1. 2- RECOMBINATION AND REPAIR OF DNA
1-PLASMIDS
2- RECOMBINATION AND REPAIR OF DNA

2. plasmids
A plasmid is a circular, self-replicating DNA molecule carrying a few, useful but non necessary genes. Occurence prokaryote organisms
eukaryotic organisms like Entamoeba histolytica, yeast etc.
Their size varies from 1 kbp to over 400 kilobase pairs (kbp).
In a single cell there are anywhere from
one copy, for large plasmids,
to hundreds of copies of the same plasmid.

3. We speaks of low and high copy number plasmids
Plasmids are easy to manipulate and isolate from bacteria (kits). After being modified, they can be integrated into other genomes, plants, protists, mammals,
thereby conferring to other organisms whatever genetic functionality they carry.
Thus, this gives the ability to introduce genes into a given organism by using bacteria to
amplify the hybrid genes that are created in vitro.
This tiny but mighty plasmid molecule is the basis of recombinant DNA technology.

4. Bacterial plasmid

5. the 25 genes required for transfert
Types Of Plasmids
1-Plasmids are classified by their ability to be transferred to other bacteria
Conjugative The sexual transfer of plasmids to another bacterium through a pilus. those plasmids possess
the 25 genes required for transfert
Non-conjugative Non-conjugative plasmids don’t initiate conjugation.
They can only be transferred with the help of conjugative plasmids.

6. Mobilisable An intermediate class of plasmids are mobilisable, and carry only a subset of the genes required for transfer. These plasmids can 'parasitise' another plasmid, transferring at high frequency
in the presence of a conjugative plasmid
Incompatibility groups:
Several types of plasmids could coexist in a single cell. On the other hand, related plasmids are often 'incompatible', resulting in the loss of one of them from the cell line.

7. Conjugative plasmids
The sexual transfer of plasmids to another bacterium through a pilus.
Those plasmids, plasmidos F, possess the 25 genes required for transfer.

8. Bacterial Conjugation

9. Rolling circle replication: Passes a plasmid through a pilus.
What’s important in rolling circle replication is to
find out the two 3’ ends used in the process

10. 2. By Function 1. Fertility-(F) plasmids,
They are capable of conjugation (they contains the genes for the pili).
Resistance-(R) plasmids,
contain gene (s) that can build resistance against one or several
antibiotics or poisons.
Col-plasmids,
contain genes coding for colicines, proteins that can kill other
bacteria.
Degradative plasmids,
able to digest unusual substances, e.g., toluene or salicylic acid.
Virulence plasmids, turn a bacterium into a pathogen.
addiction system.
These plasmids produce both a long-lived poison and a short-lived
antidote.
Daughter cells that retain a copy of the plasmid survive, while a
daughter cell that fails to inherit the plasmid dies or suffers a reduced growth-rate because of the lingering poison
from the parent cell. -

11. Episomes
are units of genetic material composed of a series of genes that sometimes has an independent existence in a host cell and at other times is integrated into a chromosome of the cell,
replicating itself along with the chromosome. Episomes exist in all organisms.
In bacteria, episomes are:
viruses: Bacteriophages in bacteria (lytic or lysogenic phages, prophages and proviruses)
Sex factors Hfr: sex factors like the F factor F+ is autonomous, extrachromosomal.
Hfr (or high frequency recombination) is the same sequence, but integrated into the host chromosome.
Plasmids:
can be integrated into a chromosome.
transposons:
Transposons are known as mobile genetic elements (they were discoverd in maize by Barbara McClintock*)
While they can also exist outside of the chromosome, they prefer to, and are designed, to integrate
into the chromosome following their movement from one cell to another.For example, Class 1 transposons encode drug resistance genes.
*As Barbara Mc Clintock was a woman she got a nobel prize 40 years after her discovery

12. Conformation
In bacteria, plamids are found in the supercoil (superenrollado) form. Supercoils and relaxed (relajado) forms can be seen under the electron
microscope,
in electrophoresis or after centrifugation.
The supercoiled form migrate and sediments quicky in in eletrophoresis and
Centrifugation respectively.
Topoisomerases help to pass from one form to the other as we saw it earlier.

13. Plasmids in molecular biology
Minimum requirements for plasmids useful for recombination technology:
1. Origin of replication (ORI). ORI enables a plasmid DNA to be duplicated
independently from the chromosome
2. Selectable marker: allow to select for cells that have your plasmids.
3. Restriction enzyme sites in non-essential regions of the plasmid.
Plasmid replication initiates in a cis-site called ori. It proceeds either by a rolling circle or
a theta replication mechanism. Some of the plasmid-encoded elements required for their replication,
such antisense RNA molecules and DNA repeated sequences located close to ori,
determine plasmid attributes like copy number and incompatibility.

14. Plasmids often contain genes or gene-cassettes that confer a selective advantage when they
are inside a bacterium:
resistance to antibiotics resistance to herbicides
insecticide production
to the bacterium harboring them, for example, the ability to make the bacterium antibiotic resistant.

15. Mutations of Genes
Mutation – change in the nucleotide base sequence of a genome; rare
Almost always deleterious
Rarely lead to a protein having a novel property that improves ability of organism and its descendents to survive and reproduce

16. Mutations of Genes Types
Point mutations (most common) – one base pair is affected
Insertions, deletions, and substitutions
Frameshift mutations – nucleotide triplets after the mutation displaced
Insertions and deletions

17. Effects of Mutation
Figure 7.21a-c

18. Effects of Mutation
Figure 7.21d-e

19. An English Example THEBOYANDTHEDOGRANFAR THE BOY AND THE DOG RAN FAR
Missense:
THE BOY AND THE HOG RAN FAR
Nonsense:
THE BOY AND .

20. English Example-Frameshift
THEBOYANDTHEDOGRANFAR
THE BOY AND THE DOG RAN FAR
DEletion:
THE BOY ADT HED OGR ANF AR
Insertion:
THE EBO YAN DTH EDO GRA NFA R

21. Mutagens Radiation Chemical mutagens
Ionizing radiation – induces breaks in chromosomes
Nonionizing radiation – induces pyrimidine dimers
Chemical mutagens
Nucleotide analogs – disrupt DNA and RNA replication and cause point mutations
Nucleotide-altering chemicals – result in base-pair substitution mutations and missense mutations
Frameshift mutagens – result in nonsense mutations

22. DNA Repair
Figure 7.25a-b

23. DNA Repair
Figure 7.25c-d

24. Genetic Recombination and Transfer
Horizontal gene transfer – donor contributes part of genome to recipient; three types
Transformation
Transduction
Bacterial Conjugation

25. Griffith’s Experiments
Figure 7.30

26. Transformation
Transforming agent was DNA; one of conclusive pieces of proof that DNA is genetic material
Cells that take up DNA are competent; results from alterations in cell wall and cytoplasmic membrane that allow DNA to enter cell

27. Transduction

28. Bacterial Conjugation
Figure 7.32a

29. Transposons and Transposition
Transposons – segments of DNA that move from one location to another in the same or different molecule
Result is a kind of frameshift insertion (transpositions)
Transposons all contain palindromic sequences at each end

30. Transposons and Transposition
Simplest transposons are insertion sequences which have no more than two inverted repeats and gene for transposase
Complex transposons contain one or more genes not connected with transposition (e.g. antibiotic resistance)