Искусственные генетические системы. Том 1 - Патрушев Л.И.
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Author preface ............................................................ 9
Part I
PRINCIPLES OF GENETIC ENGINEERING ................................ 13
Chapter 1. Introduction. Natural gene systems, their organization
and expression................................................ 13
1.1. Genes and chromosomes ........................................ 13
1.2. Genome........................................................ 16
1.2.1. Genome sizes .......................................... 17
1.2.2. Viral genome........................................... 19
1.2.3. Eubacterial genome..................................... 21
1.2.4. Archaeal genome........................................ 22
1.2.5. Eukaryotic genome ..................................... 23
1.3. Gene expression............................................... 28
1.3.1. Transcription ......................................... 29
1.3.2. Cotranscriptional and posttranscriptional RNA modifications ...................................................... 32
1.3.3. Translation ........................................... 33
1.3.4. mRNA and protein surveillance systems.................. 34
1.3.5. Cotranslational and posttranslational protein modifications ........................................................ 35
1.4. Use of genetic engineering for construction of artificial
genetic systems .............................................. 36
Chapter 2. Nucleic acid purification and the enzymes commonly used
for their manipulation........................................ 40
2.1. Basic methods for nucleic acid purification .................. 40
2.1.1. Conventional methods .................................. 40
2.1.2. Metaphase chromosome isolation by flow cytometry 43
2.2. Nucleic acid restriction and modification enzymes ............ 48
2.2.1. Classification of restriction-modification systems .... 48
2.2.2. Type II restriction endonucleases as essential tools of
genetic engineering .................................... 52
2.2.3. Universal restriction endonucleases for single-stranded
DNA..................................................... 54
2.2.4. Isoschizomers......................................... 55
2.2.5. Changing the sequence specificity of restriction
enzymes................................................ 56
2.2.6. Changing the form of DNA fragment ends ............... 58
2.2.7. DNA methyltransferases and uracil DNA glycosylases 59
2.3. DNA and RNA ligases......................................... 60
2.4. The enzymes of DNA and RNA template synthesis............... 62
2.4.1. DNA-dependent DNA polymerases ........................ 62
2.4.2. RNA-dependent DNA polymerases......................... 63
2.5. Other enzymes used in genetic engineering................... 64
Chapter 3. Vectors....................................................... 68
3.1. Plasmid vectors............................................. 68
3.1.1. Biological properties of bacterial plasmids .......... 69
3.1.2. Vectors for ligation-independent cloning ............. 76
3.1.3. Vectors for direct cloning of PCR products ........... 77
3.1.4. Use of transposons for DNA cloning ................... 78
3.2. Vectors based on phage X chromosome......................... 80
3.3. Cosmids and phasmids ....................................... 83
3.4. Artificial chromosomes...................................... 86
3.4.1. Supercapacity vectors YAC, ВАС and РАС................ 86
3.4.2. Human and animal artificial chromosomes .............. 97
3.5. Integrative and shuttle (binary) vectors ................... 101
3.5.1. Integrative vectors of gram-positive bacteria Bacillus
subtilis............................................... 101
3.5.2. Shuttle vectors....................................... 103
3.6. Engineering of expression vectors and their functioning 104
3.6.1. Factors which affect recombinant gene expression
levels in bacterial cells: transcription............... 107
3.6.2. Factors which affect recombinant gene expression
levels in bacterial cells: translation ................ Ill
3.6.3. Factors which affect recombinant gene expression levels in bacterial cells: stability of recombinant RNA
