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

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.

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.

Bacterial plasmid

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.

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.

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

Bacterial Conjugation

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

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. -

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

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.

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.

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.

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

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

Effects of Mutation Figure 7.21a-c

Effects of Mutation Figure 7.21d-e

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

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

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

DNA Repair Figure 7.25a-b

DNA Repair Figure 7.25c-d

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

Griffith’s Experiments Figure 7.30

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

Transduction

Bacterial Conjugation Figure 7.32a

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

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)