Department of Molecular Biology and Genetics

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Open Access
Effect of oxygen supply on metabolism of immobilized and suspended Escherichia coli
(John Wiley & Sons Inc, New York, NY, United States, 1996) Inanç, E.; Miller J. E.; DiBiasio, D.
The effect of reduced oxygen supply on the production of a recombinant protein (plasmid-encoded β-galactosidase) was investigated in Escherichia coli. A novel modified bubble tank reactor was used to provide a direct comparison between immobilized and suspended cells in identical environments except for the immobilization matrix. Decreased oxygen supply led to increased β-galactosidase synthesis by both immobilized and suspended cells. Immobilized cells produced similar amounts of β-galactosidase as the suspended cells. Lactose consumption and acetate production, on a per cell basis, were significantly higher in immobilized cells, suggesting that immobilized cells utilized fermentative metabolism. However, a transport analysis of the immobilized cell system showed that immobilized cells were not subject to either external or internal mass transfer gradients.The effect of reduced oxygen supply on the production of a recombinant protein (plasmid-encoded β-galactosidase) was investigated in Escherichia coli. A novel modified bubble tank reactor was used to provide a direct comparison between immobilized and suspended cells in identical environments except for the immobilization matrix. Decreased oxygen supply led to increased β-galactosidase synthesis by both immobilized and suspended cells. Immobilized cells produced similar amounts of β-galactosidase as the suspended cells. Lactose consumption and acetate production, on a per cell basis, were significantly higher in immobilized cells, suggesting that immobilized cells utilized fermentative metabolism. However, a transport analysis of the immobilized cell system showed that immobilized cells were not subject to either external or internal mass transfer gradients.
Open Access
Identification of BTG2, an antiproliferative, p53-dependent component of the DNA damage cellular response pathway
(Nature Publishing Group, 1996) Rouault, J. P.; Falette, N.; Falette, V.; Guillot, C.; Rimokh, R.; Wang, Q.; Berthet, C.; Lalle, C.; Savatier, P.; Pain, B.; Ozturk, M.; Samaru, C.; Puisi, A.
Open Access
Coagulation factor V gene mutation increases the risk of venous thrombosis in Behcet's disease
(Oxford University Press, 1996) Gül, A.; Özbek, U.; Öztürk, C.; Inanç, M.; Koniçe, M.; Özçelik, T.
We investigated the prevalence of the coagulation factor V gene G1691A mutation in 64 patients with Behcet's disease (BD) and in 107 apparently healthy individuals. The mutation was present in the heterozygous state in 37.5% of the patients with a history of deep vein thrombosis (12/32) and in 9.4% of the patients without any thrombotic event (3/32). Eleven healthy individuals were also heterozygous for the mutation (10.3%). The prevalence of the mutation in BD patients with and without thrombosis was significantly different (P = 0.0079). We conclude that the factor V gene mutation may play a major role in the development of venous thrombosis in BD.
Open Access
Multiplex systems for the amplification of short tandem repeat loci: evaluation of laser fluorescence detection
(1997) Ricciardone, M. D.; Lins, A. M.; Schumm, J. W.; Holland, M. M.
Short tandem repeat (STR) loci are ideal markers for personal identification and for genomic mapping. Two fluorescent multiplex systems, each designed for simultaneous PCR amplification of four polymorphic STR loci (HUMCSF1PO, HUMTPOX, HUMTH01 and HUMVWFA31, and HUMF13A01, HUMFESFPS, HUMBFXIII and HUMLIPOL), were evaluated on three laser fluorescence detection instruments. Concordant DNA typing results were obtained with all three detection methods. These fluorescent multiplex STR systems offer an accurate, reproducible and versatile method of DNA profiling that is well-suited for forensic identity testing and other genetic analyses.
Open Access
TP53 and hepatocellular carcinoma
(Elsevier Masson, 1997) Puisieux, A.; Ozturk, M.
Open Access
Germline hMSH2 and hMLH1 gene mutations in incomplete HNPCC families
(1997) Wang, Q.; Desseigne, F.; Lasset, C.; Saurin J. C.; Navarro, C.; Yagci, T.; Keser I.; Bagci, H.; Luleci, G.; Gelen, T.; Chayvialle, J.-A.; Puisieux, A.; Ozturk, M.
Hereditary non-polyposis colon cancer (HNPCC) is a common hereditary disease characterized by a predisposition to an early onset of colorectal cancer. The majority of the HNPCC families carry germline mutations of either hMSH2 or hMLH1 genes, whereas germline mutations of hPMS1 and hPMS2 genes have rarely been observed. Almost all of the germline mutations reported so far concern typical HNPCC families. However, there are families that display aggregations of colon cancer even though they do not fulfil all HNPCC criteria (incomplete HNPCC families) as well as sporadic cases of early onset colon cancers that could be related to germline mutations of these genes. Therefore, we screened germline mutations of hMSH2 and hMLH1 genes in 3 groups of patients from France and Turkey: typical HNPCC (n = 3), incomplete HNPCC (n = 9) and young patients without apparent familial history (n = 7). By in vitro synthesis of protein assay, heteroduplex analysis and direct genomic sequencing, we identified 1 family with hMSH2 mutation and 5 families with hMLH1 mutations. Two of the 3 HNPCC families (66%) displayed hMLH1 germline mutations. Interestingly, 4 of 9 families with incomplete HNPCC (44%) also displayed mutations of hMSH2 or hMLH1 genes. In contrast, no germline mutation of these genes was found in 7 young patients. Our results show that germline mutations of hMSH2 and hMLH1 genes contribute to a significant fraction of familial predisposition to colon cancer cases that do not fulfil all diagnostic criteria of HNPCC. © 1997 Wiley-Liss, Inc.
Open Access
Evaluation of chimerism with DNA polymorphisms in bone marrow transplantation
(International Children's Center (I C C), 1997) Özbek, U.; Vural, B.; Kalayoǧlu, S.; Soysal, T.; Bilgen, H.; Yavuz, S.; Anak, S.; Sargın, D.; Gedikoǧlu G.; Ferhanoǧlu, Burhan; Akoǧlu, T.; Tangün, Y.; Özçelik, Tayfun
Evaluation of chimeric status following allogenlc BMT is an Important tool for monitoring the replacement of host cells with donor cells and for determining the risk of relapse. Polymorphic DNA sequences can be used as powerful markers in identification of donor/recipient genotype differences, even between close relatives. Polymerase chain reaction (PCR) amplification of three variable number of tandem repeat (VNTR) loci and five single-locus polymorphisms (SLP) was used to identify chimerism in 40 recipient-donor pairs. Mixed chimerism was present in 11 patients, and complete chimerism in 29. This PCR method is a rapid and sensitive assay to detect engraftment and evaluate relapse potential, and thus is very useful in the clinical management of BMT patients.
Open Access
Prognostic value of p53 gene mutations in a large series of node-negative breast cancer patients
(1998) Falette, N.; Paperin, M. P.; Treilleux, I.; Gratadour, A. C.; Peloux, N.; Mignotte, H.; Tooke, N.; Löfman, E.; Inganäs, M.; Bremond, A.; Ozturk, M.; Puisieux, A.
The most important subgroup of breast cancer patients for which reliable prognostic factors are needed are women without axillary lymph node involvement. Although overall, these patients have a good prognosis, it is known that 20-30% will experience a recurrence of the disease. To determine the prognostic significance of P53 tumor suppressor gene mutation, specimens from 113 primary breast cancers were evaluated for the presence of P53 alterations, as detected by cDNA sequencing of the entire coding sequence of the gene. The median follow-up for patients was 105 months. P53 gene mutation was an independent prognostic marker of early relapse and death. Our results suggest that P53 gene mutations could be an important factor to identify node-negative patients who have a poor prognosis in the absence of adjuvant therapy. Prospective studies should be designed to determine which therapy should be performed in this subgroup of patients.
Open Access
DNA end-independent activation of DNA-PK mediated via association with the DNA-binding protein C1D
(1998) Yavuzer, U.; Smith, G. C. M.; Bliss, T.; Werner, D.; Jackson, S. P.
DNA-dependent protein kinase (DNA-PK), which is involved in DNA double- strand break repair and V(D)J recombination, is comprised of a DNA-targeting component termed Ku and an ~465-kD catalytic subunit, DNA-PK(cs). Although DNA-PK phosphorylates proteins in the presence of DSBs or other discontinuities in the DNA double helix in vitro, the possibility exists that it is also activated in other circumstances via its association with additional proteins. Here, through use of the yeast two-hybrid screen, we discover that the recently identified high affinity DNA binding protein C1D interacts with the putative leucine zipper region of DNA-PK(cs). Furthermore, we show that C1D can interact with DNA-PK in mammalian cells and that C1D is a very effective DNA-PK substrate in vitro. Finally, we establish that C1D directs the activation of DNA-PK in a manner that does not require DNA termini. Therefore, these studies provide a function for C1D and suggest novel mechanisms for DNA-PK activation in vivo.
Open Access
Brachyury-related transcription factor Tbx2 and repression of the melanocyte-specific TRP-1 promoter
(1998) Carreira, S.; Dexter, T. J.; Yavuzer, U.; Easty, D. J.; Goding, C. R.
Previous work has demonstrated that two key melanocyte-specific elements termed the MSEu and MSEi play critical roles in the expression of the melanocyte-specific tyrosinase-related protein 1 (TRP-1) promoter. Both the MSEu and MSEi, located at position -237 and at the initiator, respectively, bind a melanocyte-specific factor termed MSF but are also recognized by a previously uncharacterized repressor, since mutations affecting either of these elements result in strong up-regulation of TRP-1 promoter activity in melanoma cells. Here we demonstrate that repression mediated by the MSEu and MSEi also operates in melanocytes. We also report that both the MSEu and MSEi are recognized by the brachyury-related transcription factor Tbx2, a member of the recently described T-box family, and that Tbx2 is expressed in melanocyte and melanoblast cell lines but not in melanoblast precursor cells. Although Tbx2 and MSF each recognize the TRP-1 MSEu and MSEi motifs, it is binding by Tbx-2, not binding by MSF, that correlates with repression. Several lines of evidence tend to point to the brachyury-related transcription factor Tbx2 as being the repressor of TRP-1 expression: both the MSEu and MSEi bind Tbx2, and mutations in either element that result in derepression of the TRP-1 promoter diminish binding by Tbx2; the TRP-1 promoter, but not the tyrosinase, microphthalmia, or glyceraldehyde-3- phosphate dehydrogenase (G3PDH) promoter, is repressed by Tbx2 in cotransfection assays; a high-affinity consensus brachyury/Tbx2-binding site is able to constitutively repress expression of the heterologous IE110 promoter; and a low-affinity brachyury/Tbx2 binding site is able to mediate Tbx2-dependent repression of the G3PDH promoter. Although we cannot rule out the presence of an additional, as yet unidentified factor playing a role in the negative regulation of TRP-1 in vivo, the evidence presented here suggests that Tbx2 most likely is the previously unidentified repressor of TRP-1 expression and as such is likely to represent the first example of transcriptional repression by a T-box family member.
Open Access
Thymidine dinucleotides induce S phase cell cycle arrest in addition to increased melanogenesis in human melanocytes
(1998) Pedeux, R.; Al-Irani, N.; Marteau, C.; Pellicier, F.; Branche, R.; Ozturk, M.; Franchi, J.; Doré, J. F.
Although the induction of pigmentation following exposure of melanocytes to ultraviolet light in vivo and in vitro is well documented, the intracellular mechanisms involved in this response are not yet fully understood. Exposure to UV-B radiation leads to the production of DNA damage, mainly cyclobutane pyrimidine dimers, and it was recently suggested that the thymidine dinucleotide pTpT, mimicking small DNA fragments released in the course of excision repair mechanisms, could trigger melanin synthesis. We now report that the thymidine dinucleotide pTpT induces melanogenesis both in human normal adult melanocytes and in human melanoma cells. Thus, the SOS- like response suggested by Gilchrest's work to be evolutionary conserved, based primarily on work in murine cells and guinea pigs, is also apparently present in the human. Thymidine dinucleotide is non toxic to melanoma cells and does not induce apoptosis in these cells, but induces S phase cell cycle arrest and a proliferation slow down. Because thymidine excess in culture medium leads to the synchronization of cells in S phase, we investigated whether this phenomenon was involved in the increase in melanin synthesis. We show that melanin synthesis is specifically triggered by the dimeric form of the thymidine and not by the monomeric form pT. Thus, our data strongly support that thymidine dinucleotides pTpT mimic at least part of the effects of ultraviolet irradiation, and may hence represent an invaluable model in the study of the molecular events involved in melanogenesis induction triggered through DNA damage.
Open Access
Turkish population data on the HLA-DQα, LDLR, GYPA, HBGG, D7S8, and GC loci
(Springer, 1998) Vural, B.; Atlioǧlu, E.; Kolusayin, Ö.; Togan, I.; Büyükdevrim, S.; Özçelik, T.
We have determined the allele and genotype frequencies of six PCR-based genetic markers HLA-DQα, LDLR, GYPA, HBGG, D7S8 and GC in the Turkish population (n = 361 for HLA-DQα, and n = 260 for PM). All loci meet Hardy- Weinberg expectations. The frequency data can be used in forensic analyses in the Turkish population.
Open Access
Functional role of the noncatalytic domains of elongation factor tu in the interactions with ligands
(1998) Cetin, R.; Anborgh, P. H.; Cool, R. H.; Parmeggiani, A.
Elongation factor (EF) Tu from Escherichia coli contains three domains, of which domain 1 (N-terminal domain) harbors the site for nucleotide binding and GTP hydrolysis. To analyze the function of domains 2 [middle (M) domain] and 3 [C-terminal (C) domain], EF-Tu(AM) and EF-Tu(δC) were engineered as GST-fused products and purified. Circular dichroism and thermostability showed that both constructs have conserved organized structures. Though inactive in poly(Phe) synthesis the two constructs could bind GDP and GTP with comparable micromolar affinities. Therefore, like the isolated N- terminal domain, they had lost a typical feature of EF-Tu, the ≤ 100 times stronger affinity for GDP than for GTP. EF-Tu(ΔM) and EF-Tu(AC) had an intrinsic GTPase activity comparable to that of wild-type EF-Tu. Ribosomes did not stimulate the GTPase activity of either factor, while kirromycin increased the GTPase activity of both constructs, particularly of EF-Tu(ΔC), to a level, however, much lower than that of the intact molecule. The interaction with aa-tRNA of both mutants was ≤90% reduced. As a major result, their GDP-bound form could efficiently respond to EF-Ts. All four EF- Tu-specific antibiotics [kirromycin, pulvomycin, GE2270 A (=MDL 62 879), and enacyloxin IIa] retarded significantly the dissociation of EF-Tu(ΔC)·GTP, showing the same kind of effect as on EF-Tu·GTP, but they were little active on EF-Tu(ΔM)·GTP. Like EF-Tu(ΔC)·GTP, EF-Tu(ΔM)·GTP was, however, able to bind efficiently kirromycin and enacyloxin IIa, as determined via competition with EF-Ts. Together, these results enlight selective functions of domains 2 and 3, particularly toward the interaction with EF-Ts and antibiotics, and emphasize their functional cooperativity for an efficient interaction of EF-Tu with ribosomes and aa-tRNA and for maintaining the differential affinity for GTP and GDP.
Open Access
p53 mutation with frequent novel codons but not a mutator phenotype in BRCA1-and BRCA2-associated breast tumours
(Nature Publishing Group, 1998) Crook, T.; Brooks, L. A.; Crossland, S.; Osin, P.; Barker, K. T.; Waller, J.; Philp, E.; Smith, P. D.; Yulug, I.; Peto, J.; Parker, G.; Allday, M. J.; Crompton, M. R.; Gusterson, B. A.
The status of p53 was investigated in breast tumours arising in germ-line carriers of mutant alleles of BRCA1 and BRCA2 and in a control series of sporadic breast tumours. p53 expression was detected in 20/26 (77%) BRCA1-, 10/22 (45%) BRCA2-associated and 25/72 (35%) grade-matched sporadic tumours. Analysis of p53 sequence revealed that the gene was mutant in 33/50 (66%) BRCA-associated tumours, whereas 7/20 (35%) sporadic grade-matched tumours contained p53 mutation (P < 0.05). A number of the mutations detected in the BRCA-associated tumours have not been previously described in human cancer databases, whilst others occur extremely rarely. Analysis of additional genes, p16(INK4), Ki-ras and β-globin revealed absence or very low incidence of mutations, suggesting that the higher frequency of p53 mutation in the BRCA-associated tumours does not reflect a generalized increase in susceptibility to the acquisition of somatic mutation. Furthermore, absence of frameshift mutations in the polypurine tracts present in the coding sequence of the TGF β type II receptor (TGF β IIR) and Bax implies that loss of function of BRCA1 or BRCA2 does not confer a mutator phenotype such as that found in tumours with microsatellite instability (MSI). p21(Waf1) was expressed in BRCA-associated tumours regardless of p53 status and, furthermore, some tumours expressing wild-type p53 did not express detectable p21(Waf1). These data do not support, therefore, the simple model based on studies of BRCA-/- embryos, in which mutation of p53 in BRCA-associated tumours results in loss of p21(Waf1) expression and deregulated proliferation. Rather, they imply that proliferation of such tumours will be subject to multiple mechanisms of growth regulation.
Open Access
Identification of relative protein bands in Polyacrylamide Gel Electrophoresis (PAGE) using multiresolution snake algorithm
(IEEE, 1998-10) Gürcan, Metin Nafi; Koyutürk, Mehmet; Yıldız, H. Serkan; Çetin Atalay, Rengül; Çetin, A. Enis
Polyacrylamide Gel Electrophoresis (PAGE) is one of the most widely used techniques in protein research. In the protein purification process, it is important to determine the efficiency of each purification step in terms of percentage of protein of interest found in the protein mixture. This study provides a rapid and reliable way to determine this percentage. The region of interest containing the protein is detected using the snake algorithm. The iterative snake algorithm is implemented in a multiresolutional framework. The snake is initialized on a low resolution image. Then, the final position of the snake at low resolution is used as the initial position in the higher resolution image. Finally, tile area of the protein is estimated as the area enclosed by the final position of the snake.
Open Access
Pax3 and regulation of the melanocyte-specific tyrosinase-related protein-1 promoter
(1999) Galibert, M. D.; Yavuzer, U.; Dexter, T. J.; Goding, C. R.
Previous work has established that the melanocyte-specific tyrosinase- related protein-1 (TRP-1) promoter is regulated positively by the microphthalmia-associated transcription factor Mitf, acting through the conserved M box and negatively by the T-box factor Tbx2, which can bind two 'melanoeyte-specific elements' termed the MSEu and MSEi. Both the MSEu and MSEi, which share a 6-base pair GTGTGA consensus, are also recognized by a previously unidentified melanocyte-specific factor, MSF. Here we show using a combination of DNA binding assays, proteolytic clipping, and anti-Pax3 antibodies that MSF is indistinguishable from Pax3, a paired homeodomain transcription factor implicated genetically in melanocyte development and the regulation of the Mitf promoter. Consistent with Pax3 being able to bind the TRP-1 promoter, Pax3 is expressed in melanocytes and melanomas, and TRP-1 promoter activity is up-regulated by Pax3. The results identify a novel role for Pax3 in the expression of TRP-1, and the potential role of Pax3 in the melanoeyte lineage is discussed.
Open Access
A retrospective comparison of allogeneic peripheral blood stem cell and bone marrow transplantation results from a single center: a focus on the incidence of graft-vs.-host disease and relapse
(Elsevier, 1999) Üstün, C.; Arslan, Ö.; Beksaç, M.; Koç, H.; Gürman, G.; Özçelik, T.; Yilmaz, B.; İlban, O.; Akan, H.; Özcan, M.; Demirer, T.; Uysal, A.; Konuk, N.; Arat, M.; Dilek, İ.; Çelebi, H.; Coskun, H. S.
To detect the effect of the stem cell source, allogeneic peripheral blood stem cell transplantations (alloPBSCTs) performed between 1995 and 1997 from human leukocyte antigen (HLA)-identical siblings in 40 patients with acute and chronic hematological disorders were compared with a historical group of 40 patients with similar variables who had received allogeneic bone marrow transplants (alloBMTs) between 1993 and 1995. Patients in both groups were identical except that both the recipient and the donor ages were, on average, higher in the alloPBSCT group (26 vs. 36 [p = 0.005] and 27 vs. 32 [p = 0.024], respectively). Patients received similar therapy excluding posttransplant granulocyte colony-stimulating factor administration (97% in alloBMT vs. 12.5% in alloPBSCT). The median time to reach neutrophil counts >0.5×109/L and platelet counts >20×109/L was 13 and 14 days, respectively, in patients receiving alloPBSCTs compared with 19 and 27 days in patients receiving alloBMTs (p = 0.0014 and p = 0.0002). The alloPBSCT group required similar transfusions of red blood cells or platelets. The incidence of grade II-IV acute graft-vs.-host disease (aGVHD) was similar in both groups. However, chronic GVHD (cGVHD) of all grades developed in 78.1% of patients in the alloPBSCT group after a median follow-up period of 12.5 (range 0.5-34) months. In alloBMT recipients, cGVHD of all grades developed in 21.4% after a median follow-up period of 38 (range 0.5-62) months (p = 0.00001). Day 100 transplant-related mortality was also similar: 20% (8 of 40) in the alloBMT patients and 17.5% (7 of 40) in the alloPBSCT group. Although not statistically significant, a relatively higher relapse rate occurred in the alloBMT group (21.4 vs. 10.7%). The estimated disease-free survival in month 24 was 51.3% for alloBMT and 54.6% for alloPBSCT, and the estimated overall survival in month 24 was 56.1% for alloBMT and 64.6% for alloPBSCT. In conclusion, this retrospective comparison suggests that alloPBSCT from HLA-identical donors is associated with faster engraftment, fewer transfusions, and no greater incidence of aGVHD, but a high incidence of cGVHD.
Open Access
Ectopic expression of RET results in microphthalmia and tumors in the retinal pigment epithelium
(1999) Schmidt, A.; Tief, K.; Yavuzer, U.; Beermann, F.
The retinal pigment epithelium (RPE) is essential for eye development by interacting with the overlaying neuroepithelium. Regulatory sequences of the gene encoding for tyrosinase-related protein 1 (TRP-1), linked to the lacZ reporter gene, lead to strong and specific β-galactosidase expression in the RPE. We asked how the oncogene ret would affect this epithelial cell type during mouse development. We used the TRP-1 promoter to express ret in the developing RPE, and obtained transgenic mouse lines, which showed mild to severe microphthalmia. During development, the RPE changed to a stratified epithelium with reduced or absent pigmentation from E10.5 onward. In addition, proliferation of RPE cells and tumor formation were observed from E12.5 onward. These early events prevent closure of choroid fissure and lead to microphthalmia and secondary malformations after birth. We conclude that ret transgene expression in the RPE prevents normal differentiation of this epithelial layer and induces proliferation and tumor formation. The appearance of the microphthalmic phenotype underlines the requirement of a normally developed RPE for eye development.
Open Access
Smad2 and Smad4 gene mutations in hepatocellular carcinoma
(1999) Yakicier, M. C.; Irmak, M. B.; Romano, A.; Kew, M.; Ozturk, M.
TGF-β is a negative regulator of liver growth. Smad family of genes, as mediators of TGF-β pathway, are candidate tumor suppressor genes in hepatocellular carcinoma (HCC). We studied 35 HCC and non-tumour liver tissues for possible mutations in Smad2 and Smad4 genes. Three tumours displayed somatic mutations; two in Smad4 (Asp332Gly and Cys401Arg) and one in Smad2 (Gln407Arg) genes. All three mutations were A:T → G:C transitions suspected to result from oxidative stress as observed in mitochondrial DNA. These observation demonstrate that TGF-β pathway is altered in hepatocellular carcinoma.
Open Access
Factors in the success and failure of microbial agents for control of migratory pests
(1999) Lomer, C. J.
Microbial control agents generally kill insects more slowly than chemical pesticides, and fast-moving migratory pests may not at first sight appeal' to offer the most promising targets for microbial control. Operators responsible for control may need to have recourse to chemical control agents. Nevertheless, there are many occasions when pests breed and feed outside the crop and a microbial control agent can be used. Similarly, immature stages may cause little damage and early treatment in the crop can avoid damage. Microbial control agents are particularly likely to be favoured if the pest breeds in a conservation area, and if a publicly-accountable agency is responsible for control. Other key points of importance are the IPM context, in particular detection, planning and forecasting of outbreaks and the role of natural enemies. With these points in mind, we identify several locust and grasshopper systems where microbial control is becoming established; additionally, Sunn pest of wheat and Armyworm are identified as promising situations for microbials.