1Gene 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.

2Transformation

• 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.

3Transformation details

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

4Transduction

• Transfer of DNA via a virus.

More common, but still requires close relative.

5Conjugation: 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.

http://fig.cox.miami.edu/Faculty/Dana/ffactor.jpg

7Conjugation 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.

8Mutations 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 read3 at a time (codon); when the first baseis read, the “reading frame” is established.If the frame is altered at some point the bases will NOT be inthe right places, and the information will be garbled.

9

10Mutagens

• 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.

http://www.bio.cmu.edu/Courses/03441/TermPapers/96TermPapers/spontaneous/dimer-2.jpg

11Mutagens-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.

12Non-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.

13DNA damaged often repaired

• The mutation rate is variable – Typical 1^106 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.

14Using 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.

15Using 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.

16Replica platingFirst, 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.

17Result of replica plating

• Look: where did a colony NOT grow on this plate?• Go back to original plate and grow colony.

www.sp.uconn.edu/.../ lectures/genetics1.html

18Transposon 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”

19Transposon 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.

20Tools 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

21Tools 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.

http://employees.csbsju.edu/hjakubowski/classes/ch331/dna/restriction.gif

22Tools 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.

23Tools 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.

24PCR

http://members.aol.com/BearFlag45/Biology1A/LectureNotes/LNPics/Recomb/pcr.gif