oO uecture 1. Columbia University. April 6, 1964 Principles Derived from Study of Bacterial and Phage Genetic oystems I. the Organization of the Nucleus in the Bacteria: 1. DBA is not bound with protein as in higher organizims. ec. No true nucleus as in higher organisms; a "nuclear vacuole" containing the DNa, 3. No nucleolus; no nuclear membrane. wlides 1,2, 3,4 4, DNA in form of a ring. E. coli, K-12; DNA 1,100 to 1,400 microns long. 50-40 angstroms wide. 5. Replication of bacterial chromos:re: Semi conservative. Starts at one position and continues ahong chromosome: Cairns Diagram Slide s Autoradiograph Study: Photogravh: slide, “oe 6. Position of start of replication process: Examined in #. coli and B. subtilis. (a) B. subtilis: One strain: always starts at one point and goes in wy one direction: lys ind met oniy ade thr tyr his leu iluval phe ileu < Gua, > end (0) Other strain: No discovered set position. stole at deliv) pout vow (c). E. coli, K-12. Relation of start of replication to the sex factor, when present: Always starts at F factor:Slide 7 (ad). No mitdtic apparatus; cell membrane; particular position; Mode of growth of bacterium during division. Bede & when F,incorporated into bacterial chromosome - its replication system dominates bucterial system, as shown by Nagata. Significance important for sex behavior. “il. return to this. It. types of genes recognized in bacteria: Classes: Class ai, }otructural" genes; related to production of eazymes: m kWA —- transcription from Dia. Yrotein - translation from mRNA Mutant sites in the structural genes: Diagram perm—reent Craungg wn Coe, ~ Cbouga w4 ale ceed ig bee . T Class B: Only RNA produced: lau ab acu tYih (2) Ribosomal genes - 2 % of genome Os ll Tsu up sc me 4or lh TUE ist . (3) Transfer RNA - soluble RNA , # known; how acts. Cue ov age? fn Ged Due ae O ithe owt ob x ong tel (4) Regulator genes -- product not yet known. Possibly RUA attached to specific oréfein Special classes:{, (5) The super-suppressors: “ypes » DY RR pede? by (a) cup. Garen et al. Cputw - oki — pale _ Lee i ad av . i, . | p (bo) Sm certain mutants requiring streptomycin. Riler aap mcopeen ~2- a EE wig =] (6) The "operators" At initiation point of read.ng of DNA of gene or operon. Ili. The organization of the genes in the bacterial chromosome. Reg Cuskoeua - 1. The operons}, _,Qp . : L \ \ a v 7 or Op + mo 4 Regulator Examples: Histidine: Genes -enzymes fur biosynthetic pathway to production of histidine: Slide 4 Order of genes and enzymes in pathway: Position of first enzyme and the operator. Coordination. 2. Biosynthetic pathway - genes not together: lxample: Arginine genes Slide ta e The Regulator - vosition; Coordination not as above TERRE ~ Serene 3. salmonella and E. coli: related. Order of genes the same in both organisms. 4, special tyse of gene org-nigzation and control: The yy and Hy genes producing flagella antigen: “uplicate genes; only one acts at a time. vontrol mechanisms -- will cowsider along with maize control mechanisms. IV. Transformation and transduction: significance for relating bacteria to higher organisms. nn oendbo 1. Transformation: DNA ,extracted from one strain: placed in medium with another; markers present; uvtake of DNA molecules; replacement of DNA in bacterium by introduced DNA molecule. ixtraordinary process: Synapsis on the molecular level; occurs with great rapidity. 2. Transduction: occurs through participation of episones. Introduce | DNA from one bacterium to another through being carried by the episome,:4¢ %* 3. The bacterial viruses: DNA viruses. Different types. Different sizes. Hxample: The phage particle; its parts. Slides tl, ie Attachment of chuage to bacterium: Slide 13 Insertion of phage chromosone: vlide |4 { omall pPuege. Galuanty soda? ry, the order of the genes in phage T-4: slide 4% 4, Transduction: chage vicks up piece from bacterial chro.ossie: infects bacterium. Yoes not cortain its own genes. No phage reproduction. striece from one bacterium to another. ‘arkers present: wee) a oO Vote) factuniiu - L [oA \ nave ae i7Behavior of bacterium during pnsse reporduction: rlide 17 othe phage chromoso e: sambda: Slides 15) lb 16.3 microns = 5. Importance: synapsis on molecular level and exachange by "crossing-over" : ne Lo + eee Ie —— ~7 ees Ioana te, | WT asin Y 6. Mlagella genes: “tT apalo Tllustration: Importance: ec). Synaosis at meiosis: not altovetner a howologous act: initiated by homology. Abortive transduction: ohage does not beconue crossing over? Naked DNA = axanvle of SF Og. Slide 19 rece e able to nuclear vacuole; replicate!, d). Under yet unknown cor ditions in higher organisms as (see 6, below). piece of bacterial p.corporated. action: Beet ins - fr flagotla e of same type to multicly in cell. incorporated phage produces a repressor substeunce that represses first stages of phage multivlication. (b>). Phage sometimes released from bacteriab chromcsone and multiplies vegetatively as above: bacterium lysed and phage particles released. Uccurs spontanesusly or induced by U.V. or chesical treatment. lysogeny: name associnted with potential for lysing bact. 2. Position where phage is incorvorstcd into bacterial chronoso.e: lwo ty oes (a) Can enter any location in the bacterial chronosowe: Used for producing phage for transduction of different bacterial characters: Grow bacteria carrying prophage ( phe DNA in bacterial chrouosome). Treat with UV. light to release phupe. (>) Phage is Bncorporated into one particular position in bacterial chromosome. Example, Lambda shage at locus of gal genes in bacteria. (c) Induce lysis of bambémia carrying lambda. Yecasional ohave varticle thet picks up part of gal locus; loses part of its own DNA. Will transduce the gale locus at very high frequency as a consequence. Valled high frequency transducing pheges. VIII. The sex-factor. A DNA carrying episome. Does not lyse the bacterium; l. # factor exists in two different stz2tes: In cytoplasm of the bacterium or incorporated into the bacterial chromosome. 2. when in cytoplasm; divides slong with the bacteriz2l chro osoe: > Qbide or: fas its own replication system; associnted with cell membrane. ~"” 3. Nembrane association of F reinted to conjugation between bacteria carrying F and those with no F: Transfers the F factor from mile to female througn the tube: Slide 32 ad Male Female Female becomes a male when F Received. g Wh Atti. 2? _Z veo F 4a, ‘neorporation of F me bacterial ~~ ome: Positions / \- ‘a ee A Cc now Lbs # factor: its replication system,takes over cortrol of initiating i. position of repbhication of the bacterial chrouosome: "Dominant". 6. When incorvorated into bacterial chronos me: carries bacterial chrorioso.e into female during conjugation: R gE Qos ¥. eed 8 0 a, 7. Position of incorporation of F: Varies. Any one position gives high frequency of transfer of those genes near the origin. , Chrouosome entering bacterium takes 120 minutes at normal, temocrature xe Chromosome can be broken off during process; part that has entered can under go recombination "crossing-over" with chromosone of female. 8. Factor -- controlling element -- bn F factor that controls enterance of F into female, (with or without chromosome of wacteria att.ched.) if within pacterial chromosome, will carry bacterial chrowoso.:e along with it. Resembles the controlling factor in Sciara X. Te 4m IX. The sing-stranded DNA phages: Single strand in phege particle. Yuring replicetion, a double strand and a ring.shape. Double strand required for replication. X. Importance of double-strand: Required for redunlication of DNA. pall , % “ pe fs ute ound a4, Lue-pudl S aud Eo ay rab Ruegall oud -Vs 26 Foor gouol dy TL ao i -1b) Up S oh (fp Lf ‘Splut p : ~ Reagan's MB Qi > j i} ho . ) nbbe ~ Bet . / ) ty (2 “4e | “ ( >- 25 bate ib Ayn ny i. by 24 deeded vile bed. ra get ts 34 I 34 oho