Microbiology lOOA Spring 1976 Harold Vam, M.D. 1. VIRUSES AS VEHICLES OF GENETIC CHANGE R uired Readin : Chapter 46 in Davis et al., Microbiolo , 2nd edition. .*a1 reading, see Chapter 9 in Dadications of the Cold Spring Harbor Laboratory: The Bacteriophage Lambda and Pha e and the Origins of Molecular Biology ,especially chapter by Lwoff;JBD- katson, The Molecular Biology of the Gene, 3rd edition.) Highly Recommended: S.N.Cohen: Gene Manipulation, New Engl. J. Med. 294:883-889, AT 15, 1976. This lecture is intended as an introduction to mechanisms by which viruses can enter into relationships with their host cells that differ from classical lytic infections. cells, yet confer new properties upon them, and viruses can act as vehicles for transfer of stably inherited genetic information between cells. nucleic acids, both cellular and viral, can be considered "infectious" in the sense that they can introduce usable genetic information into host cells; in this respect, cellular DNA, particularly bacterial plasmid DNA, appears closely related to viral genomes, suggesting evolutionary relation- ships between viruses and plasmids. The considerations raised here pro- vide a framework for proposing approaches to genetic engineering, in par- ticular to the amplification of specific genes in bacteria ("cloning") and the more improbable therapy of genetic diseases. L so en . Lysogenic bacteria contain a viral genome in a repressed state, and + t ey may, spontaneously or after "induction," ultimately produce mature, lytic virus. The bacteriophage lambda is the best studied example of a "temperate" bacterial virus (one wFEl'iTs capable of lysogenizing cells). Thus viruses can become cryptic in infected Naked I. The life cycle of lambda Dharre: - Injection of linear viral DNA - Circularization of the DNA and - Phase of uncertainty: vegative cell lysis versus lysogenic production of "early" gene products athwa to virus production and to viral gene repression To enter the lysogenic pathway, repressor (the protein product of viral gene CI, capable of blocking transcription of "early" genes required for viral replication) dominates over other viral genes (especially the early gene - cro, which interferes with production of repressor). viral genome with the circular bacterial chromosome by a single "crossing over" event; in the case of lambda, this event normally occurs at a specific site in both DNA's; the event requires the action of viral gene(s). integrated viral genome is called the prophage. repression of other viral genes and immunity to superinfection by similar viruses. Stable lysogeny requires recombination (integration) of the circular The Maintenance of lysogeny: continuous production ofrepressor maintains 2. Induction: W radiation or cytotoxic chemicals can inactivate re- pressor by uncertain mechanisms; increased temperature can induce prophage mutants which produce heat sensitive repressor. Inactivationofrepressor is followed by: (1) (2) (3) Lysogenic cells ma contain only one new product (repressor), but in Expression of early genes: cro (augments repressor shutoff); Expression of genes adjacent to N and cro, which are required for Expression of late genes for structural proteins of phage and for - N (antiterminator: allw7transcription into adjacent genes) excision of prophage and repTicatiKof DNA cell lysis. some cases they contain -5 ot er new products; some of these are of medical at any point in the cellu -+- ar genome and thereby interrupt a cellular gene or importance (eg, diphtheria toxin, streptococcal toxins, and some other toxins are found only in lysogenic bacteria). example is provided by Mu-1 hage (mutator phage) which may integrate its DNA operon (coordinated set of genes). perhaps, certain "slow" viruses and certain "latent" infections. mation between cells (bacteria, plant, or animal), either by packaging host DNA in a viral coat or by recombining its genome with a piece of the host genome. If the recombination tends to occur at a specific site in the host genome, certain genes are likely to be transferred by that virus; this is called "specific" or "s ecialized" transduction. (Example : lambda phage transducing the gal or bio hich are close to the lambda integration site on the bactzal cfiromosome.) of Mu-1 phage), or if host DNA is randomly packaged in a viral coat (as in the case of P1 or P22 phage), the transduction of virtually any host gene may occur, and the event is called tions upon the amount of "%an DNA w IC fit inside the viral coat, viral genomes which have added cellular genes may have lost critical viral genes and have become "defective" viruses. a "helper" (non-defZEEJ virus in the same cell, supplying the product of the missing gene. PackageFZ7iost DNA in viral coats cannot replicate and are referred to as "pseudoviruses." For successful and stable transduction in this case, the transferred cellular DNA must recombine with the chromosome of the recipient host cell. 111. Plasmids and other Forms of_Infectious, Nonviral DNA. Plasmids are circluar DNA molecules, generally loo to loo dal tons in molecular weight, which can replicate autonomusly in cells; some plasmids can integrate into the host chromosome, and some can direct bacterial conjugation, but they are not normally required for survival of the bacteria; although they have been thus far studied principally in bacteria, they may well exist in eukaryotic cells as well (eg, mitochondrial DNA can be considered a plasmid). Prophage may also influence expression of bacterial genes; the best Lysogeny poses an important model for understanding tumor viruses and, 11. Transduction. Viruses can promote the exchange of genetic infor- If the recombination occurs randomly (as in the case eneralized" transduction. Since there are limita- This means they are unable to replicate without In bacteria, 3. plasmids are extremely common; most strains of some bacterial species will carry one or more plasmids. of plasmids with a wide variety of functions, and with various relation- ships with the host chromosome: Most plasmids fall into three major categories (1) F (fertility) factors: large plasmids which can direct bac- terial mating (conjugation) for transfer of F factor itself and for transfer of any bacterial genes which have recombined with it. integrate into the host chromosome; from this position, it can direct trans- fer of host genes from one bacterium to another. (2) Resistance transfer factors (RTF's): an assortment of plasmids which can render the host resistant to antibiotics (mechanisms will be con- sidered in detail in the fall quarter, and are of paramount importance in determining clinical efficacy of antibiotics) ; for transfer of these factors from cell to cell, even between species of bac- teria. (3) Toxin-producing plasmids: these produce factors toxic either for other bacteria (eg, colicins, a variety of chemicals toxic for E. coli) or for animals (eg, enterotoxins, producing diarrhea in man and otlier animals). The F factor can also they also direct conjugation - In addition to being transmissible via bacterial conjugation, naked plasmid DNA, like viral DNA or host cell DNA, is infectious. DNA contains the elements required for its replication, it need not integrate to successfully t5nfect1t a cell. Pieces of host DNA, however, cannot repli- cate and therefore must integrate into the genome of the recipient cells, as in the case of generalized transduction by pseudovirions (see above). fection" of cells by plasmid or host DNA is called "transformation" but should not be confused with the change in behaviour of animal cells (also called t'transformation'') that is induced by infection with tumor viruses (see next lecture). in cells has recently been discovered. These are relatively short segments of DNA, called "translocation sequences" or "insertion sequences ,I1 which may per- form important functions (eg, direct resistance to antibiotics),and which can jump, in a block, by unknown mechanisms, from one larger DNA molecule to another. Virus-mediated transduction, transformation by host or plasmid DNA, and migration of translocation sequences all suggest that evolution can occur much more rapidly than can be accomplished by a series of simple point mutations. The mechanisms discussed permit genes developed during evolution of one organism to be "shared" by others, thus hastening their development. The de ree to which genetic change is mediated by such mechanisms in nature "natura conditions viruses are more highly developed in that they can usually direct the synthesis of coat proteins, often have highly regulated patterns of gene expression (cf lambda), and may lyse their host cells. However, the struc- ture, replicXion, integrative capacity, and infectivity of their DNA's are very similar. When a defective, transducing virus is compared with plasmid containing host genes, the differences may seem very small indeed. these reasons, it is generally thought that plasmids and viruses are closely Since plasmid "In- A new class of DNA molecules capable of making rapid genetic changes is, 2- owever, unknown, although these mechanisms have all been observed under here are somi! obvious differences between plasmids and DNA viruses - For 4. related,perhaps having arisen similarly from host chromosomes and evolved to different degrees. IV. Gene Transfer by Genetic Engineering. We have considered some ways in which viruses and plasmids, occurring naturally, are able to effect the transfer and replication of genetic information. It is also possible in the laboratory to recombine viruses or plasmids with genetic material from virtually any source for a variety of purposes. Several technical advances have made the manufacture of such "recombinant" (or "chimeric") molecules possible (1) biochemical procedures for adding homopolymeric "tails" (eg , dAdAdAdA.. . and dTdTdTdT.. .) to DNA molecules one desires to join; (2) the discovery ofnucleases ("restriction endonucleases") capable of making site- specific (and often "staggered") cleavages of DNA which also allow joining of DNA molecules (see slides). bacteria, and plasmids; in the purification of single genes; in the synthesis of nucleic acids de novo; messenger RNA (by7rGEFse transcriptase ,I1 see tumor virus lecture) have ex- pedited the development of genetic engineering. The basic strategy in most "genetic engineering" proposals is to join some specific gene (the "donor" DNA) with at least the replication-competent part of viral or plasmid DNA (the "vector" DNA). The "hybrid" molecule is then used to l'transforml' a recipient cell, most commonly a bacterial cell. The process of "infecting" a single cell with a defined piece of DNA and growing the progeny of that single cell is referred to as the "cloning" of DNA. Since the progeny cells can be grown in large number and my contain hundreds of copies of the cloned DNA per cell, it is possible to prepare very large amounts of single genes for detailed study (eg, DNA sequencing, etc). (The human beta-globin gene has already been "cloned" for such purpose.) addition, it might be possible to produce large amounts of the protein product of the "cloned" gene in the recipient cell. might be possible to prepare "cloned" genes for delivery to patients with genetic deficiencies (gene therapy") . associated with this general strategy for cloning and gene therapy: In addition, advances in the genetics of viruses, and in the preparation of DNA from purified messenger In Lastly, and more remotely, it There are many problems and dangers, as well as potential benefits, Difficulties with cloning: (1) Could certain DNA molecules, produced in large amounts in bac- terial cells, pose a medical danger? (For example, an "oncogene" from mammalian DNA? See tumor virus lecture.) (2) Will the use of certain vectors, eg, certain plas~ds, lead to the spread of unwanted genes, eg, those determining resistance to antibiotics? (3) Can a "safe" vector and recipient cell be created which will pre- vent the possibility that cells containing cloned DNA would grow in the human intestine? Or can safe facilities be constructed for growing potentially dangerous material? Difficulties with gene ther-: (1) Can the necessary gene be identified, purified, and prepared in suf- ficient quantities? (2) Can the genetic disease really be cured by administration of a gene? (Multigenic diseases like diabetes or Mendelian dominant disorders would likely be resistant to gene therapy; recessive point mutations or gene deletions might be susceptible to it.) affect the patient? the recipient cells? it be properly controlled at transcriptional and translational levels?) DNA will be delivered and expressed? Or that the integration of new DNA within a nom1 gene will disturb the function of the normal gene (as in the example of Mu-1 phage, see above)? (3) (4) How can the gene be delivered to sufficient numbers of cells to Will the delivered gene be perpetuated and function properly in (For example, will it replicate or be integrated? Will (5) What are the chances that unwanted genetic material or mutated The consensus at present (if there is one) is that gene therapy is currently impractical and of less use than its alternative (genetic counsel- ing or other forms of treatment, eg, replacement of the missing gene roduct dicious selection of the DNA to be cloned, the vector, apd the recipient cell, and by use of the proper techniques and facilities. lines governing such research have recently been recommended by the scientific community and are the subject of public debate. or drug therapy). The dangers of cloning, however, can be minimized E- y ju- Rather stringent guide- 6. - SLIDES - COMPARISON OF EXTRACHROKISOMAL ELEMENfS DIRECTS CONJUGATION CODES FOR COAT PROTEIN DIRECFS CELL LYSIS DNA ~IR~Z~S DNA INTEGRATES DNA ~~~1~s WITH HOST DNA DNA IS 1~~1~s DNA SIZE (ranJ) TRANSDUCING VIRUS + (UNLESS DEFECTIVE) + + + + 3 - 30 x lo6 GENE TRANSFER MODE AGENT ~S~~TION NAKED DNA CONJUGATION (MEDIAED BY FREE OR r~G~~D PLASMID) TRANSDUCTION VIRUS PARTICLE "CLONING" HYBRID DNA (PLASMID OR VIRAL PLUS ANYTHING) TRANSLOCATION PLASMID SEQENCES + or - - - + (+I + + GENES TRANSFERRED HOST, PLASMID, OR VIRAL PLASMID OR BACTERIAL GENES HOST DNA (MAY BE LINKED To VIRAL DNA) ANY ANY 7. Lac Operon -v* ... LYSOGENY BY LAMBDA PHAGE Lac Operon -v* ... Mature Phage Particle INFECTION g72-!L3 43 injection and circularization of Phage DNA I I B LY SOG E NY 9 Integration of Phage DNA, production @@ & @ I lysogenic state and immunity i IN DUCTION inactivation of repressor, DNA repticotion, virus producticn (cell lysis) LYSOGENY BY MU-1 PM$.GE Ld E3 RANDOM INTEGRATION MAY INACTIVATE BACTERiAL GENES 8. Inactivation repressor of Production of cro + N g e n e prod uc ts , in h i bi t.i n g repressor production, switching on excision gen and DNA synthesis genes os Correct excision and replication of viral DNA; expression of "late" Benes for structural proteins \ 4 STRUCTURAL d PROTEINS VIRUS-MEDIATED TRANSDUCTDM & LYSOGENIC HOST CELL @ DNA 1 INFECTiON #l . / VIRAL DNA d- W LYSOGENfC c.c INFECTION #2 VIRAL DNA I -HOSY CELL 0 DNA PACKAGING OF HOST CELL DNA: A MECHANISM FOR GENERALIZED TRANSDUCTION \ #--\ I` 1 l 0 .---@A VIRION PSEUDOVIRION \HOST CELL DNA BEHAVIOR PATTERNS FOR BACTERIAL PLASMIDS Boctoriol C h ro mo I o rn e DNA 000 INDEPENDENT REPLICATION u INTEGRATION EXCISED PLASMID CONTAING HOST DNA Page 9 PLASMID-MEDIATED TPANSFER OF HOST DNA 10. GENETIC ENGINEERING: HYBRID DNA I HOMOPOLYMERIC TAILS m- m AAA- -MA II ENDONUCLEASE- GENERATED "STICKY ENDS' `CG G- -c-c- Q 4 CG- + CG -GC + - 4 CG GC CG- -GC I- (;] STRATEGY FOR GENETIC ENGINEERING Donor DNA Plasmid or Viral Hybrid DNA El DNA (vector) Transforma tion and "Cloning" Grow large Quantities s- and /or of Donor its Gene DNA Product p0-0) Transfer Donor DNA to Genetically-Deficient Host (gene therapy) Bacterial Cell (recipient)