1 Gene transfer Ways that bacteria can acquire new genetic info –Transformation Taking up of “naked DNA” from solution –Transduction Transfer of DNA one to cell to another by a virus –Conjugation “Mating”: transfer of DNA from one bacterium to another by direct contact.

2 Transformation Uptake of “naked” DNA from medium. Classic experiment from Genetics history, 1920’s. Virulent cells have genes for making capsule which assists in infection. Mutant cells lack capsule, are harmless. Griffith combined heat killed, virulent cells with live, harmless mutants. The living cells took up the DNA from solution, changed into capsule- producing, virulent bacteria.

3 Transformation details DNA must be homologous, so transformation only occurs between a few, close relatives.

4 Transduction Transfer of DNA via a virus. More common, but still requires close relative.

5 Conjugation: bacterial sex If sex is the exchange of genetic material, this is as close as bacteria get. Conjugation is widespread and does NOT require bacteria to be closely related. Bacteria attach by means of a sex pilus, hold each other close, and DNA is transferred. Plasmids such as F plasmids and R plasmids can be exchanged, leading to antibiotic-resistant bacteria.

6 “feminist’s nightmare” F+ cells (donor) are considered “male”, recipient cells (F-) are considered female. F+ cells transfer a copy of the F plasmid to the recipient, therefore making it “male” also.

7 Conjugation can result in transfer of chromosomal genes F plasmid can insert and excise, bringing with it a piece of chromosomal DNA = F’ F plasmid stays inserted, directs copying and exchange of chromosomal DNA, but not F plasmid: Hfr cell.

8 Mutations A mutation is an inheritable change in DNA. This means an alteration in a basepair or in the order of the basepairs. Mutations may affect a single basepair, (point mutation) where they may change the sequence in an RNA or protein, or not (silent mutation). During protein synthesis, bases are read 3 at a time (codon); when the first base is read, the “reading frame” is established. If the frame is altered at some point the bases will NOT be in the right places, and the information will be garbled.

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10 Mutagens Radiation –Ionizing radiation chemically alters DNA, breaks one or two DNA strands Leads to replication failure, death –Ultraviolet radiation causes thymine dimers to form Kink in DNA chain also leads to replication failure unless repaired.

11 Mutagens-2 Chemical changes –Many mutagens are chemicals that change one base to another, cause mispairing. –Others are base analogs, used instead of real base, cause mispairing or disrupt replication. Frameshifts –Flat molecules (ethidium bromide, acridine orange) intercalate into DNA (slip between flat bases). When replication occurs, extra base added, disrupts reading frame, scrambles codons.

12 Non-mutagen cause of mutations (causes background level) Copying errors –Fairly rare; enzyme messes up and doesn’t catch mistake –DNA polymerase has a 3’ to 5’ exonuclease activity that backspaces, deletes mistakes. Bases themselves undergo spontaneous change to wrong base. Repair sometimes doesn’t happen. –Cytosine to uracil change is detected, removed. –Missing purines are detected, repaired.

13 DNA damaged often repaired The mutation rate is variable –Typical 1^10 6 per gene per generation –Rare, so DNA damage must be frequently repaired. Repair of thymine dimers –Excision repair: bad spot of DNA cut out, replaced –Light repair: a photon of blue light + enzyme undoes it. Mismatch repair: if base pairs aren’t a pair –Stretch of newer DNA is cut out, replaced. SOS repair: too much damage!! –Base pair “fidelity” is relaxed to save time. More mutations produced, but cells live.

14 Using mutations-1 The Ames test –Developed by biochemist Bruce Ames to determine whether a substance is a mutagen Nearly all carcinogens are mutagens; first level screen. –His - mutant of Salmonella combined with chemical Plated onto medium without histidine, won’t grow If chemical is a mutagen, revertants will appear at high frequency, no longer need histidine in medium. Test often done with liver extract; enzymes mimic human body where metabolite may be mutagen.

15 Using mutations-2 How to get a mutant like the his - mutant? –Treat with a mutagen (UV light, chemical, etc.) –Spread survivors onto rich nutrient medium, get colonies. Many bacterial mutants that are studied have nutritional defects. –Wild type (normal) is called a prototroph –Mutant that can’t make a nutrient is called an auxotroph. This example: looking for a serine auxotroph.

16 Replica plating First, expose cells to mutagen, then spread onto defined medium containing many amino acids and other nutrients. Replica plate onto medium with same composition except NO SERINE.

17 Result of replica plating Look: where did a colony NOT grow on this plate? Go back to original plate and grow colony. lectures/genetics1.html

18 Transposon mutagenesis and positive selection of mutants Transposons –Piece of DNA that can copy itself and insert at random into the bacterial chromosome. –Contains gene for transposase, DNA sequences needed for insertion, and an antibiotic resistance gene. Transposons are useful for making mutants –Insertion is random, so there is the possibility of mutating the gene you are looking for. –Antibiotic resistance provides a “selectable marker”

19 Transposon mutagenesis-2 Use a virus or conjugating donor bacterium to introduce Tn5 into recipient. Once DNA with Tn5 is in cell, transposon jumps once into recipient’s DNA, causing a different mutation in each cell. Plate recipient cells onto kanamycin-containing agar; only cells with transposon mutations survive. –This is positive selection Mutations can now be mapped, cloned, or whatever else you wish to do.

20 Tools and Techniques of genetic engineering Reverse transcriptase: an enzyme that makes DNA from an RNA copy –Naturally produced by retroviruses –For cloning eukaryotic DNA into bacteria –Eukaryotic DNA has introns that prokaryotic cells don’t have and can’t deal with If you make DNA directly from mRNA, the introns have already been removed

21 Tools and Techniques-2 Restriction enzymes (restriction endonucleases) –Made by bacteria to destroy DNA from viruses –Recognize 6 (or 4) base DNA sequences and cut them Palindromes like GAATTC, cut staggered, leaving sticky ends. Sticky ends are the key: allow DNA from ANY source to be cut, combined, and stitched together /dna/restriction.gif

22 Tools and Techniques-3 Cloning –To work with a piece of DNA, you need enough molecules of it (identical molecules) –Vectors A way to move a piece of DNA into a cell to make multiple copies of it (clone) Usually a plasmid or something similar; cut with restriction enzyme, paste in DNA, use transformation or transduction to get it into the bacterium. Plasmid will copy itself, so will bacterium.

23 Tools and Techniques-4 Cloning-2 –PCR: polymerase chain reaction –If you know the sequence on either side of the DNA, you can use PCR. –PCR “amplifies” DNA, so you can get multiple copies of identical DNA molecules this way. –Requires dNTPs, heat stable polymerase (Taq), primers complementary to piece of DNA, and your DNA. –PCR incredibly useful for wide variety of applications.

24 PCR ag45/Biology1A/LectureNotes /LNPics/Recomb/pcr.gif