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Human gene mapping using the polymerase chain reaction / Malika Nebbali 

Public ISBD Titre : Human gene mapping using the polymerase chain reaction Type de document : texte imprimé Auteurs : Malika Nebbali, Auteur ; Stuart Brown, Directeur de thèse Editeur : Nottingham : University of Nottingham Année de publication : 1992 Importance : 153 f. Présentation : ill. Format : 30 cm. Note générale : Thèse de Doctorat : Génie Chimique : Angleterre, University of Nottingham : 1992 Bibliogr. f. 154 - 167 . Annexe [3] f Langues : Anglais (eng) Mots-clés : Gene  mapping Hybridisation  techniques DNA  polymorphism Polymerase  chain  reaction  methods Chemical  carcinogenesis Chromatographic  techniques Immunological  methods Index. décimale : D001492 Résumé : There has been strong association between cigarette smoking and the incidence of lung cancer. Cigarette smoke is a rich source of PAH and it has been reported that high AHH activities in individuals makes them at high risk to lung cancer if they smoke cigarettes. In previous work a gene necessary for AHH induction was found to segregate with chromosome 2 markers; MDH-1, IDH-1 and ACP in human x mouse hybrid cells and was mapped to 2q31 --> 2pter. However a hybrid subclone containing a small deletion 2p25 --> 2pter which did not express MDH-1, ACP-1 or AHH was isolated. This could mean that the genes for these enzymes were actually missing from the chromosome and thus are located in the small deleted region. It is also possible that they are present elsewhere on the chromosome byt in an inactive form. Whichever explanation was true mapping of the three enzymes was of interest and MDH-1 was chosen as the enzyme to be mapped. Mapping of MDH-1 would strongly suggest that the genes for the other two enzymes were located in the same region. Since no data was available concerning the human MDH-1 gene, a strategy was designed in which MDH-1 was to be purified to homogeneity from red blood cells and the purified protein was to used to raise antibodies in rabbits. The aim then was to isolate the MDH-1 gene by immunological screening of a human λgt11 cDNA expression library. The gene would be subsquently used as a probe in in-situ hybridization techniques. MDH-1 was purified from human red blood cells using polyethylene glycol fractionation. The activity precipitated between 8 and 16%. Solubilised protein was separated by DEAE-cellulose and agarose affinity chromatography. Homogeneous enzyme was prepared by removing the contaminating proteins by FPLC using a Superose 12 gel filtration column. A 5500 fold purification was achieved. The purified preparation, had a molecular weight of approximatly 76000 and 38000 after sodium dodecyl sulphate gel electrophoresis. Thus, the enzyme is composed of two subunits of equal molecular weight. Antibodies were raised by injecting a rabbit with the agarose affinity fraction, but failed to detect the gene of interest after immunological screening of a human placental cDNA expression library. A second strategy was designed which involved sequencing peptides generated from the pure protein. The data obtained was used to sythesize oligonucleotides which served as primers for the amplification of specific regions of DNA from the MDH-1 gene via the polymerase chain reaction (PCR). Four peptides were produced by cyanogen bromide cleavage, purified using HPLC and sesuenced. It was found that the enzyme is highly homologous to the mouse enzyme as only one major difference was apparent at residue 288 where leucine was replaced by phenyalanine in humans. Three oligonucleotides called, 21, 31 and 30 mers respectively were synthesized P₄ and P₅ were used in PCRs for the amplification of specific regions of the MDH-1 gene which corresponded to 872 bp and 708 bp in length for the mouse and human genomic DNA respectively. The 708 bp product was purified from the agarose gel and was subjected to direct DNA sequencing. Comparison of the data obtained with the known mouse MDH-1 gene sequence strongly suggested the 708 bp band being amplified was human MDH-1. The fact that the amplification of human and mouse genomic DNA yielded products of different size had a major impact on future work. It was realised that it would not be necessary to carry out in-situ hybridisation to dorectly test for the presence or absence of the MDH-1 gene in hybrid cells. PCR alone would be able to give the desired results in a fraction of the time. Human gene mapping using the polymerase chain reaction [texte imprimé] / Malika Nebbali, Auteur ; Stuart Brown, Directeur de thèse . - Nottingham : University of Nottingham, 1992 . - 153 f. : ill. ; 30 cm. Thèse de Doctorat : Génie Chimique : Angleterre, University of Nottingham : 1992 Bibliogr. f. 154 - 167 . Annexe [3] f Langues : Anglais (eng) Mots-clés : Gene  mapping Hybridisation  techniques DNA  polymorphism Polymerase  chain  reaction  methods Chemical  carcinogenesis Chromatographic  techniques Immunological  methods Index. décimale : D001492 Résumé : There has been strong association between cigarette smoking and the incidence of lung cancer. Cigarette smoke is a rich source of PAH and it has been reported that high AHH activities in individuals makes them at high risk to lung cancer if they smoke cigarettes. In previous work a gene necessary for AHH induction was found to segregate with chromosome 2 markers; MDH-1, IDH-1 and ACP in human x mouse hybrid cells and was mapped to 2q31 --> 2pter. However a hybrid subclone containing a small deletion 2p25 --> 2pter which did not express MDH-1, ACP-1 or AHH was isolated. This could mean that the genes for these enzymes were actually missing from the chromosome and thus are located in the small deleted region. It is also possible that they are present elsewhere on the chromosome byt in an inactive form. Whichever explanation was true mapping of the three enzymes was of interest and MDH-1 was chosen as the enzyme to be mapped. Mapping of MDH-1 would strongly suggest that the genes for the other two enzymes were located in the same region. Since no data was available concerning the human MDH-1 gene, a strategy was designed in which MDH-1 was to be purified to homogeneity from red blood cells and the purified protein was to used to raise antibodies in rabbits. The aim then was to isolate the MDH-1 gene by immunological screening of a human λgt11 cDNA expression library. The gene would be subsquently used as a probe in in-situ hybridization techniques. MDH-1 was purified from human red blood cells using polyethylene glycol fractionation. The activity precipitated between 8 and 16%. Solubilised protein was separated by DEAE-cellulose and agarose affinity chromatography. Homogeneous enzyme was prepared by removing the contaminating proteins by FPLC using a Superose 12 gel filtration column. A 5500 fold purification was achieved. The purified preparation, had a molecular weight of approximatly 76000 and 38000 after sodium dodecyl sulphate gel electrophoresis. Thus, the enzyme is composed of two subunits of equal molecular weight. Antibodies were raised by injecting a rabbit with the agarose affinity fraction, but failed to detect the gene of interest after immunological screening of a human placental cDNA expression library. A second strategy was designed which involved sequencing peptides generated from the pure protein. The data obtained was used to sythesize oligonucleotides which served as primers for the amplification of specific regions of DNA from the MDH-1 gene via the polymerase chain reaction (PCR). Four peptides were produced by cyanogen bromide cleavage, purified using HPLC and sesuenced. It was found that the enzyme is highly homologous to the mouse enzyme as only one major difference was apparent at residue 288 where leucine was replaced by phenyalanine in humans. Three oligonucleotides called, 21, 31 and 30 mers respectively were synthesized P₄ and P₅ were used in PCRs for the amplification of specific regions of the MDH-1 gene which corresponded to 872 bp and 708 bp in length for the mouse and human genomic DNA respectively. The 708 bp product was purified from the agarose gel and was subjected to direct DNA sequencing. Comparison of the data obtained with the known mouse MDH-1 gene sequence strongly suggested the 708 bp band being amplified was human MDH-1. The fact that the amplification of human and mouse genomic DNA yielded products of different size had a major impact on future work. It was realised that it would not be necessary to carry out in-situ hybridisation to dorectly test for the presence or absence of the MDH-1 gene in hybrid cells. PCR alone would be able to give the desired results in a fraction of the time.

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