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BACTERIAL GENETICS DR. GHADA YOUNIS

BACTERIAL GENETICS
Genetics is the study of inheritance and variation. All inherited characteristics are encoded in DNA, except in RNA viruses.

Who discovered DNA?

Historical Events
1869 Friedrich Miescher identified DNA, which he called nuclein, from pus cells 1889 Richard Altman renamed nuclein nucleic acid 1928 Griffith discovered that genetic information could be passed from one bacteria to another; known as the transforming principle 1944 Avery showed that the transforming material was pure DNA not protein, lipid or carbohydrate. 1952 Hershey and Chase used bacteriophage (virus) and E. coli to show that only viral DNA entered the host 1953 Watson and Crick discovered the structure of DNA was a double helix

DNA
RNA
Protein
Central Dogma of Molecular Biology

anticlockwise

clockwise
Becomes untwisted
overwinds
R


The bacterial chromosome Mw.2x 109

REPLICATION

It is accurate process that insures that the progeny cells receive identical copies from the mother cell

REPLICATION OF BACTERIAL DNA FROM A SINGLE ORIGIN

January 2008
Single replication origin in bacteria.

BACTERIAL REPLICATION AND CELL DIVISION

January 2008
No apparent chromosome condensation Origin used to separate chromosomes

DNA REPLICATION MACHINERY

DNA POLYMERASE
Unable to separate the two strands of DNAOnly elongate a pre-existing DNA or RNA (Primer)Only add nucleotides to the 3’-hyforxyl group, i.e., only 5’-3’ synthesis

GENES

Genetic code of bacteria is a series of units called genes( triplet series of nucleotides). Bacterial chromosome has only one copy of gene( haploid)

GENETIC VARIATION IN BACTERIA

G.v. occur as a result of mutation or gene transfer. Mutation : change in the base sequence of DNA : new protein( different) : altered phenotype. Either by chemical s, radiation, or viruses.

3 TYPES OF RESULTS:

1. base substitution: 2.frame shift mutation 3. insertion

E.G.: DELETION MUT.

CAT-ACT-GAG-GTT-AGT--------- genotype His- thr -glu - val---- PROTEIN CAT-ATG-AGG-TTA------ NEW SEQUENCEHis – met-arg-leu-------- NEW PROTEIN

Gene transfer Transformation Conjugation Transduction Transposition

Transformation: donor DNA molecule is taken up from the external environment and incorporated into the genome of the recipient cell Conjugation: direct contact between bacterial cells; DNA from donor to recipient Transduction: DNA goes from one bacteria to another via a phage

RECOMBINATION

Unidirectional – material flows in one directionDonor strain – fragment of chromosome involved in a recombination eventRecipient strain - strain that carries intact chromosomeRecombination occurs in the recipient strain only –Donor fragment linear; recipient chromosome circular


RECOMBINATION
A piece of DNA transferred from one cell to another ,and integrated into host( recipient) genome. Homologous recombination Non homologous rec.

PLASMIDS

Extrachromosomal DNA moleculeTransmission of plasmid DNA from one bacteria to another- produce new strains (a bacteria type is called a “strain”Circular molecules with antibiotic resistance genesTransmissible plasmidsNon-transmissible

CLINICAL RELEVANCE OF PLASMIDS

1. antibiotic resistance( R plasmids)2. colicins( anti bacterial)3.Resistance to heavy metals( mercury, silver….)4.pilli 5.exotoxins

TRANSPOSONS

Jumping genes: pieces of DNA that move from site to another , within or between DNAs of bacteria, plasmids, and bacteriophages. *Transposons can code for metabolic , or drug resistance enzymes or toxins. ** also cause mutations into the gene they insert ***alter the expression of of nearby genes.

Transposons cannot replicate independently of the recipient cells. Transposon can jump from : Host DNA to plasmid One plasmid to another Plasmid to genomic DNA.

Gene cloning Gene cloning is the artificial incorporation of one or more genes into the genome of a new host cell by various genetic recombination techniques


DNA is extracted from the source, Purified , cut into small fragments by restriction enzymes -leaving 'sticky ends'. These are then inserted into a vector DNA, first by cutting the vector DNA with the same enzyme so as to produce complementary sticky ends. The sticky ends of the vector and the candidate DNA are then ligated together using 'DNA ligases' to produce a recombinant DNA molecule. The vector used for gene transfer is usually a plasmid or a virus. The vector with the integrated DNA has to be inserted into a cell in order to obtain multiple copies of the organism that express the selected gene.



This can be done by:• transformation • electroporation - here an electric current induces poreson the cell membrane for vector entry• gene gun - tungsten or gold particles are coated with the vector and propelled into cells by a helium burst microinjection - direct manual injection of the vectorinto a cell by a glass micropipette.

DNA/RNA probes and oral microbiology a number of bacterial genera are difficult or almost impossible to culture. The introduction of DNA and RNA probes has helped us to obtain a more complete picture of the oral flora. the samples, say in paper points, could be simply sent by post to distant laboratories for identification without the fear of death of organisms

PCR
PCR – Polymerase Chain Reaction, a method that uses a high temperature DNA polymerase, DNA separation and reannealing, and repetitive cycles, to amplify fragments of DNA in vitro.

IMPORTANCE OF PCR

Allowed rapid advances in genomics Enabled detection of single genes Forensics Sequencing

PCR RESULTS

m
a
b
c
d
e
f
1
2
3
4
5
6
Lane M : size marker Lane A : B. hyodysenteriae Lane B : B. innocens / intermedia Lane C : B. pilosicoli
Lane D : B. murdochii Lane E : inhibited Lane F : Internal control



Some reasons why the use of PCR is so widespread:• To study minuscule quantities of DNA, as a singleDNA molecule is adequate for an amplification reaction(hence its use in forensic studies).• Use in rapid clinical diagnostic procedures.( The sensitivity of the PCR) . amplification of viral DNA in a patient samplecould be made within hours, and sometimes even beforethe onset of symptoms.• Amplification of RNA. Here the RNA molecule has tobe first converted to single-strand complementary DNA(cDNA) with an enzyme called reverse transcriptase

• Comparison of different genomes. Random amplificationwith short lengths of primers can be used in phylogenetics,the study of evolutionary history and lines ofdescent of species or groups of organisms. This technique is called random amplification of polymorphic DNA(RAPD).

OTHER TECHNIQUES FOR GENETIC TYPING OF MICROORGANISMS

Restriction enzyme analysis Restriction fragment length polymorphism Pulsed-field gel electrophoresis




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