7. Chromosomes, plasmids and gene transfer(1)

Question 1

What is the most common bacteria used for visualisation techniques?

Answer 1

E. coli

Question 2

What are the differences between the chromosomes of eukaryotes and prokaryotes?

Answer 2

Eukaryotes contain genes, introns, genome-wide repeat and (human pseudogene in humans)

Prokaryotes contain genes and genome wide repeats

Question 3

What percentage of chromosomes in the human are non coding sequences?

Answer 3

95%

Question 4

What are the 3 parts of the genetic organisation of bacteria?

Answer 4

Genes, operons and regulons

Question 5

What kind of message does a single even make in bacteria?

Answer 5

MONOcistronic

Question 6

What kind of operon makes a POLYcistronic message in bacteria?

Answer 6

An operon of 3 genes

Question 7

What can genes and operons form in a chromosome?

Answer 7

regulon controlled by regulatory protein

Question 8

How many domain loops are there in DNA after release from a bacteria cell?
(left side)

Answer 8

30-100

Question 9

Describe the structure on the right side of genetic organisation in bacteria?

Answer 9

RIGHT – supercoiling is relaxed in a nicked stra

Question 10

Describe the eugenic organisation of bacteria?

Answer 10

Double helix in each is helical, i.e. the structure is supercoiled. Think of a coiled phone wire that loops on itself. This wire becomes more compact just as the chromosome becomes compact.

Question 11

Describe DNA replication from a single origin? (5)

Answer 11

1) Replication starts at origin
2) Replication bubble forms. Forks progress in opposite directions
3) One strand at each fork is synthesised continuously, 5’ to 3’
4) Second strand is made discontinuously 5’ to 3’ in Okazaki fragments
5) Replication ends at terminus

Question 12

How is DNA replication terminated? (4)

Answer 12

1) Terminator regions for DNA replication on the E. coli chromosome.
2) There are >8 arrest sites at the terminus (Mirkin & Mirkin 2007, Microbiol Mol Biol Rev 71, 13).
3) Replication forks moving clockwise are trapped by terB, C, F, G, J.
4) Counterclockwise-moving forks are trapped by terA, D, E, H, I.

Question 13

How are linked rings seated in DNBA replication? (4)

like 2 key chain rings

Answer 13

1) Because of the topology of the chromosome, DNA replication gives a catenane of linked rings.
2) Two mechanisms can resolve the rings. One involves topoisomerase IV (not covered here).
3) The second mechanism involves enzymes XerC (site-specific tyrosin-recombinase) and XerD that recognise a dif site on both molecules and catalyse cutting and rejoining steps.
4) When the strands are separated and segregated towards opposite ends of the cell, division can occur.

Question 14

Describe Chromosome of the model bacterium Escherichia coli K-12?

Answer 14

• The chromosome is circular
• 4,288 genes
• Some genes and operons are shown outside
• Order of transcription can be CW or CCW
• Replication proceeds in both directions from the origin at 84.3 min
• Restriction sites shown inside are for Not1
• Some Hfr origins are also shown

Question 15

What is a common cloning vector or plasmid?

Answer 15

pBR322

Question 16

Describe pBR322?

Answer 16

1) This is widely used in molecular cloning

2) It possesses:
An origin of replication

Two antibiotic resistance genes

Six unique 6-bp restriction sites

Question 17

What is the mechanism of plasmid replication?

Answer 17

the rolling circle model

Question 18

Describe plasmid replication? (4)

Answer 18

1) Replication starts at origin but moves in only ONE direction
2) As polymerase (the enzyme that does the replicating) moves, it displaces one strand and replicates the other progress in opposite directions
3) At the end of a round of replication, the polymerase releases the displaced strand as a single-stranded circle
4) Finally, this circle is then replicated

Question 19

How are resistance mutations seen and selected? (3)

Answer 19

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

Question 20

What is the nomenclature of genotype?

Answer 20

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

A wild-type gene or strain is written cydA+

If there are several alleles, we write cydA1, cydA2 etc

Question 21

What is the nomenclature of phenotype?

Answer 21

A plus or minus is used to designate presence or absence of a property, e.g. Cyd+, Cyd-. NB: not in italics, only the first letter is in capitals

Question 22

What is the nomenclature of an encoded protein?

Answer 22

The CydA protein, CydD etc. NB: no sub/superscript, not in italics, only the first letter is in capitals.

Question 23

What is the difference between selecting and screening?

Answer 23

selecting (finding the primary property, like antibiotic-resistance)

screening (finding the ones we want, like a nutritional deficiency, from many contenders

Question 24

How are mutants screened?

Answer 24

Mutants don’t grow in culture

Question 25

What kind of MRNAs do Genes and Operons make?

Answer 25

Genes make monocistronic mRNAs

Operons make polycistronic mRNAs

Question 26

What is used to release supercoiling in Bacterial DNA?

Answer 26

topoisomerase