Bacterial Genetics Ch 18, 19

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1 Bacterial Genetics Ch 18, 19 1

2 Genomics First genome sequenced = human mitochondra = 16, 159 base pairs 2

3 Mitochondrial DNA 37 genes 2 rrnas 22 trnas 13 polypeptides 3

4 Bacterial Genomics Chapter 10, page 251 First sequenced was Haemophilus influenzae 1.83 million bp 4

5 Escherichia coli (1997) Located in the lower intestines of animals Pathogenic strains (ex. E. coli 0157) Genome 4.6 megabases ~ 4000 genes, ~88 % of genome open reading frames 5

6 6

7 Single circular chromosome 7

8 E. coli biology Prokaryote nucleoid region contains the chromosome Neisseria gonorrhoeae. 8

9 E. coli reproduction Bacteria reproduce by binary fission -> Exponential growth ~ 3 microns in length = 3 millionths of a meter 9

10 Bacterial growth colony - visible cluster of clones about 1 million cells /colony Growth on agar plate lawn entire plate is covered, no individual colonies visible 10

11 Growth of bacteria (E. coli) Lag phase - slow or no apparent growth Log phase double every 20 to 1 X 10 9 /ml Stationary phase nutrient and/or oxygen limited Cell number remains constant Death phase Nutrients gone, toxic products build up, cells die 11

12 Bacterial growth curve 12

13 Generation times for bacteria vary Escherichia coli Glucose-salts 17 min. Streptococcus lactis Milk 26 Streptococcus lactis Lactose broth 48 cheese Staphylococcus aureus Heart infusion broth infectious Rhizobium japonicum Mannitol-salts-yeast extract soil Mycobacterium tuberculosis Synthetic Treponema pallidum Rabbit testes

14 Growth media minimal media =only essentials provided Sugar (carbon source) + salts bacteria synthesize aa, nucleotides, vitamins complete media selective media Allows one species to grow while selecting against another 14

15 Solid and liquid culture Growth in liquid media Growth on agar plate 15

16 Phenotypes Prototroph can synthesize requirements from minimal media Auxotroph nutritional mutant Requires one or more supplements to grow 16

17 Bacterial phenotypes Resistant to ampicillin = Amp r Sensitivity to streptomycin = Str s auxotroph mutant requires tryptophan = Trp - trp - leu - thi + tet r? 17

18 18

19 Bacterial mutants Nutritional mutants Auxotrophs that require supplement to grow Conditional mutants The mutation is only expressed in a certain condition Resistance mutant Antibiotic resistance in bacteria 19

20 How do bacteria undergo genetic recombination? 20

21 Noble Prize for bacterial genetics Lederberg, Beadle and Tatum 1946> Nobel

22 Conjugation parasexual mating one-way transfer of genetic information from male to female bacteria 22

23 E. coli nutritional mutants demonstrate conjugation Mix auxotrophs alone cannot grow on minimal media: Strain A met- bio- thr+ leu+ Strain B met+ bio+ thr- leu- OBTAIN ---> a few prototrophs that grow on minimal media: What would the genotype of this prototroph be? 23

24 Fig 18.2 Its rare! 1 /10,000,000 Genetic recombination 24

25 Fig Davis U-tube showed that conjugation requires cell/cell contact met- bio- thr- leu- filter Note the filter Media can pass but cells can t no prototrophs obtained Show that cell-cell contact is required 25

26 F factor (plasmid) carries DNA from male to female bacterium F factor circular, episomally maintained piece of DNA Encodes F pilus on donor cell Donor cell is F+ 26

27 F pilus fig Donor Recipient 27

28 Conjugation fig F+ + F- = 2F+ Steps: Pilus -> nick -> transfer -> double stranded -> break pilus 28

29 F factor is a plasmid 94,000 bp Must have an origin of replication (ori) to be maintained self-mobilizable -- can transfer to other cells. (Pili cannot attach to other donor cells due to the presence of the proteins coded by the tras and trat genes -- this is called surface exclusion) 29

30 30

31 Recombination (rare): Integration of F factor into chromosome requires insertion sequences (IS) Hfr strain fig

32 Hfr conjugation: F factor would transfer last The first DNA to be transferred is the chromosomal DNA Pilus is broken before F factor is transferred Recipient cell remains F- 32

33 genetic recombination with Hfr The transferred DNA may degrade or undergo homologous recombination 33

34 Comparing an Hfr to F+ strain F+ x F- recipients are F+ Low frequency of recombinants upon conjugation Hfr x F- recipients are F- High frequency of recombinants upon conjugation 34

35 Hfr strains allow mapping of the E. coli chromosome! Site of integration and orientation of plasmid integration in the Hfr bacterial DNA is random Linear transfer of genes So, the time it takes for a particular gene to transfer depends on where its located on the chromosome 35

36 Lederberg s experiment explained fig

37 Interrupted mating technique to map genes on E. coli 1. Mix donor and recipient cells. Hfr str s + F- str r 2. Incubate to allow conjugation to get started 3. At time t, blend the culture in the kitchen blender. This disrupts the cell pairs but does not break the individual cells. 4. Plate recipient cells (use streptomycin selection why?). 5. Screen for recombinant markers. Elie Wollman & François Jacob 37

38 The mating: Hfr H (azi R ton R lac+gal+str S ) x F- (azi S ton S lac-gal-str R ) 38

39 Fig

40 E. coli minute map = 4.7 million bp (4377 genes) Clock face... Gene controlling Noon+ threonine synthesis 1 o'clock lactose degradation (lac-operon) 2 o'clock galactose -> glucose (gal-operon) 3 o'clock tryptophan synthesis (trp-operon) 5 o'clock histidine synthesis (his-operon) 7 o'clock lysine synthesis 8 o'clock streptomycin resistance 9 o'clock mannitol degradation 10 o'clock Place where chomosome synthesis begins in both directions ("OriC") 11 o'clock methionine synthesis Noon- "F"-episome (where "F" is inserted) 40

41 41

42 42

43 Map genes using different Hfr strains In E. coli, four Hfr strains donate the genetic markers shown in the order given: STRAIN 1: QWDMT STRAIN 3: BNCAX STRAIN 2: AXPTM STRAIN 4: BQWDM What is the order of these markers on the circular chromosome of the original F+? What is the location and orientation of the F factor integration in the bacterial chromosome? 43

44 Transduction phage mediated transfer of genes into bacteria Phage a virus that infects bacteria Salmonella typhimurium bacteria and P22 virus U-tube experiment mix 2 auxotrophs prototrophs appear (low rate) 44

45 Filter prevents cell contact, transduction still occurs 45

46 Viral infection 1. Virus adsorbs to cell and injects DNA 46

47 47

48 2. normal bacterial activity is shut down and bacterium becomes a phage factory 48

49 49

50 3. host DNA broken into pieces, new viruses released to infect new cells 50

51 chromosomal DNA is chopped as viruses destroy cell 51

52 Faulty head stuffing As chromosomal DNA is broken, a piece can get packaged into a virus. This virus can infect a new cell and transfer genes from the first bacterium 52

53 Gene therapy with virus Ch 9, pg 231 Objective : insert a normal gene into human DNA Use virus as vector 53

54 Remove viral replication genes insert human gene Infect the human 54

55 Gene Therapy ADA 1990 Defect in T cells Remove T cells Engineer in lab Infuse into patient Repeat 55

56 56

57 Status of gene therapy in US The FDA has not approved any gene therapy 1999 set back with death of Jesse Gelsinger Gene therapy trials are going on Aggressive brain cancer Arthritis Blindness (dogs) Hemophilia (dogs) Liver disease (mice) 57

58 Bacteriophage phenotypes virulent phage - always lytic, cannot become a prophage temperate phage - lysogenic 58

59 Temperate phage 59

60 Transformation Naked DNA enters bacterial cell. Brings new genes Plasmids are extrachromosomally maintained 60

61 Plasmids are cloning vectors (ch 8 pg 179) puc19 plasmid, a cloning vector amp r gene ori restriction sites (multiple cloning site) 61

62 Amp r Ori arac GFP 62

63 Transformation in the laboratory Make cells competent by calcium chloride 42 degree C heat shock facilitates uptake To be continued in lab 63

64 The Lac Operon 1961, Jacob and Monod E. coli and other bacteria Bacterial Genes Many genes are constitutively expressed these are housekeeping genes Other genes are regulated Can be turned on, or off depending on cell needs 64

65 Operon group of coordinately regulated genes One promoter for a number of genes Polycistronic mrna 1 mrna molecule has info from multiple genes 65

66 E. Coli Lac Operon E. coli cells can convert lactose to glucose and galactose 66

67 The Lac Operon allows for coordinate gene expression Note: 1 mrna, promoter 67

68 3 STUCTURAL GENES = Z, Y, A Lac Z gene encodes b-galactosidase enzyme b-gal lactose glucose + galactose 68

69 LacZ gene is only transcribed when lactose sugar is present b- gal is an inducible enzyme (induced by lactose from 5 copies enzyme to 1000s) 69

70 This only occurs in the presence of lactose, the inducer Fig hydrolysis 70

71 DNA -> Proteins -> promoter = regulates transcription of ZYA operator = must be unbound for P to be open 71

72 omit Lac Y gene encodes a permease that transports lactose into the cell Lac A encodes a transacetylase 72

73 REPRESSOR PROTEIN (I) Encoded by Lac I gene Binds to operator Prevents RNA pol from binding to promoter 73

74 Is this operon ON or OFF? Is lactose PRESENT or ABSENT? Lac I, P, O, ZYA genes are CIS elements 74

75 INDUCER (LACTOSE SUGAR) LACTOSE PRESENT Lactose enters Binds repressor protein (I) causing a conformational change This pulls the repressor off the operator RNA polymerase transcribes genes Cell metabolizes lactose 75

76 Lactose (the inducer) enters the cell Binds repressor protein causing a conformational change 76

77 No lactose: repressor binds to operator polymerase cannot bind promoter no transcription of ZYA genes 77

78 NO LACTOSE 78

79 Why study the lac operon? The lac operon is one of the most basic examples of gene regulation. Gene regulation is an important area of study in medicine as many diseases and conditions are as a result of deficiencies in gene regulation. Cancer is one such disease that results in the inability of a cell to control the genes that regulate its growth. Many systems of gene regulation in humans are quite complex and not understood by biologists and researchers. In studying simple models of gene regulation, we hope to perhaps gain some insight into how more complex gene regulatory systems work. 79

80 Operon mutants Mutant Mutant Phenotype lac I- constitutive expression because O c constitutive expression because P- no expression of operon because lac Z-? 80

81 Operon on, or off in the absence of lactose? Presence of lactose? Lac I- (I - P + O + Z + Y + A + ) Lac O c (I + P + O c Z + Y + A + ) 81

82 Remember, repressor and polymerase are proteins which are diffusible These proteins bind DNA They act in TRANS The promoter, operator, and ZYA and I are genes and cannot move They act in CIS 82

83 Operon inducible? Always on? Never on? Genes may be carried on plasmid (F ) F I + I - P + O + Z + Y + A + - F Z + Y + A + I + P + O + Z - Y - A 83

84 GFP use as a reporter for the expression of other genes 84

85 Cloning a gene from a jellyfish into bacteria using plasmid transformation

86 Terms and concepts Plasmid transformation Arabinose operon Reporter gene Gene cloning Recombinant DNA 86

87 Engineering the plasmid, pglo 1. Isolate jellyfish DNA 2. Use restriction enzymes to cut out GFP gene 3. Purify GFP gene 4. Ligate GFP into plasmid 87

88 Aequorea victoria source of the GFP gene 88

89

90 Transform E. coli with recombinant plasmid E. coli bacterium plasmids E. coli DNA Cell membrane 90

91 E. coli bacteria strain K12/HB101 Host for plasmid DNA The strain used in lab is non pathogenic 91

92 Use the arabinose operon to regulate expression of GFP in bacteria pglo plasmid contains ori replication of plasmid Amp r (bla)- ampicillin resistance Only transformed bacteria can grow in presence of amp GFP gene Ara C GFP gene expressed in presence of arabinose sugar 92

93 Transformation in the lab : Cells must be competent to take up plasmids treat with calcium chloride on ice 42 o C heat shock facilitates uptake of plasmid Bacteria then plated (100 ul on solid agar) 93

94 Grow in the presence of ampicillin + arabinose 94

95 GFP gene cloned into plants Arabidopsis thaliana seedlings Reporter gene 95

96 C. elegans GFP a reporter for olfactory receptor gene expressed when worms sense the odorant, diacetyl 96

97 M. musculus (mouse) GFP reporter for MHC gene 97

98 GFP embryo GFP mother with GFPminus embryo 98

99 Every cell has GFP 99

100 100

101 Anopheles gambiae cells GFP and the reaper apoptosis gene 101

102 Hoxc13-GFP fusion protein expression in nails of embryonic day 14.5 mouse 102

103 GFP and YFP reporter for stem cells 103

104 Glow fish pets 104

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