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Title of Thesis

Azra Sajid
Institute/University/Department Details
University Of The Punjab
Molecular Biology
Number of Pages
Keywords (Extracted from title, table of contents and abstract of thesis)
restriction enzymes, bacterial strain, dna sequence, dna methyaltransferase, cloned type ii r-m system, bacillus species

Type II restriction end nuclease are defined as double strand nucleases that recognize specific DNA sequence and cleave at a defined point within or close to that sequence. They require only Mg++ as a cofactor. These are the basic tools in genetic engineering and molecular biology.

Type II restriction enzymes are wide gapped in nature. Almost restriction enzymes are known representing specificities. Vast microfloras have been extensively examined for the presence of these end nucleases but still this seems to many more theoretical sites for which specific enzymes must exist in nature.

The present study was aimed to discover new type II restriction endonculeases (REases) and clone interesting restriction modification(R €“ M) systems into E. coli to study the primary structure of restriction modification proteins.

During the study bacterial strains, collected from different ecological environments were screened for the presence of type II specificity and ten strains were found positive for presence of endonuelease activity. Sex enzymes were highly purified by a combination of ion exchange and affinity chromatography techniques. The purified enzymes were characterized for recognition sequences and cleavage sites. All enzymes purified and characterized during the study proved to be isoschizomers of already known specificities. The purified enzymes were characterized for different biochemical properties.

One of the R-M systems, Bspli an isoschizomer of NtalV, was selected for detailed study. An attempt was made to clone BspLi R-M system in E. coli through shotgun cloning technique. Selections were made on the basis of functional methyl transferae selection. Not a single clone was found positive for BspLI methylase activity during selection process. Alternative strategy was devised to PCR amplify conserved region of Bspli methlase gene. Based on the conserved motifs PCQ (motif IV) and ENV (motif VI) present in 5-methl cytosine methlases, a set of degenerated oilgonucleotide primers were designed. The PCR amplified product was cloned into the vector, MI3 mp 18 and its DNA sequence was determined. 171 nt. Sequence of BspLI M8 fragment showed 84-87% identity with four already existing R-M genes including Ntaiv R-M system, an isoschizomer of BspLI. Amino acid translation of the sequence showed presence of motifs, specific for 5 methyl cytosine methyl transferees which confirms that a conserved part of BspLI methylase gene have been successfully cloned. In future it can be used as a probe to clone complete BspLI R-M genes.

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2426.9 KB
S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
173.93 KB
2 1 General Introduction 1
44.83 KB
3 2 Type Ii Restriction Enzyme 4
690.21 KB
  2.1 Introduction 4
  2.2 Practical Consideration For The Use Of Restriction Enzyme 7
  2.3 Assay For Purity Of Restriction Enzyme Preparation 15
  2.4 Analytical Application 23
  2.5 Biological Methylation 27
  2.6 Kinetic Parameters 29
  2.7 Specificity Of Restriction Enzyme 30
  2.8 Inhibition 33
  2.9 Star Activity 35
  2.10 Effect Of Site Specifie Methysation On Methl Transferase And Restriction Enzyme 36
  2.11 Protein Structure 38
  2.12 Reaction Mechanisms 40
  2.13 Mega Base Mapping 44
  2.14 Dna Methyaltransferase 46
  2.15 Application Of Dna Methyltransferases 48
  2.16 Cloned Type Ii R-M System 51
4 3 Methodology 56
367.02 KB
  3.1 Bacterial Strain 60
  3.2 Screening Of Bacterial Isolates For Presence Of Restriction End Nuclease Mini-Scale 60
  3.3 Assay For Endonuclease Activity 60
  3.4 Purification Of Restriction Endonuclease 61
  3.5 Standard Assay Procedure 64
  3.6 Determination Of The Salt Concentration And Incubation Temperature 65
  3.7 Determination Of Cleavage Site 66
  3.8 Isolation Of Chromosomal Dna 69
  3.9 Isolation Of Plasmid Dna 69
  3.10 Cloning Of The R-M Gene From Thermophilie Bacillus Species L8 70
5 4 Results
716.31 KB
  4.1 Purification And Characterization Of Newly Isolated Enzyme 78
  4.2 Purification Of Bspli Enzyme Bacillus Species Camb#2884 78
  4.3 Purification Of Bspli Enzyme Thermophilie Bacillus Species L8 84
  4.4 Purification Of Bspk 21 Enzyme From Bacillus Species Camb#2884 90
  4.5 Purification Of Bspmi Enzyme Thermophilie Bacillus Species Mo 28 95
  4.6 Purification Of Bsuai Enzyme From Bacillus Subtilis Atcc#1204 99
  4.7 Purification Of Ssoai Enzyme From Strepto Sporangium Atcc#23818 106
  4.8 Cloning Of Restriction Modification Gene Of Bacillus Species L8 112
6 5 Discussion 124
75.46 KB
7 6 References 129
293.26 KB
8 7 Appendixes 152
46.95 KB