北京大学：《微生物学》课程PPT教学课件（英文版）Distinct Properties of M13

Single-stranded Filamentous DNA Bacteriophages M13, fI and fd Distinct Properties of M13 Infect Escherichia coli Infect only male cells through attachment to the male-specific pilus Have circular single-stranded dnA Can be used as a cloning vector and dnA sequencing vehicle in genetic engineering Infect but not kill the cells

Single-Stranded Filamentous DNA Bacteriophages M13, f1 and fd Distinct Properties of M13 – Infect Escherichia coli – Infect only male cells through attachment to the male-specific pilus – Have circular single-stranded DNA – Can be used as a cloning vector and DNA sequencing vehicle in genetic engineering – Infect but not kill the cells

M13 leaves an infected cell without lysis Cytoplasmic memb Cell Coat proteins embedded in cytoplasmic membrane 00 e dNA A-protein ss circle C-p Environment Cytoplasm

M13 leaves an infected cell without lysis

Double-Stranded DNA Bacteriophage: the first discovered phage and the most extensively studied ds dna viruses. Example: T7 1. Linear double-stranded DNA with 39,936 base pairs 2.92% DNA codes for protein 3. Gene overlap through translation in different reading frame and through internal translational reinitiation as well as internal frame shifts 4. One of the coded proteins inhibits the host restriction system, it is synthesized before the entire t7 genome enters the cells

Double-Stranded DNA Bacteriophage: the first discovered phage and the most extensively studied ds DNA viruses. Example: T7 1. Linear double-stranded DNA with 39,936 base pairs 2. 92% DNA codes for protein 3. Gene overlap through translation in different reading frame and through internal translational reinitiation as well as internal frame shifts. 4. One of the coded proteins inhibits the host restriction system, it is synthesized before the entire T7 genome enters the cells

Left end Earty promoters Overcomes host restriction Protein kinase Genetic Early transcript RNA polymerase 1.1 Unknown Origin of DNA replication Map of T7 DNA lease 1.7 Nonessential Inactivate host RNA polymeraseProteins Early transcription occurs Endonuclease 3.5 Lysozyme for DNA replication when left end enters cells Helicase, pr and host lysis DNA polymerase A host rna polymerase Protein in phage part is used just to copy the Head prot first few genes and to 9 Head assembly prot make the mrNa that Tail protein codes for the phage- Tail protein specific rna polymerase Protein in phage partick 14 Head protein structural Head protein and Two other early mrnA maturation 16 code for proteins which stop the action of host RNa polymerase DNA m

Genetic Map of T7 Early transcription occurs when left end enters cells A host RNA polymerase is used just to copy the first few genes and to make the mRNA that codes for the phage￾specific RNA polymerase Two other early mRNA code for proteins which stop the action of host RNA polymerase

DNA Replication in T7 Origin of In both direction. an RNa primer is involved Rightward: RNA primer is synthesized by T7 Eye"form 5 RNa polymerase Leftward: RNa primer is "Y form synthesized by primase Both primers are elongated by T7 DNA polymerase Completed strands

DNA Replication in T7 In both direction, an RNA primer is involved. Rightward: RNA primer is synthesized by T7 RNA polymerase Leftward: RNA primer is synthesized by T7 primase Both primers are elongated by T7 DNA polymerase

DNA Replication in T7 DNA ligase acts GA BC D E F G GAB'CDE′FG Pairing of unreplicated terminal repeats GA B C D E F G GAB′ C D E F G Joining of new and old molecules by DNA ligase, forming a concatamer

DNA Replication in T7

DNA Replication in t7 BCDEFGABCDEF+GABcD T has a much B DNA polymerase more complex GG AA replication Cutting cc cIC enzyme DID DID scheme than EIE G DNA that seen for the other bacterial Cutting enzyme DNA polymerase Mature T7 (arrows) makes completes the molecule viruses single-stranded with terminal cuts cuts

DNA Replication in T7 T7 has a much more complex replication scheme than that seen for the other bacterial viruses

Large double-Stranded DNA bacteriophages the T-even Phages T2, t4 and t6 T4(as example) 1.7X 105 base pairs Have a unique base, 5-hydroxymethylcytosine instead of cytosine The above hydroxyl group is modified by addition of glucosyl residues This glucosylated dna is resistant to virtually all restriction endonucleases of the host

Large Double-Stranded DNA bacteriophages: the T-even Phages T2, T4 and T6 T4 (as example) – 1.7 X 105 base pairs – Have a unique base, 5-hydroxymethylcytosine instead of cytosine – The above hydroxyl group is modified by addition of glucosyl residues – This glucosylated DNA is resistant to virtually all restriction endonucleases of the host

DNA replication in T4 O0 Ta. Nucleases DNA polymerase Phage head and tail fiber Mature phage partice New sigma factors T4 lysozyme Phage DNA replication Ea arly mRNA Middle mRNA Late mRNA Set assembly Lysis Minutes Time course of events in phage t4 infection

DNA replication in T4 Time course of events in phage T4 infection

Enzyme DNA Replication in T4 GABcDEFGABcDEFGABcDEFGABcDEFGAB BcDEFGABcDEFGABcDEFGABcDEFG The cutting enzyme which forms virus sized fragments does not recognize specifIc locations on the long molecule. but cuts off DNA irrespective of the sequence Thus each viruS dNa sequences at the end of Long DNA Molecules different molecules are molecules attacked b different cutting

DNA Replication in T4 The cutting enzyme which forms virus￾sized fragments does not recognize specific locations on the long molecule, but cuts off DNA irrespective of the sequence. Thus, each virus DNA sequences at the end of different molecules are different