Thesis--Wisconsin. / Vita. Includes bibliographical references.

Isolation of DNase II from bovine small intestine by chromatography on DEAE cellulose of a 105,000 xg supernatant solution prepared from an homogenate of the mucosa in Krebs Ringer phosphate buffer appeared to yield two activities, a major activity which was eluted from the column with 20 mM phosphate buffer and a minor activity which was eluted with a potassium chloride gradient. The two DNase II activities differed in their response to increasing ionic strength, pH, sulfate ion concentration and temperature for the hydrolysis of DNA. The major activity degraded native DNA more rapidly than denatured DBA whereas the minor activity degraded both at the same rate. Previous investigators have reported the presence of two DNases lis with different properties in other tissues. In bovine intestinal DNase II, the minor activity, upon rechromatography on DEAE cellulose, eluted in the same position as the major DNase II and it was concluded; that the appearance of the minor DNase II activity was an artifact of the chromatography. It is likely that a small quantity of DNase II was bound to endogenous DNA on the DEAE cellulose column in the 20 mM phosphate buffer and later eluted from the column along with some of the DNA. with the potassium chloride gradient. DNA present in the minor DNase II preparation probably caused the apparent differences in properties of the two DNase lis by interfering in the enzymic reactions. Intestinal DNase II was partially purified by ion exchange chromatography and gel filtration and had properties similar to DNase lis from other tissues. The enzyme hydrolysed calf thymus DNA endonucleolytically at acid pH in the absence of a divalent metal ion to oligonucleotides with 3'-phosphate and 5'-hydroxyl terminals. The activation energy for the reaction was 19 kcal/mole; that for denaturation of DNase II itself, k3 kcal/mole. Michaelis-Menton kinetics were observed for the reaction of DNase II with Escherichia coli DNA—the Michaelis constant was 2.42 x 10⁻⁷ M DNA-phosphate. The molecular weight of DNase II was estimated to be 41,000 by gel filtration on Sephadex G100. The early stages of the digestion of DNA by DNase II were investigated by labelling the reaction products with ³²P at their 5'-terminals using polynucleotide kinase and [x-³²P]ATP and at their 3'-terminals using terminal deoxynucleotidyl transferase and [⍺-³²P]ATP. The mode of cleavage of native DNA by DNase II was determined by comparing the polynucleotide-catalysed incorporation of 32p from [x-³²P]ATP into native and denatured DNase II reaction products. Since single-strand cleavage of DNA by DNase II released 5'-hydroxyl terminals that were inaccessible to polynucleotide kinase as long as the DNase II reaction products remained double-stranded, incorporation of ³²P into native products was proportional to the number of double-strand cleavages while incorporation of ^P into denatured products was proportional to the number of double-strand cleavages plus single-strand cleavages. It was found that DNase II degraded native DNA primarily by a double-strand cleavage mechanism. After DNase II catalysed hydrolysis of DNA each of the four bases present in DNA was found at the 5'- and 3'-terminals of the reaction products. Thus DNase II did not have an exclusive preference for one or two bases at either terminal, and likely cleaved a large number of different base sequences in the DNA.. The most susceptible internucleotide linkage was GpG; the most resistant, CpT. The base specificity at the 5'-terminal changed during the reaction, especially in the initial and terminal phases. In the initial phase the proportion of guanine was elevated and the order of cytosine and adenine was reversed compared to later stages in the reaction. These changes could reflect the presence of a preferred sequence that was selectively degraded and exhausted during the initial phase of the reaction. Different proportions of terminal bases in cleavage products of DNA from diverse species indicated that susceptible sequences occurred with different frequencies in the various DNAs. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Unknown

No description available.

Thesis (Ph. D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1989. / Includes bibliographical references (leaves 143-172).

by Go Tong-Ming, Thomas. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 86-95). / Chapter CHAPTER 1: --- INTRODUCTION --- p.1 / Chapter CHAPTER 2: --- PURIFICATION OF α- AND β-MMCs --- p.23 / Chapter CHAPTER 3: --- DEOXYRIBONUCLEASE ACTIVITY OF αAND β-MMCs --- p.44 / REFERENCES --- p.86

The studies reported herein indicate that, under the conditions commonly employed for cultivating Neisseria gonorrhoeae, colonial type T1 and T2 cultures of the organism dissociate to type T3 and T4 forms, and that this occurs both among populations of the organism grown in liquid media as well as in individual, well-isolated colonies grown on solid media.

Hepatitis B virus (HBV) is a major global public health burden, with over 350 million people chronically infected. This results in approximately 600,000 liver cancer-related deaths annually. Chronic HBV infections are normally managed with long-term anti-HBV therapeutics, such as reverse transcription inhibitors, which target post-transcriptional viral processes without affecting the cccDNA. Treatment failure however is largely as a result of the stability of this episomal viral DNA. The cccDNA minichromosome serves as a reservoir of HBV DNA and is capable of re-establishing viral replication following withdrawal of treatment. Designer nucleases, like transcription activator-like effector nucleases (TALENs), have recently been used to create double stranded breaks (DSBs) at target-specific endogenous DNA loci. These nucleases are designed as pairs, which upon dimerisation cleave double-stranded DNA. Subsequent activation of the cellular non-homologous end-joining (NHEJ) pathway often results in targeted mutagenesis at the DSB site. As TALENs may be designed to bind to any DNA sequence, they are commonly used as genetic engineering agents. Inactivation of HBV cccDNA, using these engineered TALENs, presents a unique approach to disabling viral replication permanently. To investigate this, a panel of TALENs targeting the core (C), surface (S) and two different polymerase (P1 and P2) regions of HBV cccDNA were generated using a Golden gate modular assembly approach. TALENs were initially tested in two liver-derived cell lines. Firstly as transient co-transfections in Huh7 cells using a HBV replication competent plasmid, followed by long term investigations in HepG2.2.15 cells which model HBV replication in vitro. Inactivation of HBV was determined by measuring markers of viral replication, whilst TALEN-mediated targeted disruption was verified by T7 endonuclease 1 (T7E1) or CELI endonuclease assays and sequencing. In vitro, the S TALEN inhibited HBsAg secretion by 80% in Huh7 cells and 60% in HepG2.2.15 cells. Furthermore, S TALEN-mediated targeted disruption occurred in 35-47% of cccDNA copies, whilst the C TALEN resulted in 11% targeted disruption of cccDNA in without inhibition of HBsAg expression. The P2 TALEN showed no anti-HBV efficacy, however the P1 TALEN inhibited HBsAg expression by up to 60% without any evidence of site-directed cleavage. As this TALEN binding site spans the HBV Enhancer I sequence, knock-down of HBsAg expression is most likely to occur as a result of transient transcriptional repression. To confirm whether permanent repression of HBV transcription could be achieved, a KRAB-based transcription activator-like repressor (rTALE) targeting the HBV pre-S2 promoter was generated. Using an in vitro reporter gene assay, the pre-S2 rTALE inhibited luciferase expression by up to 90%. However this was only achieved using high molar concentrations of the repressor, suggesting multiple rTALEs may improve HBV transcriptional repression. As the S and C TALENs displayed significant anti-HBV efficiency in vitro, they were tested in a murine hydrodynamic injection model of HBV replication. In vivo, the S TALEN inhibited HBsAg secretion by 95% and induced disruption in 77–87% of HBV DNA targets. In addition the C TALEN inhibited HBcAg expression and induced disruption in 78-93% of HBV DNA targets. Additionally, serological analysis showed a reduction in circulating virions and no apparent liver toxicity, as determined by real-time PCR (qPCR) and aspartate transaminase (AST)/ alanine aminotransferase (ALT) liver function tests respectively. Deep sequencing at the S and C TALEN binding sites showed targeted mutagenesis of HBV DNA in samples extracted from murine hepatocytes transfected with TALENs, however wild-type sequences were exclusively detected in mice that had not been treated with anti-HBV TALENs. Furthermore, frameshift deletions were predominantly detected indicating major disruptions in the HBV surface and core sequences. These results indicate that TALENs designed to disable and silence HBV cccDNA are effective both in vitro and in vivo and as such provide a promising therapeutic approach to treat this serious infection.

Thesis (M.S.)--University of Texas at El Paso, 2007. / Title from title screen. Vita. CD-ROM. Includes bibliographical references. Also available online.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 142-167).

Mok Yu-Keung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (Leaves 195-201). / Abstract --- p.i / Acknowledgements --- p.iii / List of Abbreviations --- p.iv / Table of contents --- p.v / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- The need to increase the specificity and variety of restriction endonucleases --- p.1 / Chapter 1.2 --- Classification of methods used for increasing the specificity and variety of restriction endonculeases --- p.2 / Chapter 1.3 --- Isolation and characterization of restriction endonucleases from natural sources --- p.3 / Chapter 1.4 --- Modification of DNA substrate to produce new cleavage specificities --- p.6 / Chapter 1.4.1 --- Methylation of the DNA substrate --- p.6 / Chapter 1.4.1.1 --- Achilles' hell cleavage-The use of canonical methylation to produce novel specificities --- p.10 / Chapter 1.4.1.2 --- Cross protection-The use of non-canonical methylation to generate new cleavage specificity --- p.14 / Chapter 1.4.1.2.1 --- Recognition sequence of a restriction endonuclease and a methylase partially overlap --- p.14 / Chapter 1.4.1.2.2 --- Methylase recognizing a subset of the degenerate sequence of the restriction endonuclease --- p.16 / Chapter 1.4.1.2.3 --- Methylase-limited partial digestion --- p.16 / Chapter 1.4.1.3 --- The use of methylation dependent restriction endonucleases and methylases to generate new specificity --- p.17 / Chapter 1.4.1.4 --- Sequential double-methylation-A two step methylation procedure to generate new specificities --- p.20 / Chapter 1.4.2 --- The generation of a universal restriction endonuclease by combining a Type IIS restriction enzyme moiety and an oligonucleotide adaptor --- p.22 / Chapter 1.4.2.1 --- General principle for generating a universal restriction endonuclease --- p.22 / Chapter 1.4.2.2 --- Factors that affect the cleavage efficiency of universal restriction endonuclease --- p.25 / Chapter 1.4.2.3 --- Modifications and potential applications of the universal restriction endonuclease --- p.29 / Chapter 1.4.3 --- DNA triple helix formation-enhance restriction enzyme specificity by site-specific inhibition of restriction/modification enzymes --- p.32 / Chapter 1.5 --- Modification of the cleaving agent to produce new specificities --- p.36 / Chapter 1.5.1 --- Sequence-specific artificial endonucleases --- p.36 / Chapter 1.5.1.1 --- Oligonucleotides as sequence-specific ligand --- p.37 / Chapter 1.5.1.2 --- Protein or peptide as sequence-specific ligand --- p.40 / Chapter 1.5.1.3 --- General limitations and applications of artificial endonucleases --- p.42 / Chapter 1.5.2 --- Molecular cloning and protein engineering of the restriction-modification system of bacteria --- p.43 / Chapter 1.5.2.1 --- Molecular cloning of the bacterial restriction-modification systems --- p.43 / Chapter 1.5.2.1.1 --- The strategies used to clone and screen restriction-modification systems --- p.45 / Chapter 1.5.2.2 --- Protein engineering of the restriction-modification systems of bacteria --- p.50 / Chapter 1.5.2.2.1 --- Pre-requisites for protein engineering on the restriction-modification systems --- p.51 / Chapter 1.5.2.2.2 --- Effects of protein engineering on the activity and specificity of restriction endonuclease and methylase --- p.53 / Chapter 1.6 --- Variation of restriction endonuclease specificity by altering the reaction condition --- p.56 / Chapter 1.6.1 --- Effects of organic solvents --- p.57 / Chapter 1.6.2 --- Effects of pH and ionic environment on restriction endonuclease specificity --- p.58 / Chapter 1.6.3 --- Remarks on the use of star activity to introduce new specificity --- p.59 / Chapter 1.7 --- Aim of study --- p.59 / Chapter Chapter 2 --- Purification and characterization of thermophilic restriction endonucleases from soil Bacillus spp / Chapter 2.1 --- Materials and methods --- p.61 / Chapter 2.1.1 --- Purification of thermophilic restriction endonucleases from soil Bacillus spp --- p.61 / Chapter 2.1.1.1 --- Preparation of crude enzyme extract --- p.61 / Chapter 2.1.1.2 --- Purification of BsiB I and BsiE 1 --- p.63 / Chapter 2.1.1.3 --- Purification of BsiY I --- p.63 / Chapter 2.1.1.4 --- Preparation of BsiG I and BsiU I --- p.64 / Chapter 2.1.1.5 --- Concentration and storage of the purified restriction endonucleases --- p.64 / Chapter 2.1.1.6 --- Regeneration of the columns --- p.64 / Chapter 2.1.2 --- Characterization of restriction endonucleases --- p.65 / Chapter 2.1.2.1 --- Assay for the working temperature and ionic requirement for the restriction enzymes --- p.65 / Chapter 2.1.2.2 --- Unit determination of the restriction endonucleases --- p.66 / Chapter 2.1.2.3 --- Assay for the purities of restriction endonucleases --- p.66 / Chapter 2.1.2.4 --- Determination of recognition specificity --- p.67 / Chapter 2.1.2.5 --- Determination of the restriction endonuclease's sensitivity to dam and dcm methylation --- p.68 / Chapter 2.1.2.6 --- Determination of the cleavage specificities of restriction endonucleases --- p.70 / Chapter 2.1.2.7 --- Sequencing using Deaza dGTP --- p.73 / Chapter 2.2 --- Results --- p.73 / Chapter 2.2.1 --- Purification of thermophilic restriction endonucleases from soil Bacillus spp --- p.73 / Chapter 2.2.1.1 --- Strain identification --- p.74 / Chapter 2.2.1.2 --- Elution properties of the restriction endonucleases from columns --- p.74 / Chapter 2.2.1.2.1 --- BsiB I --- p.74 / Chapter 2.2.1.2.2 --- BsiE I --- p.77 / Chapter 2.2.1.2.3 --- BsiY 1 --- p.78 / Chapter 2.2.1.3 --- The working digestion temperature and ionic strength requirement --- p.81 / Chapter 2.2.1.4 --- Unit determination --- p.82 / Chapter 2.2.1.5 --- Purities of the purified restriction endonucleases --- p.83 / Chapter 2.2.1.6 --- Recognition sites of the purified restriction endonucleases --- p.83 / Chapter 2.2.1.6.1 --- BsiB I --- p.83 / Chapter 2.2.1.6.2 --- BsiE I --- p.85 / Chapter 2.2.1.6.3 --- BsiY 1 --- p.87 / Chapter 2.2.1.6.4 --- BsiU I and BsiG I --- p.88 / Chapter 2.2.1.7 --- Sensitivity of restriction endonucleases to dam and dcm methylation --- p.90 / Chapter 2.2.1.8 --- Cleavage specificities of the purified restriction endonucleases --- p.91 / Chapter 2.2.1.8.1 --- BsiB I --- p.91 / Chapter 2.2.1.8.2 --- BsiE I --- p.92 / Chapter 2.2.1.8.3 --- BsiY I --- p.93 / Chapter 2.2.1.9 --- Sequencing of a wrongly sequenced site in pACYC177 using Deaza-dGTP --- p.94 / Chapter Chapter 3 --- The use of Xcm I and BsiY I as an universal restriction endonuclease / Chapter 3.1 --- Materials and methods --- p.98 / Chapter 3.1.1 --- Assay of universal restriction endonuclease using ss DNAs --- p.98 / Chapter 3.1.1.1 --- Annealing reaction between adaptors and ss DNAs --- p.99 / Chapter 3.1.1.2 --- Digestion of the annealed DNA complex --- p.100 / Chapter 3.1.1.3 --- Assay of the digested ss DNA on alkaline denaturing agarose gel --- p.100 / Chapter 3.1.2 --- Assay system involving 5' end-labelled oligonucleotide --- p.101 / Chapter 3.1.2.1 --- Purification of oligonucleotides using preparative polyacrylamide gel electrophoresis --- p.102 / Chapter 3.1.2.2 --- 5'end-labelling of the oligonucleotide DNA substrate --- p.104 / Chapter 3.1.2.3 --- The annealing between adaptors and oligonucleotide DNA substrate and the digestion condition --- p.104 / Chapter 3.1.2.4 --- Assay of the labelled oligonucleotides in polyacrylamide gel after digestion --- p.105 / Chapter 3.2 --- Results --- p.106 / Chapter 3.2.1 --- Xcm I adaptors #2 and #4 --- p.106 / Chapter 3.2.1.1 --- Assay conditions used for the universal restriction endonucleases --- p.107 / Chapter 3.2.1.1.1 --- Conditions used for hybridization --- p.107 / Chapter 3.2.1.1.2 --- Conditions used for digestion --- p.108 / Chapter 3.2.1.2 --- Methods used to maximize the cleavage of M13mp7 with Xcm I adaptor #4 --- p.110 / Chapter 3.2.1.2.1 --- Methods used to optimize the hybridization process --- p.110 / Chapter 3.2.1.2.2 --- Methods used to relax the secondary DNA structures --- p.112 / Chapter 3.2.1.2.2.1 --- Linearization of M13mp7 with BamH I befor annealing the adaptor --- p.113 / Chapter 3.2.1.2.2.2 --- Relaxation of secondary structure using boiling and NaOH denaturation --- p.114 / Chapter 3.2.1.2.3 --- Methods used to optimize the digestion process --- p.115 / Chapter 3.2.1.2.3.1 --- Addition of BSA --- p.115 / Chapter 3.2.1.2.3.2 --- Addition of the restriction endonuclease in separate batches --- p.115 / Chapter 3.2.1.3 --- Digestion of ss M13mpl8 and ssM13mpl9 DNA using Xcm I adaptor #2 and adaptor #4 --- p.116 / Chapter 3.2.2 --- Xcm I adaptor #1 and #3 --- p.118 / Chapter 3.2.2.1 --- Methods used to maximize the cleavage of M13mp7 with Xcm I adaptor #1 and adaptor #3 --- p.119 / Chapter 3.2.2.1.1 --- Methods used to relax the secondary structure --- p.119 / Chapter 3.2.2.1.1.1 --- Linearization of M13mp7 with BamH I before the annealing reaction --- p.120 / Chapter 3.2.2.1.1.2 --- Relaxation of secondary structure by NaOH denaturation --- p.121 / Chapter 3.2.2.1.1.3 --- Relaxation of secondary structure by adding DMSO and urea --- p.122 / Chapter 3.2.2.1.2 --- Methods used to optimize the digestion and hybridization processes --- p.123 / Chapter 3.2.2.1.2.1 --- Annealing of M13mp7 with a different amount of adaptor #3 and digesting the DNA complex with Xcm I at different temperatures --- p.123 / Chapter 3.2.2.1.2.2 --- Optimization of digestion by adding Xcm I in separate batches --- p.124 / Chapter 3.2.3 --- BsiY I adaptor --- p.124 / Chapter 3.2.3.1 --- Methods used to optimize the cleavage of M13mp7-BsiY I adaptor complex with BsiY I --- p.126 / Chapter 3.2.3.1.1 --- Optimization of hybridization using various concentrations of NaCl during the annealing reaction --- p.126 / Chapter 3.2.3.1.2 --- Optimization of digestion by binding BsiY I to the BsiY I adaptor before annealing --- p.127 / Chapter 3.2.4 --- The use of 5' end-labelled oligonucleotide DNA substrates for digestion with universal restriction endonuclease --- p.128 / Chapter Chapter 4 --- Molecular cloning of the BsiY I restriction-modification system / Chapter 4.1 --- Materials and methods --- p.132 / Chapter 4.1.1 --- Preparation of chromosomal DNA from BsiY I producing Bacillus stearothermophilus --- p.132 / Chapter 4.1.1.1 --- Restriction digestion of the chromosomal DNA --- p.134 / Chapter 4.1.1.2 --- Southern hybridization to locate the position of the DNA fragment coding for the restriction-modification system --- p.135 / Chapter 4.1.1.2.1 --- Southern transfer of DNA fragments onto nitro-cellulose paper --- p.135 / Chapter 4.1.1.2.2 --- Labelling of the probes by Nick-translation --- p.136 / Chapter 4.1.1.2.3 --- Hybridization of the nick-translated probes onto the DNA fragments fixed on NC paper --- p.137 / Chapter 4.1.2 --- Large-scale preparation of the cloning vector --- p.137 / Chapter 4.1.2.1 --- Restriction endonuclease digestion and dephosphorylation of the vector ´Ø.… --- p.139 / Chapter 4.1.3 --- Ligation between vector and DNA inserts --- p.139 / Chapter 4.1.4 --- Transformation of the ligated DNA into competent cells --- p.140 / Chapter 4.1.4.1 --- Preparation of competent cells --- p.140 / Chapter 4.1.4.2 --- Transformation of the ligated vector and insert DNA into competent cells --- p.142 / Chapter 4.1.5 --- Rapid alkaline lysis method for screening transformants that contains an insert --- p.143 / Chapter 4.1.6 --- Preparation of the genomic library and its plasmid DNA --- p.144 / Chapter 4.1.7 --- Screening procedures used to clone the BsiY I restriction-modification system --- p.144 / Chapter 4.1.7.1 --- In vitro selection using Hungarian Trick --- p.145 / Chapter 4.1.7.2 --- In vivo selection using the host strain AP1-200 and AP1-200-9 --- p.145 / Chapter 4.1.7.2.1 --- Preparation of competent AP1-200 and AP1-200-9 cells --- p.146 / Chapter 4.1.7.2.2 --- Transformation of the genomic library plasmid into competent AP 1-200 and AP1-200-9 cells --- p.146 / Chapter 4.1.8 --- Assay of BsiY I restriction endonuclease and methylase activities in the suspecting clones --- p.147 / Chapter 4.1.8.1 --- Assay to BsiY I methylase activity - resistance of the plasmid to BsiY I digestion --- p.147 / Chapter 4.1.8.2 --- Assay of BsiY I methylase activity - ability to incorporate H3-methyl group from H3-SAM into DNA substrate molecules --- p.148 / Chapter 4.1.8.3 --- Assay of BsiY I restriction endonuclease activity - ability of crude enzyme extract to cleave DNA --- p.149 / Chapter 4.2 --- Results --- p.150 / Chapter 4.2.1 --- Construction of the BamH I genomic library --- p.150 / Chapter 4.2.1.1 --- Vector and insert used --- p.150 / Chapter 4.2.1.2 --- Optimization of the ligation and transformation process --- p.151 / Chapter 4.2.1.3 --- Preparation of the BamH I library --- p.153 / Chapter 4.2.1.4 --- Methods used to screen the restriction-modification system from the plasmid library --- p.155 / Chapter 4.2.1.4.1 --- The Hungarian Trick --- p.155 / Chapter 4.2.1.4.2 --- Screening of the restriction-modification system using the strains API-200 and AP1-200-9 --- p.159 / Chapter 4.2.2 --- Construction of the Hind III library --- p.161 / Chapter 4.2.2.1 --- Vector and insert used --- p.161 / Chapter 4.2.2.2 --- Optimization of the ligation and transformation process --- p.162 / Chapter 4.2.2.3 --- Preparation of the Hind III library --- p.164 / Chapter 4.2.2.4 --- Methods used to screen the restriction-modification system from the plasmid library --- p.165 / Chapter 4.2.2.4.1 --- The Hungarian Trick --- p.165 / Chapter 4.2.2.4.2 --- Screening of the restriction-modification system using the strain AP1-200 and AP1-200-9 --- p.168 / Chapter 4.2.2.5 --- Assay of methylase activity using H3-SAM --- p.170 / Chapter 4.2.3 --- The use of Southern blotting and hybridization to find if two available probes have homology to the BsiY I restriction-modification system --- p.173 / Chapter Chapter 5 --- Discussion / Chapter 5.1 --- Purification and characterization of restriction endonucleases from Bacillus spp --- p.176 / Chapter 5.1.1 --- Methods used to purify the restriction endonuclease --- p.177 / Chapter 5.1.2 --- Characterization of the restriction endonucleases --- p.179 / Chapter 5.1.2.1 --- Determination of the purities of the purified restriction endonucleases --- p.179 / Chapter 5.1.2.2 --- Determination of the recognition site --- p.179 / Chapter 5.1.2.3 --- Determination of the cleavage site --- p.180 / Chapter 5.1.2.4 --- Sequencing using Deaza-dGTP --- p.181 / Chapter 5.2 --- The use of Xcm I and BsiY I as universal restriction endonucleases --- p.182 / Chapter 5.2.1 --- The adverse effects of hair-pin loop on the cleavage with universal restriction enzymes --- p.183 / Chapter 5.3 --- Molecular cloning of the BsiY I restriction-modification system --- p.187 / Chapter 5.3.1 --- Construction of the genomic library --- p.187 / Chapter 5.3.1.1 --- Preparation of the insert and vector --- p.188 / Chapter 5.3.1.2 --- Optimization of the ligation and transformation processes --- p.188 / Chapter 5.3.2 --- Screening strategies used to clone the BsiY I restriction-modification system --- p.189 / Chapter 5.3.2.1 --- The Hungarian Trick --- p.189 / Chapter 5.3.2.2 --- Screening using the strains AP1-200 and AP1-200-9 cells --- p.191 / Chapter 5.3.3 --- Assay of the gene products from the cloned restriction-modification system --- p.192 / Chapter 5.3.3.1 --- Methylase activity --- p.192 / Chapter 5.3.3.2 --- Restriction endonuclease activity --- p.193 / Chapter 5.4 --- Future prospects --- p.193 / References --- p.195 / Appendix --- p.201