Prokaryotic Genetics-45 Flashcards
Intro
Why do we study bacterial genetics?
-Understanding basics of molecular biology and genomics.
-Understanding bacterial ecology, plant and microbe interactions.
-Application in biotechnology.
-Understanding human health and disease (prokaryotic genetics).
Timeline of genetics
-Lamarck 1800: species are not fixed, change over time.
-Darwin 1831-1836: voyage of the Beagle.
-Darwin 1859: origin of the species.
-Mendel 1866: published work on peas.
-Beadle and Tatum 1941: one gene, one enzyme.
-Luria Delbruck 1943: bacterial inheritance.
-Hershey and Chase 1953: DNA is functional.
-Nirenberg 1961: genetic code.
-1970s: recombinant DNA technologies.
-1982: approval of recombinant human insulin for diabetics.
-1995: first genome of free-living organism sequences Haemophilus influenzae.
-2003: the human genome.
-present: now live in the post genome era.
What is bacterial genetics?
The study of the mechanisms of heritable information in bacteria, their chromosomes, plasmids, transposons and phages.
How many bacterial cells does the human body contain compared to eukaryotic cells?
The human body contains approximately as many bacterial cells as eukaryotic cells.
Bacteria makes up ~0.2kg.
What are techniques that have enabled bacterial genetics?
Culture in defined media, replica plating, mutagenesis, transformation, conjugation and transduction.
What percentage of DNA on Earth does bacteria DNA make up?
~30%
Bacteria and Archaea both make up Bacteria DNA.
Why are bacteria good model organisms?
Haploid- carry only one copy of chromosome.
Asexual reproduction- simply reproduce by division & mother and daughter cells are identical.
Short generation time.
Grows on plates and liquid culture with defined media- can grow in lab conditions.
Easy to store stocks.
Easy to manipulate genetically- e.g. plasmid DNA
Describe the bacterial genome
A single circular, double stranded DNA chromosome.
Little inter-gene space compared to eukaryotes.
Introns are extremely rare.
Functionally related genes often grouped (in eukaryotes they are regulated individually) in operons (regulates genes).
Often carry plasmids- circular extra-chromosomal DNA replicating independently.
Describe binary fission.
Asexual reproductions- grow longer and DNA replicated.
Common in prokaryotes, used by a few simple eukaryotes.
Cell elongates and all its content increased.
DNA replicated and segregated.
2 identical daughter cells arise (next generation), makes it so easy to study.
Describe how binary fission works.
Once cell is about twice original length.
Septum starts forming in the middle.
Grows from both sides of the cell.
What is the generation time of bacteria?
E. coli under optimal conditions ~20 min.
Clostridium perfringens ~ 10 min (one of the fastest known).
Some extremely slow growing bacteria may only divide once in 1000s of years.
Many bacteria are unculturable, won’t grow under lab conditions.
When was E. coli adapted as a model organism? What are its growth requirements?
In the 1940s.
Capable of synthesising all cellular components from simple inorganic nutrients and a carbon/energy source.
What is the typical minimum medium composition for E.coli with glucose?
K2HPO4, KH2PO4, (NH4)2SO4, MgSO4, CaCl2, trace materials and glucose.
Phosphate and pH control- needed for making DNA and RNA & is a buffer.
Nitrogen- for amino acids.
Binds to nucleotides, nucleic acids and needed in proteins.
Often important in protein function.
Carbon and energy- no oxygen, just in glycolysis.
What is a prototroph?
A wild type E. coli that does not require special nutritional factors. The growth is prototrophic.
What are auxotrophic mutants?
They are impaired in some metabolic capabilities. Can no longer do something and need to supply something to grow.
E.g. tryptophan auxotroph requires tryptophan to be able to grow.
What are biosynthetic auxotrophs?
Biosynthetic auxotrophs require additional nutrients in order to grow.
Auxotrophs can be affected in their ability to synthesis an amino acid, a nucleotide or a vitamin.
E.g. a strain with a mutation preventing the synthesis of the amino acid histidine (His-) would require histidine in its growth medium- a histidine auxotroph.
What are catabolic auxotrophs?
Catabolic auxotrophs have lost the ability to catabolise some carbon source.
Rarely a problem when cultured as glucose is often carbon source of choice.
Any mutation in the ability to metabolise glucose is usually fatal.
E.g. an arabinose mutation (Ara-) would be unable to grow on the monosaccharide arabinose and pointless using this as a carbon source.
What are housekeeping genes?
Some genes are essential for E. coli survival.
Mutants in these genes are lethal.
E.g. genes involved in: DNA replication, transcription and translation, cell division and glycolysis.
What are conditional lethal mutants?
A mutation that is lethal in some conditions (repressive conditions) but not in other conditions (permissive conditions).
What are temperature sensitive mutants?
They grow only at permissive temperature (usually 30 degrees for E.coli) and not at a restrictive temperature (37 degrees).
Enough mutant protein folds correctly at the lower temperature due to the lower amount of energy in the system.
Cold sensitive mutants are the opposite, e.g. they might grow at 37 degrees but not at 30 degrees.
What happens if a gene cannot grow?
Cannot grow, cannot isolate, cannot study.
What is the nomenclature of gene annotation?
-Three lower case letters: indicating a biochemical pathway of process in which the gene product is involved.
-Capital letter: denoting the actual gene.
-Sometimes followed by a number to denote allele.
Letters and numbers in italics.
Describe nomenclature of proteins.
Terminology also used to refer to protein encoded by a particular gene (not the protein itself):
dnaA = dnaA gene
DnaA = protein encoded by a dnaA gene
Always distinguish clearly between genes and the product of the gene. E.g. you cannot clone a protein, you clone a gene expressing a protein.
Describe nomenclature of phenotype.
Strain phenotype described using same three letter mnemonic as genotype.
First letter is capitalised, three letters not italicised.
Mutant phenotype shown by supercript minus sign (-):
THr- = requiring threonine
Leu+ = not requiring leucine
What nomenclature do we need to know?
Three letter code for:
-amino acids
-common carbon sources
-nucleotides
-vitamins
Make educated ‘guess’ on other functions
e.g. dnaA- gene involved in DNA synthesis
What are the minimum growth requirements of the strain: E. coli his trp ara?
Carries mutations in genes involved in histidine and tryptophan biosynthesis and arabinose degradation.
Growth requirement: histidine, tryptophan, as 37 degrees in minimal medium with glucose as a carbon source.
Define wild type, mutant and phenotype.
Wild type- normal species.
Mutant- organism whose genome carries a mutation with respect to the wild type- referred to as a strain.
Phenotype- all observable properties.
Define mutation, genotype and allele.
Mutation- inheritable change in the base sequence of nucleic acid.
Genotype- defined by actual sequence of DNA.
Allele- sequence variant of a gene.
Define mutagenesis and mutagens.
Mutagenesis- process by which mutants are produced.
Mutagens- chemical and physical agents which cause mutations. Many are also harmful to humans.
What can a loss of anabolic pathways lead to?
Losing the ability to make molecules.
What is a shared pathway?
Some pathways produce metabolites as precursors of more than one pathway. Loss of one enzyme leads to requirement for more than one amino acid.
E.g. shared pathway for isoleucine and valine (Ilv)
Pyruvate -> last shared intermediate -> valine / -> isoleucine
What can a loss of catabolic pathways lead to?
Losing the ability to break down the molecules.
What do carbon sources refer to?
Refer to catabolic genes, i.e. the organism can no longer use this carbon source to grow.
E.g. ara- arabinose
man- mannose
xyl- xylose
gal- galactose
mel- melibiose
lac- lactose
rha- rhamnose
mal- maltose
What is the nomenclature for super and sub scripts?
ts: temperature-sensitive
cs: cold-sensitive
am: amber mutation
oc: ochre mutation
um: umber = (op opal) mutation
stop codons are traditionally known as amber UAG, ochre UAA and opal UGA.
When would you assume it is a wild type and a gene mutant?
If gene is not specified, it is usually wild type.
If neither + or - sign used, it is usually gene mutant.
