Lecture 11 - Chromosome of the model bacterium - E. coli

Question 1

Describe the chromosome of the model bacterium - E. coli

Answer 1

• the chromosome is circular
• 4,288 genes
• some genes & operons are shown outside
• order of transcription can be CW (clockwise) or CCW (counterclockwise)
• replication proceeds in both direction from the origin at 84.3 min
• restriction sites shown inside are for Not1
• some Hfr origins are also shown

Question 2

What are used to locate genes via translation (in the olden days)?

Answer 2

minutes - it would be based on how long it takes for the gene to be transcribed

Question 3

How many bp is rare?

Answer 3

Proteins over 1000bp - this is because it is easier to create then assemble proteins than create a long gene

• anything shorter than 30 amino acids isn’t considered a protein

Question 4

What happens to the plasmid DNA to make it visible?

Answer 4

it is coated

Question 5

How many chromosomes do bacteria have?

Answer 5

Usually 1 chromosome & plasmids - some bacteria have 2 chromosomes, however it is rare

Question 6

What is a common cloning vector (widely used in molecular cloning)?

Answer 6

1. An origin of replication
2. Two antibiotic resistance genes
3. Six unique 6bp restriction sites

Question 7

Describe plasmid replication - the rolling circle model

Answer 7

• replication starts at origin but moves in only ONE direction
• as polymerase (the enzyme that does the replicating moves, it displaced one strand and replicates the other progress in opposite directions
• at the end of a round of replication, the polymerase releases the displaced strand as a single-stranded circle

If you have plasmid that only exists in 1 copy there are often special mechanism that leads to each daughter cell receives some plasmid

The plasmid can create something that only allows it to live if it has the plasmid in

Question 8

What are examples of plasmid-conferred phenotypes?

Answer 8

• antibiotic production
• metabolism of substrate sugars/toxins etc. (e.g. herbicides)

Question 9

What are some plasmids that are recombined into the chromosome involved in?

Answer 9

resistance & maintenance of the chromosome

Question 10

How are resistance mutations seen and selected?

Answer 10

A mutation is:
- heritable
- rare
- good, bad or neutral
- usually ‘small’

1. Antibiotic-resistance colonies growing around a filter disk impregnated with the compound.
2. Spontaneous mutants of Asperrigillus (wild type = green)
3. Colonies of Halobacterium. Orange colony is a mutant that lacks gas vesicles, which obscure colour in the wild type

Question 11

Describe the nomenclature rules in bacterial genetics

Answer 11

• the gene name is given as 3 or 4 letters, all except the 4th in lower case, all italicised e.g. cydA, cydD, hmp etc.

Question 12

How are mutants screened?

Answer 12

There is a difference between selecting (finding the primary property like antibiotic resistance), and screening (finding the ones we want, like a nutritional deficiency from many contenders)

Question 13

What kinds of mutants may be isolated?

Answer 13

depends what you want - e.g. choice of characteristics - e.g. motile

Sequencing of bacterial DNA is leading to testing of mutants (bioinformatics) instead of choosing

Question 14

What is the effects of base-pair substitution on the protein produced?

Answer 14

• the genetic code is degenerate - i.e. >1 codon can specify an amino acid
• different types of mutant protein (or normal ones) may result
• example: sickle-cell anaemia (GAG to GUG)

Question 15

What is the effect of insertion or deletion?

Answer 15

leads to frameshift - often leading to non-functional proteins or the introduction of a stop codon