Žmogaus baltymo p14.5 geno struktūra bei funkcija normaliose ir onkoproliferuojančiose ląstelėse ; Human protein p14.5 gene structure and function in normal and oncoproliferating cells
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Žmogaus baltymo p14.5 geno struktūra bei funkcija normaliose ir onkoproliferuojančiose ląstelėse ; Human protein p14.5 gene structure and function in normal and oncoproliferating cells

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Natalija POZDNIAKOVAIT Ė HUMAN PROTEIN p14.5 GENE STRUCTURE AND FUNCTION IN NORMAL AND ONCOPROLIFERATING CELLS Summary of Doctoral Dissertation Technological Sciences, Chemical Engineering (05T), Biotechnology (T490) 1273 Vilnius „Technika“ 2006 VILNIUS GEDIMINAS TECHNICAL UNIVERSITY INSTITUTE OF BIOTECHNOLOGY Natalija POZDNIAKOVAIT Ė HUMAN PROTEIN p14.5 GENE STRUCTURE AND FUNCTION IN NORMAL AND ONCOPROLIFERATING CELLS Summary of Doctoral Dissertation Technological Sciences, Chemical Engineering (05T), Biotechnology (T490) Vilnius „Technika“ 2006 Doctoral dissertation was prepared at Institute of Biotechnology 1999–2006. The dissertation is defended as an external work.

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    Natalija POZDNIAKOVAIT Ė      HUMAN PROTEIN p14.5 GENE STRUCTURE AND FUNCTION IN NORMAL AND ONCOPROLIFERATING CELLS    Summary of Doctoral Dissertation Technological Sciences, Chemical Engineering (05T), Biotechnology (T490)       Vilnius Technika 2006
 
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VILNIUS GEDIMINAS TECHNICAL UNIVERSITY INSTITUTE OF BIOTECHNOLOGY       Natalija POZDNIAKOVAIT Ė      HUMAN PROTEIN p14.5 GENE STRUCTURE AND FUNCTION IN NORMAL AND ONCOPROLIFERATING CELLS      Summary of Doctoral Dissertation Technological Sciences, Chemical Engineering (05T), Biotechnology (T490)        Vilnius Technika 2006
 
 
Doctoral dissertation was prepared at Institute of Biotechnology 19992006. The dissertation is defended as an external work.  Scientific Consultant Dr Violeta POPENDIKYT Ė  (Biomedical Research Center, Technological Sciences, Chemical Engineering  05T)  The Dissertation is being defended at the Council of Scientific Field of Chemical Engineering at Vilnius Gediminas Technical University: Chairman   Prof Dr Habil Juozas KULYS (Vilnius Gediminas Technical University, Technological Sciences, Chemical Engineering  05T) Members: Prof Dr Habil Algirdas EMAITAITIS (Kaunas University of Technology, Technological Sciences, Chemical Engineering  05T) Dr Jolanta SEREIKAIT Ė  (Vilnius Gediminas Technical University, Technological Sciences, Chemical Engineering  05T)  Dr Aurelija VIRBLIEN Ė  (Institute of Biotechnology, Physical Sciences, Biochemistry  04P) Dr Ar ū nas LAGUNAVI Č IUS  (Fermentas, Physical Sciences, Biochemistry  04P) Opponents: Dr Alma GEDVILAIT Ė  (Institute of Biotechnology, Technological Sciences, Chemical Engineering  05T) Dr R ū ta NAVAKAUSKIEN Ė  (Institute of Biochemistry, Biomedical Sciences, Biology  01B)  The dissertation will be defended at the public meeting of the Council of Scientific Field of Chemical Engineering in the Senate Hall of Vilnius Gediminas Technical University at 1 p. m. on 16 June 2006. Address: Saul ė tekio al. 11, LT-10223 Vilnius-40, Lithuania Tel.: +370 5 274 49 52, +370 5 274 49 56; fax +370 5 270 01 12; e-mail doktor@adm.vtu.lt  The summary of the doctoral dissertation was  distributed on 16 May 2006 A copy of the doctoral dissertation is available for review at the Libraries of Vilnius Gediminas Technical University (Saul ė tekio al. 14, Vilnius, Lithuania) and Institute of Biotechnology (Graiciuno 8, Vilnius, Lithuania)  © Natalija Pozdniakovait ė , 2006  
 
 
 
 
 VILNIAUS GEDIMINO TECHNIKOS UNIVERSITETAS BIOTECHNOLOGIJOS INSTITUTAS       Natalija POZDNIAKOVAIT Ė     MOGAUS BALTYMO p14.5 IR JO GENO STRUKT Ū RA BEI FUNKCIJA NORMALIOSE IR ONKOPROLIFERUOJAN Č IOSE L Ą STEL Ė SE      Daktaro disertacijos santrauka Technologijos mokslai, chemijos ininerija (05T), biotechnologija (T490)       Vilnius Technika 2006  
 
 
Disertacija  rengta  1999 2 006  metais  Biotechnologijos institute. Disertacija ginama eksternu.  Mokslinis konsultantas dr. Violeta POPENDIKYT Ė  (Biomedicinos tyrim ų  centras, technologijos mokslai, chemijos ininerija  05T).  Disertacija ginama Vilniaus Gedimino technikos universiteto Chemijos ininerijos mokslo krypties taryboje: Pirmininkas prof. habil. dr. Juozas KULYS  (Vilniaus Gedimino technikos universitetas, technologijos mokslai, chemijos ininerija  05T). Nariai: prof. habil. dr. Algirdas EMAITAITIS  (Kauno technologijos universitetas, technologijos mokslai, chemijos ininerija  05T), dr. Jolanta SEREIKAIT Ė  (Vilniaus Gedimino technikos universitetas, technologijos mokslai, chemijos ininerija  05T), dr. Aurelija VIRBLIEN Ė (Biotechnologijos institutas, fiziniai mokslai, biochemija  04P), dr. Ar ū nas LAGUNAVI Č IUS (Fermentas, fiziniai mokslai, biochemija  04P). Oponentai: dr. Alma GEDVILAIT Ė  (Biotechnologijos institutas, technologijos mokslai, chemijos ininerija  05T), dr. R ū ta NAVAKAUSKIEN Ė  (Biochemijos institutas, biomedicinos mokslai, biologija  01B).  Disertacija bus ginama vieame Chemijos ininerijos mokslo krypties tarybos pos ė dyje 2006 m. birelio 16 d. 13 val. Vilniaus Gedimino technikos universiteto senato pos ė di ų sal ė je. Adresas: Saul ė tekio al. 11, LT-10223 Vilnius-40, Lietuva. Tel. +370 5 274 49 52, +370 5 274 49 56, faksas +370 5 270 01 12, el. patas doktor@adm.vtu.lt  Disertacijos santrauka isiuntin ė ta 2006 m. gegu ė s 16 d. Disertacij ą  galima peri ū r ė ti Vilniaus Gedimino technikos universiteto (Saul ė tekio al. 14, Vilnius, Lietuva) ir Biotechnologijos instituto (V. A. Grai č i ū no g. 8, Vilnius, Lietuva) bibliotekose. VGTU leidyklos Technika 1273 mokslo literat ū ros knyga. © Natalija Pozdniakovait ė , 2006
 
 
INTRODUCTION  Topicality of the problem.  Human p14.5 perchloric acid-soluble protein (PSP) is a member of family (YER057c/YJGF family) of small proteins. The highly conserved throughout evolution proteins are characterized as multifunctional proteins comprised of ~ 80 homologues exhibiting varying degrees of sequence homology from 38 % to 93 %. The proteins are involved in different cellular pathways of lower and higher eukaryotes. The biological role of these proteins is not yet well characterized. Human p14.5 protein is homologue of UK114 protein extracted from goat liver and characterized as tumor antigen. Antibodies raised against UK114 mediate in vitro complement-depended cytolysis of human cancer cells. This suggests that the expression of human p14.5 protein on the cells membrane might be an event related to neoplasmic transformation. Available literature data demonstrate that human p14.5 gene acts as human cancer antigen and is involved into cellular proliferation events. Human p14.5 gene has been characterized by a differentiation-dependent mRNA and protein expression based on the observation of a low expression in a variety of liver and kidney tumor cells and a high expression in fully differentiated cells. At the beginning of our studies no data about human p14.5 gene full structure, coding protein p14.5 structure, biological functions of target protein in normal and tumor cells were available. The research study was initiated and supported by biopharmaceutical company UAB Sicor-Biotech and granted by Lithuanian State Science and Studies Foundation. Aim and tasks of the work 1. Human p14.5 gene DNA structure determination. 2. Analysis of genetic alterations in coding and exon-intron junction sequences of human p14.5 gene in normal and tumor cells. 3. Construction and expression of human p14.5 protein mutants by site-directed mutagenesis designed for detailed protein structure analysis. 4. Determination of metabolic pathways of Hmf1p protein (human p14.5 homologue in yeast) using differential display analysis of mRNA expression. 5. Analysis of DNA methylation differences in human p14.5 gene promoter region in normal and proliferating cells. Scientific novelty Complete nucleotide sequence and exon-intron structure of human p14.5 gene have been determined (GenBank, accession number: AY026764).  Polymorphic nucleotides have been determinated in human p14.5 gene in normal and tumour cells by using bidirectional fingerprinting of DNA fragments of target gene.
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 Different expression of twelve genes associated with HMF1  (human p14.5 homologue in yeast) protein has been determinated in yeast S. cerevisiae cells. High degree of DNA methylation differences of human p14.5 gene promoter sequences in several human normal tissues and tumor cells lines has been established.  DNA methylation changes associated with the cells differentiation has been investigated in p14.5 5-flanking region by using DNA samples of freshly isolated monocytes and macrophages. Defended propositions 1. Complete structure of chromosomal human p14.5 gene consist of 6466 base pairs, contains 5 exons and 4 introns. 2. Human p14.5 gene is a very conservative in normal and cancer cell. 3. Trimeric structure of protein p14.5 is essential for stoichiometric small ligand binding activity and oligomeric structure of p14. 4. Deficiency of Hmf1p protein (p14.5 homologue) affected expression of twelve genes of yeast S. cerevisiae.  5. 5-flanking region of human p14.5 gene is significantly methylated in cancer cells and unmethylated in normal tissues. 6. Cellular differentiation processes from monocytes to macrofages are related with the elevated degree of DNA methylation of p14.5 gene. Practical value The obtained research results extended knowledge about p14.5 gene structure, functions in normal cells and expression of gene regulation during cells proliferation and differentiation. Determination of new markers of cancer cells created possibility to establish additional genetics and epigenetics means to study cells oncoproliferation. The scope of the scientific work The dissertation (in Lithuania) consists from the following parts: Introduction, Investigation Review, Materials and Methods, Results and Discussion, List of References (111), Tables (10) and Figures (26). Total 98 pages.  MATERIALS AND METHODS  Tissues and cell cultures: DNA samples were extracted from human liver and kidney tissue samples post-mortem from four individuals (Centre of Pathology, Vilnius, Lithuania), from following human cancer cells: Huh7 (hepatoma), HeLa (cervix carcinoma), Jurkat (acute T-cell leukemia), A549 (lung carcinoma), TF1 (bone marrow erythroleukemia), K562 (chronic myelogenous leukemia), COLO (colon adenocarcinoma), HuT cells (T-cell leukemia). DNA
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samples have been extracted from freshly isolated mononuclear particles (MNP, monocytes).  DNA preparation:  DNA samples were extracted by using standard phenol-chloroform DNA extraction procedure. Briefly, cells (1-3x10 6 cells) and tissues were suspended in a buffer, containing 50 mM NaCl, 20 mM Tris-HCl, pH 8,0, 20 mM EDTA (tissue samples (0,1-0,2 g) were additionally homogenized in liquid nitrogen before suspension), lysed by adding SDS solution up to final concentration 1 %, extracted twice by equal volume of phenol/chloroform mixture (1:1) and ethanol precipitated. p14.5 cDNA cloning and expression: p14.5 cDNA was obtained by PCR from human liver cell cDNA library. The PCR product through HindIII and NdeI restriction endonuclease sites was cloned into pUC57/T plasmid DNA, sequenced and re-cloned into expression vector pET-28. Site-directed mutagenesis of p14.5 gene:  Amino acid substitutions in the human p14.5 protein sequence were made by using PCR and specific primers with the sites for BpiI restriction endonuclease at the 5 ends. Plasmids DNA were extracted from several clones after each site-directed mutagenesis and made alterations of nucleotides in the p14.5 gene structure were confirmed by sequencing.   RNA extraction from  S. cerevisiae cells:  total RNA preparations have been prepared according to the protocol (Schimtt et al. 1990). Preparation of cDNA: cDNA was synthesized by using RevertAid TM H Minus First Strand cDNA Synthesis Kit. End-labelling of oligo d(T) 13 N primer:  primers were labeled by using T4 polynucleotide kinase and [ γ -33 P]  ATP. Amplification of cDNA: cDNA was amplified by using P 33  labeled oligod(T) 13 N primer and two or three arbitrary primers. Extraction, amplification and transformation of DNA sequences:  DNA bands were cut out from a gel, DNA was eluted and amplified by using the same primers, cloned into pUC57T vector and transformed into E. coli XL Blue-1 strain. Analysis of sequences of differentially expressed DNA:  Plasmid DNA samples were extracted and nucleotide sequences of inserts were determined using CycleReader TM  DNA Sequencing kit. Specific yeast genes or noncoding sequences were identified according to yeast S. cerevisiae  Gene bank database. Verification of differentially expressed genes:  Differences in genes expression were estimated as percentage ratios of quantity (labeled PCR products) of tested gene and control one (yeast actin gene) in w.t. and mutated yeast cells.
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Bisulphite DNA modification: DNA (0.5-0.7 µg) samples were digested with restriction endonuclease BglII and modified by sodium bisulphite according to protocol (Olek et al. 1996). PCR amplifications of bisulphate treated DNA : human p14.5 promoter region was amplified using hot start PCR and nested  primers. The PCR products were separated in agarose gel, extracted from the gel and cloned into pUC57/T vector. Recombinant plasmid DNA samples were extracted and nucleotide sequences of inserts were determined.  RESULTS AND DISCUSSION  1. Determination of human p14.5 gene nucleotide sequence:  According to p14.5 gene cDNA sequence six pairs of primers have been designed (approximately in 100 bp distances) for p14.5 genomic sequences amplification. Three pairs of primers resulted in high molecular weight products and were cloned into E. coli cells with the following nucleotide sequence determination. Comparison of nucleotide sequences of obtained PCR products with the cDNA sequence of target gene cDNA allowed to determine the positions and number of intron sequences of human p14.5 gene. Exon-intron splising sites in genes of eukaryotic cells are very conservative. Data obtained in the research study confirmed that exon-intron splising sites in human p14.5 gene correspond to same sequences of eukaryotic cells. All intron sequences of human p14.5 gene start with GT and finish with AG nucleotides. The length of target gene without 3- untranslated region is 5954 base pairs. Human p14.5 gene contains 5 exons and 4 introns. Data of human p14.5 gene structure was submitted to Gene data bank, accession number AY026764. 2. Analysis of genetic alterations in human p14.5 gene in normal and tumor cells by using bidirectional fingerprinting of DNA fragments of target gene:  The aim of that part of research study was to elucidate whether the expression of p14.5 protein epitopes on the human tumor cells membranes are associated with the genetic alterations of target gene in normal and proliferating cells. DNA samples were extracted from human liver normal and above-mentioned human cancer cells. Analysis of genetic alterations in normal and tumor cells by using bidirectional fingerprinting of DNA fragments of target gene was applied to coding sequences, exon-intron splising regions, 5-UTR and 3-UTR (UTR- untranslated region) of human p14.5 gene. Obtained data indicated very conservative human p14.5 gene exons sequences in normal and cancer cells. Several polymorphic alleles have been detected in 5- UTR and first intron. A/C polymorphism at -105 nucleotide in the promoter region of
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p14.5 gene matches with the putative binding site for transcription factor alpha-PAL, responsible for cells growth and proliferation. 3. Structure based ligand binding sites of protein p14.5, a member of protein family YER057c/YIL051c/ YJGF:  Important point of the research study  the description of structure-based ligand binding sites of human p14.5 protein. By using site-specific mutagenesis procedure amino acids into p14.5 protein monomer:monomer sites of interaction were altered. 22 mutants with one, two or three amino acids substitutions of human protein p14.5 have been prepared. Normal p14.5 gene and all mutated variants of p14.5 were overexpressed after traditional procedure of induction by IPTG. All mutants of p14.5 protein were used for structure-functional relation studies and ligand binding analysis using cross-linking by TAT, size exclusion chromatography, free fatty acid and ANS binding assays (structure-functional relation studies on the basis of p14.5 mutants were done by dr. E. Mistiniene, Institute of Biotechnology, Vilnius). Arginine 107 has been indicated as the most accessible amino acid in the cleft. Trimeric structure of protein p14.5 has been proven as essential for stoichiometric small ligand binding activity and oligomeric structure of p14.5. Ligand binding activity may be related with biological functions of these proteins, which still are not understood well. Detailed structure analysis of p14.5 protein and its variants indicated that single amino acid substitutions had no significant effect, only Arg107 proved to be important for stabilization of trimeric structure of p14.5. 4. Identification and characterization of differentially expressed genes in yeast S. cerevisiae  cells with inactivated Mmf1p and Hmf1p, members of proteins family YERO57c/YJGF: Determination of differences in gene expression in normal and knock-outed cells is a common experimental model in numerous research studies and might clear a role of proteins family in the regulation of cells metabolic pathways, transcriptional regulation and other properties of genes of interest. DD is powerful method that employs random reverse-transcription of RNA species, PCR and electrophoresis for comparative analysis of two or more transcriptomes. Two members  of the p14.5 family are present in the budding yeast S. cerevisiae . There we present experimental data obtained by DD by using normal S. cerevisiae  cells and cells with the inactivated genes MMF1 , HMF1  and both of them - homologues of p14.5. Mammalian protein  human p14.5 is functionally related to Mmf1p.  Human p14.5 localizes in yeast mitochondria and rescues the mmf1-associated  phenotypes. S. cerevisiae  yeast strain AH/22  derivatives: w. t. , mmf1 , hmf1,  mmf1  hmf1 were kindly provided by Dr Alma Gedvilaite (Laboratory of Eukaryotes Genetic Engineering, Institute of Biotechnology, Vilnius). Yeast RNA samples preparation was followed by reverse transcription of RNA targets
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and controls samples. cDNA samples were amplified by using modified oligod(T) 13 (N) primers and two or three upstream random primers. Side-by-side comparisons of amplified target cDNA patterns reveal differences in gene expression. Differentially expressed DNA bands were excited from the gel, re-amplified, cloned, sequenced and sequences queried against National Center for Biotechnology Information (NCBI) Yeast genome databases. A mach was defined as > 97% identit of bases over a stretch of 30-100 bases.
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