大学分子生物学经典双语课件C4-Gene_mutation_and_exchange

1、Chapter Gene mutation and exchange4.1 Mutation Mutations are heritable permanent changes (Addition, deletion, transition etc.) in the base sequence of DNA. It is the main factor in biological evolution.1 Types of gene mutation (1) Induced mutation and spontaneous mutation(2) Single-point mutation an

2、d multi-point mutation(3) Synonymous mutation and missense mutation(4) Nonsense mutation, stop codon mutation and suppressor mutation (5) Other types2 Mechanism of mutagenesis(1) Spontaneous mutation Radiation and the environment； Harmful metabolites； DNA replication error（2）Induced mutationTwo adja

3、cent TT by ultraviolet radiation to produce the dimerBase chemical modifiersbase mutationbase mutation by alkylation agentbase mutationbase mutation by hydroxylaminehydroxylamine base mutation by hydroxylamine Structure of base analogStructure of base analog5-BU caused the A-T to the G-C conversionk

4、eto form enol form Pairing of 2 amino purine and cytosineConversion the A-T to the G-C caused by 2 aminopurine(2-AP)Some mutagenicities intercalate agent Mutation mechanism induced by intercalate agentintercalate agent3 Hot spot of mutation Definition: A region of DNA that exhibits an unusually high

5、 propensity to mutate. One of the main reasons is the modifications of the bases, that deamination of 5- methylcytosine forming thymine.Comparing of 5 - methyl cytosine deamination and cytosine deamination repair4.2 DNA repair1 Mismatch repair2 Uracil-N-glucosidic bond systemGlycosylaseGlycosylase糖基

6、酶糖基酶Mismatch baseMismatch base3 PhotoreactivationThe pyrimidine dimer blocks DNA replicationphotolyase bindingreleaseCut C-C bondenergy 4 Excision repair(dark repair )5 Recombination repair Recombination repair6 SOS (Emergency repair system) 4.3 Recombination Genetic recombination is a process by wh

7、ich a molecule of nucleic acid is broken and then joined to a different one. In a broad sense, any cause of genotype changes in gene exchange is recombination. According to the difference of the mechanisms and protein factors required,recombination can be divided into three types： Homologous recombi

8、nation； Site-specific recombination； Transpositional recombination 4.3.1 Homologous recombinantion Also known as general recomination, this process involve the exchange of homologous regions between two DNA molecules. Example: The eukaryotic nonsister chromatid exchange; Transformation of bacteria;

9、bacterial transduction, etc. Holliday model Meslson-Radding model There are 7 steps： （1）Nicking （2）Strand displacement （3）Single-strand invasion （4）Loop cleavage （5）Strand assimilation (6) Isomerization (7）Branch migrationMeslson-RaddingModel2 Site-specific recombination This involves

10、the exchange of nonhomologous but specific pieces of DNA and is mediated by proteins (enzyme) that recognize specific DNA sequences.Schematic of phage integrated4.4 Transposition Replicated copies of transposable DNA elements can insert themselves anywhere in the genome. All transposons encode a tra

11、nsposase which catalyzes the insertion.4.4.1 Transposons and their structural characteristics Insertion sequences, ISComposite transposonTnA family IS element Insertion sequences are small relative to other transposable elements and only code for proteins (usually the transposase ) implicated

12、 in the transposition activity, allowing the IS to move, and also one regulatory protein which either stimulates or inhibits the transposition activity. l IS target sitetarget sequence repeats target sequence repeats directly flanked thedirectly flanked the ISIS General pattern of DNA transposon4.4.

13、1.2 Composite transposon A composite transposon has protein coding DNA segments flanked by inverted, repeated sequences that can be recognized by transposase enzymes. Composite transposons will also often carry one or more genes conferring antibiotic resistance. TnA family TnA family is about

14、 5000bp, which is much TnA family is about 5000bp, which is much greater than the insertion sequence. The same as greater than the insertion sequence. The same as composite transposon, TnA family also carries the composite transposon, TnA family also carries the gene who is responsible for its own t

15、ransposition gene who is responsible for its own transposition and other gene such as resistance gene -amine acyl and other gene such as resistance gene -amine acyl enzyme (Ampenzyme (AmpR R) . It has no IS, but there are terminal ) . It has no IS, but there are terminal repeat sequences of about 37

16、-38bp in the end.repeat sequences of about 37-38bp in the end.4.4.2 Transposons in eukaryotes Transposons in maize Autonomous Autonomous elementelement NonautonomousNonautonomous elemenelement t（1）Ac-Ds system11bpAcDstransposable element 是引起玉米糊粉层花斑不稳定是引起玉米糊粉层花斑不稳定 现象的遗传因子现象的遗传因子 SH Bz Wx Ac

17、ci Bz Wx Acchr.9 CI SH Bz Wx large colored sectorsDsDs CI CI ci SH sh Spm/dSpm has two genes. tnpA consists of 11 exons that are transcribed into a spliced 2500 base mRNA. tnpB may consist of a 6000 base mRNA containing ORF1 + ORF2.（2）Spm-dSpm system13bp Transposons in Drosophila Drosophila m

18、elanogastermelanogaster There are two There are two kinds:kinds: P P CopiaCopia 31bp The copia element is 5000 bp long, with identical direct terminal repeats of 276 bp. Each of the direct repeats itself ends in related inverted repeats. A direct repeat of 5 bp of target DNA is generated at the site

19、 of insertion. The divergence between individual members of the copia family is slight, 5%; variants often contain small deletions. Transcripts of copia are found as abundant poly(A)+ mRNAs, representing both full-length and part-length transcripts. The mRNAs have a common 5 terminus. Several protei

20、ns are produced, probably involving events such as splicing of RNA. Retrotransposons(Virus superfamily)nonvirusviral superfamily Members of the viral superfamily code for reverse transcriptase and/or integrase activities. Like other retroposons, they reproduce like retroviruses but differ fro

21、m them in not forming an independent infectious particle . nonviral superfamilyMembers of the nonviral superfamily are identified by external and internal features that suggest that they originated in RNA sequences. They were targets for a transposition event by an enzyme system coded elsewhere, tha

22、t is, they are always nonautonomous. They do not code for proteins that have transposition functions. 4.4.3 Mechanism of transposition4.4.4 Genetic effect （1 1）insertional mutagenesisinsertional mutagenesis （2 2）new genenew gene （3 3）host chromosomal DNA recombinationhost chromosomal DNA recombinati

23、on （4 4）Host phenotypic changesHost phenotypic changes Main effect in molecular biology: gene transfer who is difficult to screen, gene mapping tag, screening of insertion mutant, construction of special strains, cloning the genes who are difficult to identify by phenotype, etc.4.5 Genetic Diversity

24、 Widely, Genetic Diversity, the level of biodiversity, refers to the total number of genetic characteristics in the genetic makeup of a species.Narrowly, Genetic Diversity is the variation of heritable characteristics present in a population of the same species. 4.5.1 Causes for genetic diversity Ge

25、netic diversity is mainly caused by the variation of genetic material , including:Mutation (chromosome and gene)Mutation (chromosome and gene)RecominationRecomination4.5.2 Measures of genetic diversityMorphological markersCytological markersBiochemical markersMolecular markers Molecular mark

26、ers （1）Molecular foundation of RFLPRFLP:Restriction Fragment Length Polymorphism(限制性片段长度多态性）限制性片段长度多态性） RFLP is a difference in homologous DNA sequences that can be detected by the presence of fragments of different lengths after digestion of the DNA samples in question with specific restriction end

27、onucleases. RFLPR RF FLPLP（2 2）DNA marker basing on PDNA marker basing on PCRCR Simple sequence repeat，SSR Random amplified polymorphisms DNA，RAPD Inter-simple sequence repeat, ISSRSSRSSR, also called microsatellites, are stretches of 2 to 6 nucleotide units repeated in tandem and randomly spread in

28、 eucaryotic genomes. SSR are very polymorphic due to the high mutation rate affecting the number of repeat units. Such length-polymorphisms can be easily detected on high resolution gels , by running PCR amplified fragments obtained using a unique pair of primers flanking the repeat .Di-nucleotide r

29、epeat: CACACACATri-nucleotide repeat: ATGATGATGATG RAPDRAPDRAPDRAPD stands for random amplification of random amplification of polymorphic DNApolymorphic DNA. It is a type of PCR reaction, but the segments of DNA that are amplified are random. The scientist performing RAPD creates several arbitrary,

30、 short primers (812 nucleotides), then proceeds with the PCR using a large template of genomic DNA, hoping that fragments will amplify. By resolving the resulting patterns, a semi-unique profile can be gleaned from a RAPD reaction. Principle of RAPD markerISSRISSR technique is similar to RAPD, excep

31、t that ISSR primers consist of a di or trinucleotide simple sequence repeat with a 5 or 3 anchoring sequence of 13 nucleotides.Compared with RAPD primers, the ISSR primer sequence is usually larger(16-18bp),allowing for a higher primer annealing temperature, which results in greater band reproducibi

32、lity than RAPD markers.Di-nucleotide repeat: CACACACATri-nucleotide repeat: ATGATGATGATG （3 3）DNA marker based on the technique of PCR DNA marker based on the technique of PCR and Restriction Enzyme and Restriction Enzyme Amplified fragment length polymorphism, AFLP Cleaved amplified polymorphic sequences, CAPSAFLP markerAFLP（4）Single nucleotide polymorphism Single nucleotide polymorphism (SNP): DNA DNA sequence polymorphism at the genomic level sequence polymor