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Title of Thesis

Shahnaz Choudhry
Institute/University/Department Details
University Of The Punjab/ National Centre Of Excellence In Molecular Biology
Molecular Biology
Number of Pages
Keywords (Extracted from title, table of contents and abstract of thesis)
type restriction, megabase genome, dna, methylation, cloning, bacterial strains, enzymes

In this study, search for the presence of new type II restriction endonucleases has been carried out from fifty-two bacterial strains. Thirty-nine were characterized by CEMB culture collection, two were provided by Persian Type Culture Collection (PTCC) and eleven were obtained from American Type Culture Collection (ATCC). The aim of this study is to make available a wide variety of new restriction enzymes for use by the research community and to add these to the international data base of already existing enzymes.

Among the searched microbes, eighteen are reported positive for the type II enzyme activity. Eight of these strains namely Bacillus thuringiensis D4, Bacillus schlegellii S3, Bacillus Species 3T, Bacillus species CL 14.6, Streptomyces aureomonopodiates, Arthrobacter picolinophilus, Pseudomonas aeruginosa Q2, and an unidentified bacterium PS5(2) contain BthDI, BscA1, Bspt1, Bsp14.61, Sar1, Apil, PaeQ1, and Uba51 restriction enzymes, respectively. These enzymes have beenpurified by column chromatography, using gelfilteration, ion exchange and affinity columns. Enzymes have been characterized on different substrate DNAs, lambda, T7 phage DNA, puc 19, pBR322, PhiX174, M13mp 19RFI, SV40 and Adenovirus-2.

The precise sites of cleavage within the recognition sequences for seven enzymes have been determined by primed synthesis method. All recognize and cleave a known 4-6 nucleotide long stretch of DNA and have the same recognition and cleavage sites as those for their isoschizomers. Among these, a neoschizomer named as BscA1 from Bacillus Schlegellii S3 has been found. It recognizes a non palindromic pentanucleotide sequence 5’-GCATC-3’ and is the only known isoschizomer of its prototype enzyme SfaNI. Cleavage sites for both enzymes have been determined by using different Adenovirus-2 fragments cloned in M13. BscA1 cleaves double stranded DNA four nucleotides away from its recognition sequence and produces a two nucleotide 5’ extension GCATC(4/6), which is GCATC(5/9) in case of SfaNI. The difference in DNA sequence between the recognition site and cleavage site does not affect the enzyme cleavage specificity.

Heat inactivation property of newly isolated enzymes has been studied and compared with their isoschizomers. All enzymes are sensitive to heat, except one enzyme, PaeQI. Methylation studies regarding the methylation sensitivities of seven enzymes have been carried out. New enzymes have been tested for their ability to cleave DNA modified against the action of the old enzyme or vice versa. All enzymes reported here have the same methylation sensitivities as those for their isoschizomers. These enzymes show properties of easy purification from their host strains, simple optimal reaction conditions,and easy growth and maintenance of their source strains. Screening, partial purification and complete characterization of these enzymes have been discussed in detail in the thesis.

Moreover, the gene specifying a sequence specific modification methylase of Enterobacter cloacae J (M.Ec/JI) has been cloned in Escherichia coli by shotgun metod, using the restriction endonuclease Hindlll and the plasmid pUC19. The selection was based on detection of methylation properties rendering recombinant plasmids carrying the methylase gene nonsusceptible to Pvul/Ec/JI endonuclease cleavage. Methylation sensitivity of Ec/JI restriction enzyme recognizing CGATCG sequence has been checked which is the same as that for its isoschizomer Pvul. Two recombinant clones pEcl1 and pEc12 have been found resistant to Ec/JI/Pvul endonuclease attack. Prescence of Ec/JI methylase activity has been confirmed by carrying out 3H-methyl incorporation assays and DNA protection assays by the methylase.

Download Full Thesis
1929.35 KB
S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
113.85 KB
2 1 General Introduction 1
61.06 KB
3 2 Biology Of Restriction And Modification
287.25 KB
  2.1 Introduction 6
  2.2 Type Restriction And Modification Systems 7
  2.3 Type III Restriction And Modification Systems 10
  2.4 Type IV & V Restriction And Modification Systems 12
  2.5 Type II Restriction And Modification System 13
  2.6 Recognition Sequences And Cleavage Properties 15
  2.7 Purification And Characterization Of Restriction Enzymes 22
  2.8 Reaction Mechanism 24
  2.9 Mega base Genome Mapping 28
4 3 DNA Methylation
260.57 KB
  3.1 Introduction 31
  3.2 Methylated Bases In Prokaryotic DNA 36
  3.3 DNA Cytosine Methylation 38
  3.4 DNA Adenine Methylation 41
  3.5 Sensitivity Of Restriction And Modification Enzymes To DNA Methylation 42
  3.6 Restriction Systems Specific For Modified DNA 44
  3.7 Cloned Restriction Modification Systems 46
5 4 Materials And Methods
308.05 KB
  4.1 Bacterial Stains And Media 53
  4.2 Enzymes And Chemical Reagents 55
  4.3 Preparation Of Substrate DNAs 56
  4.4 Standard Assay Procedure 58
  4.5 Screening Of Bacterial Isolates For Type Ii Restriction Enzymes 59
  4.6 Isolation And Purification Of Type Ii Restriction Enzymes 61
  4.7 Characterization Of Type Ii Restriction Enzymes 66
  4.8 Sequencing And Cleavage Site Determination 68
  4.9 Cloning Of Ec/Ji Methylase Gene 72
  4.10 Transformation Of Escherichia Coli 75
  4.11 Identification Of Bacterial Colonies Containing Recombinant Plasmid 76
  4.12 In Vitro Assay Of The Methylase 77
6 6 Results
114.11 KB
  5.1 Screening For Type Ii Restriction Enzymes 80
  5.2 Purification And Characterization Of Bsca1 A Novel Isoschizomer Of Sfani 84
  5.3 Purification And Characterization Of Sar1 93
  5.4 Purification And Characterization Of Paeq1 103
  5.5 Purification And Characterization Of Bthd1 108
  5.6 Purification And Characterization Of Apil 113
  5.7 Purification And Characterization Of Bspt1 118
  5.8 Purification And Characterization Of Uba51 126
  5.9 Purification And Characterization Of Bsp14.61 133
  5.10 Methylation Sensitivity Of Ec/Ji Restriction Enzyme 136
  5.11 Cloning Of Ec/Ji Methylase Gene 136
7 6 Discussion 150
114.11 KB
8 7 References 159
318.57 KB