to be a DNA damage response gene that controls the genome stability in mammalian cells. We are interested in using yeast cells to study human p53-mediated cellular events after DNA damage. In this study we established an experimental model as an initial step. For construction of this system pAK31 plasmid expressing a human mutant p53-248W as well as control plasmid pL3 were used to obtain two different yeast cell populations, one expressing the mutant p53 248W protein and the other without p53 expression. Wild type p53 expression vector was avoided because of its known growth inhibitory effects in yeast cells. In this experimental system initially p53 expression at pAK31 transformed yeast cells was shown. Then, the effect of mutant p53-248W expression to growth rate of yeast cells was analysed and no growth rate difference was detected between the cells expressing and non-expressing mutant p53-248W protein. To test the participation of mutant p53-248W protein in DNA damage response in yeast cells, cells were exposed to DNA damaging agents; UVC and cisplatin that were reported to induce wild type p53 protein in mammalian cells. Codon 248 is a common site of ‘hot spot’mutation and the arginine residue that corresponds to codon 248 encoded by the wild type p53 sequence is in the DNA interacting face of the p53 protein. Mutant p53-248 (arg->trp) is defective in specific DNA binding and it has lost the ability to act as a transcription factor. Although there are not many reports about mutant p53-248W response to DNA damaging agents, it was shown that mutant p53- 248W exhibit decreased repair of active genes upon UV radiation. In this study cell survival was analysed in mutant p53-248W protein expressing yeast cells in parallel to mutant p53 protein levels following to DNA damage and no effect of mutant p53 248W expression on cell survival upon DNA damage was detected. Also no difference was detected in mutant p53 protein levels following DNA damage.
Open Access
Development of a non-radioactive diagnostic test for the detection of microsatellite instability in colorectal cancer
(Bilkent University, 1997) Vata, Korkut; Özçelik, Tayfun
Stepwise accumulation o f mutations in the human genome is the initial step in carcinogenesis. Microsatellites are the regions which are first hit by the mutations resulting from mismatch repair deficiency. Until today microsatellite alterations have been shown mainly in hereditary non-polyposis colon cancer (HNPCC) and several other cancer types. Recent advances indicate that microsatellite alterations can also be detected in DNA samples (such as blood, urine, etc.), which are shed fiOm tumors. This is an important finding for the early diagnosis o f cancer since malignant cells can be detected in tissues other than the primary tumor. Therefore microsatellite analysis, when coupled with an easy, powerful screening technique could have a high diagnostic value for cancer types other than colorectal cancer. Despite its drawbacks the most common microsatellite screening method is based on the use of radioisotopes and autoradiography. However in the clinical setting non-radioactive detection methods are preferred. The aim o f this thesis is development o f a non-radioactive diagnostic test for the detection o f microsatellite instability in genomic DNA which can be used for the early detection o f some forms o f cancer. Therefore we have optimized the PCR conditions for eight microsatellite markers which are: i. mononucleotide repeats BAT25 and intragenic repeat region o f BAX gene, ii. dinucleotide repeats D5S105, D6S291, D11S904, D13S175, D17S855, and in. tetranucleotide repeat FGA. In addition, we have analyzed the mononucleotide repeat markers in blood, paraffin embedded and fi"esh tumor samples o f six colorectal cancer patients with polyacrylamide gel electrophoresis and silver staining
Open Access
Production of recombinant human BRCA2 encoded proteins in Ecoli
(Bilkent University, 1997) Sayan, Emre; Öztürk, Mehmet
Breast cancer is known to be the most common cancer among women in the world. It is assumed that one in ten women will develop breast cancer till the age 80. Heredity is the major risk determinant in breast cancer. 30% of the women with breast cancer have a positive family history and 10% of women is defined to have high risk of early onset breast cancer who have multiple family members of breast and ovarian cancer. A novel breast cancer susceptibility gene, BRCA2 was confirmed to account more than 40% of the mutations in familial breast cancer patients. BRCA2 tumor supressor gene encodes a 3418 amino acid protein with little or no homology to other known proteins. There is no significant data concerning the cellular functions of BRCA2 and many facts about BRCA2 are waiting to be uncovered. The gaps in the knowledge about BRCA2 must be filled by generating new points of views and technical approaches. Such studies require the cloning of BRCA2 and the presence of purified protein products. In this study we cloned a fragment of exon 11 and exons 19 to 27 as 2 overlapping fragments. Since more than 80% of the mutations result in the loss of the C-terminal of the protein and produce cancer phenotype, we expressed and purified the extreme C-terminus of the BRCA2 protein.
Open Access
Analysis of p73 gene in hepatocellular carcinoma
(Bilkent University, 1998) Fındıklı, Necati; Öztürk, Mehmet
Hepatocellular carcinoma (HCC) is the eighth most frequent cancer worldwide, iipidemiologically-studied risk factors include hepatitis B virus (greater than 80%), hepatitis C virus and aflatoxins. Molecular mechanisms of hepatocarcinogenesis are poorly understood. The only gene known to be consistently involved in these tumors is the p53 tumor suppressor gene. However, this gene was found to be mutated or ifiactivated in about 30% of HCC. There is a need to study additional genes in order to fully understand hepatocellular carcinogenesis. p73 has been identified recently as a p5i -homolog gene. In this study, we analyzed the possible involvement of this gene in HCC. We investigated both the expression and structure of p73 gene in HCC for possible alterations.We first developed a novel method to analyze the expression of alternatively spliced transcripts of p73 (p73a and p73p) simultaneously. This technique, based on RT-PCR, allows the analysis of p73 transcripts semi-quantitatively. We found that p73a was expressed ubiquituously in 8 cell lines derived from normal liver or HCC tumors. Interestingly, p73|3 was present only in 5 differentiated but not in 3 undifferentiated cell lines. The differentiation status of these cell lines were tested by the analysis of albumin and a-fetoprotein transcripts by RT-PCR. These transcripts were present in 3/5 differentiated but not in 3 undifferentiated cell lines. Next, we screened 25 HCC samples for possible mutations of p73 gene at selected exons with non-radioactive heteroduplex test, radioactive SSCP analysis, restriction enzyme analysis and DNA sequencing. No alterations were found in exons homologous to those of p53 known to harbor mutational hotspots.From these observations, we conclude that i) /3 gene is not mutated in HCC, but it may play a critical role in hepatocellular differentiation. As p73p was found in differentiated cell lines, this form may be involved in transcriptional regulation of liver-specific genes. Additional studues are needed to confirm this hypothesis.
Open Access
Molecular analyses of hematological malignancies
(Bilkent University, 1998) Yılmaz, Z. Buket; Özçelik, Tayfun
Normal hematopoiesis is known to be disrupted in hematological malignancies. As research advances, many new treatment techniques are introduced, among which allogeneic transplantation is the most effective. In patients who undergo such treatment, different hematopoietic chimeric states may result which can be detected by indirect analyses using DNA polymorphisms. MRD (minimal residual disease) describes leukemia cells present at a level below that is detectable by conventional means. The detection of MRD has gained special significance in transplantation patients. The detection of fusion transcripts that are generated by chromosomal rearrangements in various hematological malignancies is the direct and the most powerful approach for the detection of MRD. It not only has a prognostic significance but also has a diagnostic importance for patients who have not taken any treatment. The aim of this thesis is the development of PCR-based tests for the diagnosis and monitoring of patients with different hematological malignancies. Therefore the chimerism status of 22 recipient-donor pairs have been evaluated by PCR amplification of STR and VNTR polymorphisms followed by polyacrylamide gel electrophoresis and silver staining. In addition, 44 patients with acute and chronic leukemias have been analyzed for the detection of fusion transcripts generated by t (9;22) (q34;qll), t (8;21) (q22;q22), t (15;17) (q22;q21), inv 16 (pl3;q22), t (4;11) (q21;q23), and t (1;19) (q23;p23) chromosomal rearrangements with RT-PCR.
Open Access
Expression and purification of the hepatitis C virus core protein in Ecoli and testing of human sera with this core antigen
(Bilkent University, 1998) Eroğlu, Çağla; Pınarbaşı, Ergün
The hepatitis C virus (HCV) infection is an important cause of morbidity and mortality world wide. Infection with HCV becomes chronic in more than 80% of the cases and it accounts for 20%of all cases of acute hepatitis. Hepatitis C virus was first identified by the molecular cloning of the virus genome in 1989. It is an enveloped, positive strand RNA virus with a genome size of around 9.5 Idlobases. The single stranded RNA genome of the virus contains a large open reading frame codes for a large poly-protein of 3,010 to 3,033 amino acids which is shown to be processed by a combination of host and viral proteinases to produce at least ten proteins post-translationally. The proteins that are closer to the amino terminal of the poly-protein are termed as structural and the rest closer to the carboxy terminal are called non-structural (NS) proteins. The core protein is the putative nucleocapsid component of the virion, and it is highly basic in nature. Core protein is the most highly conserved region of the hepatitis C virus open reading frame and it is shown to be highly immunogenic. Also, as the core protein is the putative capsid protein of the hepatitis C virus, antibodies against core antigen most probably arise much earlier than the antibodies against nonstructural proteins. In this study, the core protein of the hepatitis C virus was cloned, expressed and purified in order to establish an ELISA system to test the human sera with this viral antigen. It was shown that in 86% of the patients, diagnosed previously with the third generation enzyme immunoassays to be infected with hepatitis C, have antibodies against this core antigen. The core antigen gave no false positive results when tested with the negative control samples which were found to be Anti-HCV negative previously.
Open Access
Genetic analysis of Smad2 gene in hepatocellular carcinoma
(Bilkent University, 1998) Romano, Alper; Yakıcıer, Cengiz
Hepatocellular carcinoma is one of the most malignant cancers and is the most frequent one in some regions in the world. Although it is a multistage disease, its genetic composition is not well understood. TGF(3 is shown to be a strong inhibitor of cell growth and during hepatocellular carcinogenesis there is an escape from the anti-proliferative effect of TGF(3. Smad2 protein is the mediator of response to TGFP and its gene is mutated in several cancers. To clarify the role of Smad2 in TGFP signalling in hepatocellular carcinoma we performed single-strandconformation-polymorphism (SSCP) analysis in five exons of Smad2 for 35 tumor samples and in C-terminal region for five hepatoma cell lines. Two alterations were found out of 35 samples and no abnormal expression or big deletions were observed in cell lines. Thus Smad2 might be involved at least a part of hepatocellular carcinomas.
Open Access
Analysis of tumor suppressor genes in testis cancer
(Bilkent University, 1998) Ünal, Resat; Yuluğ, Işık G.
At least two classes of genes are involved directly or indirectly in the regulation of cell growth and differentiation. One group of these genes, known as tumor suppressor genes, is involved in cellular regulation by inhibiting uncontrolled proliferation. The most frequent genetic alteration in tumor suppressor genes is loss of one of their alleles and this is called “loss of heterozygosity” (LOH). Through several studies it is found that LOH in tumor suppressor genes is associated with uncontrolled cellular proliferation in many cancers. Testis cancer is a common cancer among young men. The disease is lethal for 20-30% of the young patients and the risk of having testis cancer increases in industrialized countries. Turkish population is a young population where testis cancer might be an important threat. The relation between distinct genetic alterations and testis cancer has been studied. LOH studies in testis cancer also have been performed however the number of such studies is very low. In this study the relation between testis cancer and the genes BRCA\, BRCAl and PTEN is investigated on 18 tumor samples of 10 individuals by using 8 highly polymorphic intragenic and extragenic markers with a PCR based LOH assay. LOH in BRCA1 was observed in two tumors of two individuals, and LOH in BRCAl was observed in five tumors of two individuals. No LOH was found within the PTEN gene where only one intragenic marker was used for studying LOH status.
Open Access
Genetic analysis of Smad4 gene in TGF-Beta signalling pathway in human liver cancer
(Bilkent University, 1998) Irmak, Meliha Burcu; Yakıcıer, Cengiz
HCC is a multistep genetic disease in which many genomic changes occur as a result of uncontrolled proliferation of hepatocytes. Molecular events leading to HCC is still unclear. Until now, neither an oncogene nor a tumor suppressor gene has been shown to be prefentially altered in HCC. Genetic alterations other than p53, pl6, BRCA2 (Breast Carcinoma Associated Protein), M6P/IGFIIR (lyiannose 6 Phosphate/ Insulin Like Growth Factor II Receptor), Rb (Retinoblastoma), PRLTS (Platelet Derived Growth Factor Receptor-|3-Like Tumor Suppressor Gene), and Tg737 (Candidate polycyctic kidney disease gene) genes remain unknown. TGF-P is a strong inhibitor of hepatocyte proliferation. In HCC and cirrhosis increased levels of TGF-P is observed, so this shows that the presence of high levels of TGF-3 does not avoid hepatocyte proliferation. Thus, there may be a disruption in the signalling pathway of TGF-p. The common mediator Smad4 gene, which is among the genes located in TGF-P signalling pathway, is found to be mutated in many cancer types. We decided to do the mutational analysis of Smad4 gene, which is located in the signalling pathway of the hepatocyte antiproliferative factor, TGF-p. Exons 8, 9, 10, and 11 which are in MH2 region, and exon 2 which is in MHl region of Smad4 is mutationally analysed by SSCP for 35 HCC cases. In the 35 HCC tumors, 5 alterations were observed (14%), 3 of them being in exon 8, one of them being in exon 9a, and the last one being in exon 10 of Smad4 gene. In the samples we tested, no big deletions were observed, but the alterations observed are probably single base changes. Also HCC cell lines namely, HepG2, Hep3B, Huh-7, FOCUS, Mahlavu, and PLC/PRF/5 were checked for their mutations and cell lines other then PLC/PRF/5 were analysed for their mRNA transcription. There were no big deletions or alterations in Nand C- terminals of the cell lines and we have shown mRNA transcription for all cell lines except Hep3B in which PCR has revealed very weak amplification. Our results suggest that Smad4 might be involved in at least a part of primary HCC tumor development.
Open Access
Analysis of hMLH1 germline mutations in three Turkish hereditary nonpolyposis colorectal cancer kindreds
(Bilkent University, 1998) Akyerli, Cemaliye; Ricciardone, Marie D.
Hereditary nonpolyposis colorectal cancer (HNPCC) is one of the most common genetic diseases in Western world. It is a clinical syndrome characterized by an inherited predisposition to early onset colorectal and an increased incidence of other cancers. The disease is caused by a germline defect in one of five human DNA mismatch repair genes, hMLHl, hMSH2, hPMSl, hPMS2, and hMSH6. Defects in hMLHl and hMSH2 account for the majority of mutations found in HNPCC families. In this study, a variety of mutation detection methods were used to identify hMLHl germline mutations in three Turkish HNPCC kindreds. Restriction enzyme analysis of genomic DNA was used to analyze five members of an HNPCC family with a previously described G884C mutation. The genotypes of all five individuals were determined by Dde\ digestion and the results were confirmed by DNA sequence analysis. Hph\ restriction enzyme analysis was used to analyze twenty-nine members of an unrelated HNPCC family for a previously identified A1652C mutation. Genotypes were determined for all individuals and the results were confirmed by DNA sequence analysis. Both of these restriction enzyme analyses are reliable, cost-effective methods that can be used in mutation screening programs for family members who request genetic counseling. Single strand conformation polymorphism analysis (SSCP) was used to screen for unknown germline mutations. Nine DN A samples with defined mutations in the hMLHl gene were analyzed using several gel formulations and electrophoretic conditions to determine the most sensitive protocols. These protocols were then used for routine mutation detection. In a third HNPCC family, for whom no mutation has yet been defined, the complete coding sequence of the hMLHl gene was screened by SSCP. Two exons, 7 and 15, showed an altered mobility compared to control sequences. The nucleotide sequence of these two exons was determined by automated fluorescence DNA sequence analysis. The differential mobility observed for exon 15 appears to be due to an intonic polymorphism in the control sample. Preliminary results for exon 7 show no difference between proband and control nucleotide sequences. Thus, the DNA mismatch repair defect in this kindred appears not to be in hMLHl. Further studies will focus on the analysis of hMSH2.
Open Access
Identification of the role of p33ING1 protein in the cellular activities of p53 tumor suppressor protein
(Bilkent University, 1999) Emre, N. C. Tolga; Çetin-Atalay, Rengül
p53 is a tumor suppressor gene which is mutated in about 50% of human cancers. The product of p53 gene encodes a sequence-specific transcription factor. The genes are transactivated by p53 code for proteins that are implicated in the negative regulation of cell proliferation (via apoptosis or cell cycle arrest) and DNA damage repair. p53 protein interacts with several viral and cellular proteins and these interactions are important in the regulation and dysregulation of the functions of p53. Another gene, named ING1 (for "Inhibitor of Growth 1"), was identified as a candidate tumor suppressor gene due to its functions in apoptosis and cell cycle arrest. p33ING1, the protein product of ING1, was shown to enhance the growth suppressive functions of p53. Furthermore, a physical association between p53 and p33ING1 proteins has been detected by immunoprecipitation. In this study, we investigated the physical interaction between p53 and p33ING1 using in vitro methods in order to determine the region of p53 that enabled this interaction. As a preliminary step for the study, the ING1 cDNA was amplified from total cDNA of a cell line by PCR and cloned into expression vectors. The recombinant p33ING1 protein was overexpressed in E.coli and purified as a fusion protein with GST. The wild-type p53 cDNA and its several deletion mutant constructs were subcloned into a suitable expression vector to enable subsequent in vitro transcription-translation reactions. In vitro transcription-translation products of these constructs were used in GST pulldown assays with purified GST p53 protein to map the interacting region on the human p53 protein. The results of the study suggest that the primary determinant on p53 protein in its interaction with p33ING1, is the C-terminal domain while there may be other regions that are involved in the interaction.
Open Access
Analysis of the function of the nuclear matrix-associated protein C1D
(Bilkent University, 1999) Bilican, Bilada; Yavuzer, Uğur
DNA Double-strand breaks (DSBs) are generated as intermediate stnictures during V(D)J recombination, as a consequence of oxidative metabolism, or can be induced by exogenous factors such as gamma irradiation and radiomimetic drugs. Mutational studies identified the serine/threonine kinase, DNA-PK, as an essential component of DNA DSB repair machinery. The activation of the multi-component DNA-PK complex requires either free DNA ends or an association with the nuclear-matrix associated protein CID, which facilitates the activation of DNA-PK in a DNA end-independent fashion. The activation of DNA-PK through its interaction with CID, joins an increasing body of evidence which suggests a role for higher order nuclear organisation in the orchestration of complex cellular processes such as transcription, RNA splicing, nucleotide excision repair, replication and double-strand DNA break repair. In this study, the yeast two hybrid system was employed to screen a B-cell cDNA library to identify the interacting proteins with CID, and the interactions determined were further characterised. It was found that, CID interacts specifically with the recombinational hotspot binding protein Translin and Translin associated factor X, TRAX, both in vitro and in vivo, providing evidence that C 1D may play a critical role in DNA repair and recombination. Interestingly, an interaction between TRAX and DNAPKcs has also been identified under in vivo conditions. Tlie interaction of TRAX with DNA-PKcs and C ID indicates a connection between DNA double-strand break repair, recombination, and dynamic nuclear architecture.
Open Access
Molecular cloning and characterization of the common 1b subtype of HCV from Turkey
(Bilkent University, 1999) Öztan, Aslı; Öztürk, Mehmet
Hepatitis C Virus is a major cause of acute and chronic hepatitis worldwide. 80-90% of Hepatitis C Virus infections become chronic and 75% of these cases lead to liver disease, including cirrhosis, liver failure and hepatocellular carcinoma. Hepatitis C Virus was first identified by molecular cloning of the viral genome in 1989. Hepatitis C Virus is an enveloped virus containing a positive stranded RNA genome with a size of around 9.5 kilobases. In terms of genomic organization, it was accepted as a member of Flaviviridae family as a new genus named Hepaciviruses. The single-stranded RNA genome encodes a single open reading frame, which is transcribed into a single polypeptide of 3010 or 3030 amino acids and cleaved into viral proteins Core, E l, E2/p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B by host and viral proteases. Sequencing, serotyping and RFLP studies indicate that Hepatitis C Virus genome is highly variable. There are six distinct genotypes and at least 74 subtypes with different distributions between the geographic areas. Variability is not equally distributed throughout the genome. 5' UTR, some parts of the 3' UTR and capsid protein are the most conserved regions. The predominant genotype in the Turkish population was found to be lb by sequencing of the 5-UTR. In this study, the entire sequence encompassing the complete coding region and partial non coding regions of the genotype lb obtained from a HCV-infected Turkish patient was cloned to investigate its evolutionary relationship with other genotypes and to study its overall genome organization,. In order to characterize the viral genome, viral RNA was extracted from the serum, cDNA was synthesized, the HCV genome was amplified by PCR in 7 overlapping fragments, PCR fragments were cloned into bacterial vectors and cloned inserts were sequenced by automated sequencing methodology. The partial sequence data covering 70% of the cloned HCV genome indicate that the Turkish lb genotype displays high homology to other lb genotypes, but differs from others by distinct amino acid changes. To our knowledge, this is the first report about the HCV genome structure from Turkey. The HCV subgenomic fragments obtained here will serve to further molecular and immunologic studies on this dominant form of HCV found in Turkish patients.
Open Access
Characterization of programmed cell death induced by oxidative stress in selenium - deficient hepatocellular carcinoma cells
(Bilkent University, 1999) İnce, Gülayşe; Öztürk, Mehmet
Selenium (Se) plays an important role in eliminating the reactive oxygen species generated during oxidative stress. Se-containing selenocysteine is an essential amino acid required for the synthesis of many selenoproteins, such as glutathione peroxidase. Se-dependent glutathione peroxidase is the major en2yme that metabolizes organic and hydrogen peroxides in the cell. It appears that Se manifests its antioxidant effect through the selenoproteins. Se deficiency causes a wide range of pathological conditions. Keshan disease, characterized by cardiomyocyte and liver cell death, defined as "necrosis", was found to be associated with Se deficiency. Se deficiency is also observed in liver cirrhosis, alcoholic liver disease, HIV positive and AIDS patients, thyroid hormone abnormalities, etc. Moreover, epidemiological studies show that Se deficiency increases the risk of many cancer types, including hepatocellular carcinoma (HCC). The molecular mechanisms of the pathologies associated with Se deficiency are mostly unknown. In this study, the in vitro responses of human hepatocellular carcinoma cells, to Se deficiency have been examined. Apoptotic changes (nuclear condensation, positive annexin V staining, genomic DNA breaks) are detected in Huh7, HepG2, and Mahlavu cells under Se-deficient culture conditions. Hydrogen peroxide addition into the culture medium aggravates apoptosis. Such changes are prevented by the supplementation of Se. The Se-dependent glutathione peroxidase enzyme is shown to be reduced in cells grown under Se-deficient conditions, explaining the build up of intracellular oxidative stress. It is proposed that the cell death observed in many pathologies associated with Se deficiency, is the result of the programmed cell death, triggered by an increase in intracellular oxidative stress, as opposed to necrotic cell death. Hep3B, Hep3B-TR, Hep40, PLC/PRF/5 and BC1/R14 cell lines have not displayed cell death when grown in Se-deficient conditions and were resistant to hydrogen peroxide, as well. The presence of such cell lines and the differential sensitivities manifested by HepG2, Huh7 and Mahlavu suggest a complete or partial gained resistance for apoptosis, which may contribute to the onset or progression of HCC, consistent with the increased risk of HCC in Se deficiency.
Open Access
NF1 mutation analysis in a child homozygous for MLH1 mutation
(Bilkent University, 1999) Al-Otaibi, Hani S. M.
Open Access
Analysis of BRCA1 and BRCA2 genes in Turkish breast cancer patients
(Bilkent University, 2000) Özdağ, Hilal; Özçelik, Tayfun
Breast cancer is the most frequent cancer type and the second cause of death among women. It is estimated that 10 to 15% of breast cancer cases are hereditary. The majority of hereditary breast cancers can be attributed to germ-line mutations in ^Jgeast CAncer susceptibility genes BRCAl and BRCA2. In this study, germ-line BRCAl and/or BRCA2 gene mutations were screened in 50 Turkish breast and/or ovarian cancer patients divided into four groups of hereditary, familial, early onset, and male cancer by heteroduplex analysis and DNA sequencing. Two BRCA2 mutations, one novel (6880insG) and one previously reported (3034delAAAC), were found in the hereditary group. A novel BRCAl (1200insA) mutation was formd in the early onset group. All three mutations cause premature- termination codons. In addition, five BRCAl sequence variants have been identified in 23 patients. K654E (2080 A—>G), D693N (2196 G->A), P871L (2731 C—>T), and K1183R (3667 A—>G) result in a change of amino acids. 1013 T—^>C and 2201 C—>T are silent mutations. One patient in the early onset group was compound heterozygote for K654E and D693N. These results indicate that BRCAl and BRCA2 genes are involved in some but not all hereditary breast cancers in the Turkish population.
Open Access
Tumor suppressor functions of p53 gene
(Bilkent University, 2000) Ünsal, Keziban; Öztürk, Mehmet