Lecture 23

Question 1

RNA visits have ____ genomes

Answer 1

Small
Mammals pre big

Question 2

Genome size correlates with….

Answer 2

Protein coding genes

Question 3

Genome size correlates with….

Answer 3

Protein coding genes

Question 4

Genomes vary in size, even among microbes

Question 5

Higher genome size =
Lower genonome size =

Answer 5

Lower mutation rate
Higher mutation rate

Question 6

Genomes of viruses really dont waste space - wdym by this?

Answer 6

• many small DNA viruse, such as Circoviruses contain extensive overlapping reading frames
• suggests that they are under strong selective pressure to maximise the diversity from a restricted genomic space
• RNA virus genomes contain very few, if any, non-coding RNA
• Although the genomes of large DNA viruses Captian may regions that don’t close for proteins, these still may encode promontory, transcription termination signals, functional RNA molecules or other functional elements

Question 7

Features of the minimal genome: the genetic bare necessities

Answer 7

• a genome that contains only the genes required to sustain free-living self replication
• technically this means cellular life - not viruses
• a functional minimal genome would help broaden understanding of the requirements for life
• demonstrate the ability to build synthetic organisms
• lead to biotechnological applications

A MINIMAL GENOME CONTAINS ONLY THE GENES REQUIRED TO SUSTAIN LIFE

Question 8

Can genes evolve to become smaller?

Answer 8

Yeah some pathogen genomes have gotten smaller since diverging from a common ansester and adapting to specialised pathogenic life style

(Viruses Can become smaller given they can still infect and replicate)

Question 9

What are essential genes

Answer 9

Genes essential for growth and survival
Therefore mutation of esssential gene = no growth
Some genes will always be essential (e.g DNA replication, transcription, translation)

Question 10

What happens if there is a mutation of an essential gene

Answer 10

No growth

Question 11

What are the two types of essential genes?

Answer 11

Conditially essential (essential depending on what conditions are used for growth)
Essential but redundant ( some genes can do the same essential function but deletion of either gene is tolerated)

Question 12

What is conditially essential

Answer 12

essential depending on what conditions are used for growth

Question 13

Essential but redundant

Answer 13

some genes can do the same essential function but deletion of either gene is tolerated

Question 14

How you test for essentiality will vary the outcome

Answer 14

Yes it will
There are 4 approaches to testing essentiality

Question 15

4 approaches to define the minimal genome (and test what genes are essential)

Answer 15

1. Look in nature for the smallest genomes
2. Compare distantly related genomes computationally to identity conserved (essential?) genes
3. Take a small natural genome and mutate genes to find out which ones are not essential
4. Create a synthetic organism

Question 16

1. Look at the smallest genomes in nature

Answer 16

The natural minimal genome of free-living bacterium, pelagibacter ubique

Question 17

P. Unique’s small genome

Answer 17

• grows as free cells in the oceans (large abundance - easy to study)
• utilises dissolved organic carbon in ocean
• group of bacteria account for 25-50% of all bacteria in oceans
• genome is 1 million bp and encodes 1354 proteins
  
  • (this genome doesn’t have much wasted compared to M.leprae where there are 3 million bp and encode 1604 proteins)
• the genome has no junk
• encodes the basic functions for growth
• contains almost non-functional / redundant DNA
• no gene duplications
• has the smallest median intervening spaces of 3bp

Gens are packed very tightly in the genome

Question 18

2. Estimation of minimal genome by genome comparison

Answer 18

• first two genomes H.influenzae (1740) and mycoplasma genitalium (480) compared
• conserved genes were identified and predicted to encode essential products
• this defined a minimal genome of 256 genes
• similar studies have now used more genomes to refine this number

Question 19

2. Estimation of minimal genome by genome comparison - WHAT ARE THE CAVEATS

Answer 19

1. Some proteins share no sequence or strucutureal similarity but can perform same function (no sequence homology so you can’t compare them when looking at genome comparison)
2. These are omitted from these analyses
3. More then one different metabolism / nutrient can support life

Question 20

Estimation of minimal genome by mutagenesis

Answer 20

• use a very small genome (580 kb) such as M. Genitalium (517 genes)
• they lack cell walls
• intracellular parasites of humans
• urogenital infections
• mutagenesis was used to identify nonessential genes in an effort to learn whether the naturally gene complement is a true minimal genome under labraroty growth conditions
• transposon mutagenesis utilises transposonable genetic elements that integrate into a recipient genome to generate random inertion mutations which are easily identified
• the analysis suggests that 265 - 350 of the 480 protein-coding genes of M. Genitalium are essential under laboratory growth conditions including about 100 genes of unknown function

This is all under lab conditions

Question 21

Estimation of minimal genome by mutagenesis - MYCOPLASMA MUTAGENESIS METHOD

Answer 21

• Performed transposon mutagenesis on M genitalium
• only mutants that could survive could grow
  E.g mutations in essential genes would result in no growth

The insterion sites of transposon in the survivors were sequenced and non-essential genes identified

Question 22

Estimation of minimal genome by mutagenesis - MYCOPLASMA MUTAGENESIS RESULTS

Answer 22

No transposon insterions identified in genes involved in:
1. DNA metabolism
2. Transcription
3. Translation

This is a good proof-of-proof principle that it has worked
Many predicted essential genes have no function
Essential processes of life are not fully understood

…they estimate a minimal genome of 265-350 genes

Question 23

4. Designing and building a minimal genome - a synthetic bacteria

Answer 23

• a goal in biology is to understand the molecular and biological function of every gene in a cell
• one way to approach this is to build a minimal genome that includes only the genes essential for life
• more complex than it might seem
  
  • essential genes
  • nonessential genes
  • quasi-essential genes (required for robust growth)

JVCI-sync3.0: minimal genome, maximal knowledge
- first truly synthetic organism
- 531,560bp and 473 genes
- a genome shorter then any in nature

Question 24

4. Designing and building a minimal genome - a synthetic bacteria - THREE STEPS

Answer 24

1. Synthesis the genome
2. Transplant the genome
3. Optimise and minimise the genome

Question 25

Summary of the minimal genome

Answer 25

This is the concept of what the minimal gene set is for replication of cellular life And estimates made using various methods 1. Sequencing small genomes (P. Ubique) 2. Comparing conserves genes across genomes 3. Mutation analysis to determine essential genes 4. Synthesis a minimal genome

Question 26