I= CLONING OF MICROCOCCUS LUTEUS REPAIR GENES
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Title of Thesis
CLONING OF MICROCOCCUS LUTEUS REPAIR GENES

Author(s)
Bushra Chaudhry
Institute/University/Department Details
Centre for Advanced Molecular Biology/ University of the Punjab
Session
1991
Subject
Molecular Biology
Number of Pages
270
Keywords (Extracted from title, table of contents and abstract of thesis)
micrococcus luteus, genes, dna, repair enzymes, adp-ribosylation, padparp activity, mnng, psr75, psr99, m.luteus

Abstract
No Abstract

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1385.62 KB
S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
79.07 KB
2 1 General Introduction 1
54.88 KB
3 2 Dna Repair Enzymes 7
204.82 KB
  2.1 Perspectives 7
  2.2 Base Excision Repair Enzymes 7
  2.3 Genetics Of Base Excision Repair Enzymes 15
  2.4 Direct Repair 16
  2.5 Genetics Of Adaptive Repair Pathway 20
  2.6 Regulation Of Adaptive Repair Pathway 23
  2.7 Nucleotide Excise Repair 25
  2.8 Genetics Of Nucleotide Excision Repair 25
  2.9 Mismatch Repair 27
  2.10 Sos Response 28
4 3 Adp-Ribosylation 29
70.92 KB
  3.1 Perspectives 29
  3.2 The Poly ( Adp-Ribosyl ) Polymerase 29
  3.3 Genetics And Cloning Of Poly ( Adp -Ribose) Polymerase 30
  3.4 Padp-Activity Is Modulated By Dna Strand Breakage 31
  3.5 Specificity Of Poly( Adp -Ribose) Polymerase Inhibitors 32
  3.6 Padprp Participates In Dna Excision Repair 33
  3.7 Poly ( Adp -Ribose) Acceptor Proteins 34
5 4 Materials And Methods 36
217.42 KB
  4.1 Chemicals And Regents 36
  4.2 Radiochemicals Mutagens And Enzymes 36
  4.3 Bacterials Strains And Media 36
  4.4 General Method Of Dna Manipulation 39
  4.5 Isolation Of Chromosomal Dna 39
  4.6 Isolation Of Plasmid Dna 39
  4.7 Transformation Of E. Coli 40
  4.8 Elution Of Dna Fragments 40
  4.9 Cloning Of M. Luteus Genes In Pbr322 41
  4.10 Cell Survival 42
  4.11 Adaptation 42
  4.12 Preparation Of Protein Extract 42
  4.13 Preparation Of Substrate Dna 43
  4.14 In Vitro Removal Of Modified Bases 44
  4.15 Separation Of Released Bases 44
  4.16 Construction Of Partial Restriction Map Of Psr75 And Psr99 45
  4.17 Expression In Maxi Cells 45
  4.18 Sub-Cloning Psr75 Plasmid 45
  4.19 Construction Of Ubiquitin Fused Padprp Vector 46
  4.20 Screening For Hybrid Plasmids 54
  4.21 Gene Expression 57
  4.22 Sample Preparation And Sds -Page Electrophoresis 57
  4.23 Western Blotting 58
  4.24 Padparp Activity 59
  4.25 Activity Gel Analysis 59
  4.26 Survival Of Uv Irradiated Cells 60
  4.27 In Vitro Interaction Between Mnng And Ubpadprp Polymerase Protein 61
  4.28 Gamma Radiations Survival Studies 61
6 5 Results 62
453.63 KB
  5.1 Construction Of Mnng Resistant Hybrid Plasmid 62
  5.2 Functional Activity Of Cloned Gene Products 62
  5.3 Biochemical Characterizations 64
  5.4 Methyltransferase Activity 69
  5.5 In Vitro Repair Of Alkylated Bases 71
  5.6 Induction Of Increased Resistance To The Killing Effects Of Mnng 71
  5.7 Identification Of Plasmid Encoded Protein 73
  5.8 Partial Restriction Map Of Psr75 And Psr99 Plasmid 86
  5.9 Homology Of Psr75 And Psr99 Plasmids 76
  5.10 Sub-Cloning Of Psr75 Gene Fragment 76
  5.11 Construction Of Poly ( Adp -Ribose) Polymerase Expression Vectors 82
  5.12 Expression Of Recombinant Protein 82
  5.13 Catalytic Activity Of Eukaryotic Polymerase In E.Coli Extracts 93
  5.14 Biochemical Characterization 97
  5.15 Activity Gel Analysis 97
  5.16 In Vitro Poly (Ado-Ribose) Polymerase Effect On Uv Irradiation Cells 102
  5.17 Functional Activity Of Padprp From Uv Treated Cells 107
  5.18 Effect Of Poly ( Adp -Ribose) Polymerase On Mnng Damaged Cells 111
  5.19 Functional Activity Of Poly ( Adp -Ribose) Polymerase Of Gamma Radiation Damaged Cells 111
  5.20 Poly ( Apd -Ribose) Polymerase Participate In Repair Of Gamma Radiation Damaged Cells 111
  5.21 Poly( Adp -Ribose) Acceptor Proteins In Bacteria 114
7 6 Discussion 119
63.44 KB
  6.1 Cloning Of M.Luteus Gl 119
  6.2 Cloning Of M.Luteus Gll 119
  6.3 Cloning Of Ada Like Gene Of M Luteus 120
  6.4 Ubiquitin Fused Poly ( Adp -Ribose) Polymerase Expression In Prokaryotes 121
  6.5 Poly ( Adp -Ribose) Polymerase Response To Dna Damage In Cells 123
8 7 References 125
224.98 KB