Chapter 1 Long questions

Question 1

Contrast between the challenges of gene identification in prokaryotes vs eukaryotes

Answer 1

• Gene identification is easier in prokaryotes than in eukaryotes
  -Prokaryotes have smaller genomes, fewer genes, contiguous genes that lack introns and small intergenic regions
• Eukaryotes have sparsley distributed genes with most having introns and alternative splicing also complicates gene identification

Question 2

Distinguish between two general methods of gene identification

Answer 2

• Priori method: recognize sequence patterns within expressed genes and the regions flanking them
• Been there seen that method: recognize regions corresponding to previously known genes

Question 3

Describe useful features of gene identification in addition to codon usage, what to look for in the beginning, middle and end of genes

Answer 3

• Beginning of a gene (5’ end):
  > 5’ exons start with a transcription start site, preceded by a core promotor (TATA box), they are free of in-frame stop codons and they end immediately before a GT splice signal
• Middle of a gene
  >Internal exons begin immediately after an AG splice signal, they are free of in-frame stop codons and they end immediately before a GT splice signal
• End of a gene 3’ exon
  > Starts immediatly after a AG splice signal and ends with a stop codon (TAA or TGA), followed by a polyadenylation (Poly-A) signal sequence

Question 4

Reasons for sequencing non-human genomes

Answer 4

-Reveal and illuminate the processes of evolution
- Help us understand the functions of different regions in the human genome
- Help us understand the genomes of pathogens that exhibit antibiotic resistance for better human welfare
- Improving plants and animals
- conservation of endangered species
Clinical applications in humans, genetic testing for diseases, geneology, law enforcement, mutation discovery

Question 5

The relationship between SNPs and haplotypes, How does recombination affect this relationship, what factors affect the incidence of recombination

Answer 5

• SNPs, sine nucleotiode polymorphism, a mutation at a single base position in a genetic sequence
• Haplotypes are local combinations of genetic polymorphisms that tend to be co-inherited as a block
• Some SNPs are co-inherited as blocks
  >Discrete combinations of SNPs in recombination-poor regions define a haplotype
• Mutations on different chromosomes will be separated by independent assortment within one generation
• Mutations on the dame chromosome will be separated by recombination
  -Recombination is affected by distance and vary in the genome, hot and cold hot-spots for recombination

Question 6

International HapMap project - describe and elaborate each main finding

Answer 6

• Most variations appear in all populations sampled
  >Some of the inter-population differences reflect different relative amounts of the same SNPs
• Very few SNPs are unique to specific populations
  >11 were constantly different between all individuals of European origin and all Chinese and Japanese origin
  -Genomes of individuals from Japan and China are very similar
  >They have a more recent common ancestor
• The X-chromosome varied the most between different populations than others
  > X-chromosomes recombine in females only ( Faster-X-Effect)
  -The length of haplotype blocks varied among different sources of samples
  >They tend to be shorter in African populations, the older the population the greater the chance of recombination. (Out of Africa Theory)

Question 7

Implications of little vs high genetic variation in populations

Answer 7

Population - an interacting and interbreeding group of individuals of the same species inhabiting the same geographical area
- A population that has passed through a bottleneck or that has developed in isolation from a small “founder” group will show very little variation
- A population with relatively high variation is likely to have a longer evolutionary history (Out of Africa theory)

Question 8

Role of genomics in delineating species

Answer 8

• Genomics raised opportunities and challenges to the idea of species
  -Pro: DNA sequences of members of different species differ more than the variation among individuals of the same species
  -Con: Extent of sequence diversity to distinguish species is quite variable across taxa, genomic distance also doesn’t help
• e.g in microbiology bacterial species are those that maintain > 97% sequence identity in 16s rRNA

Question 9

Causes of extinction

Answer 9

• Excessive hunting
• Pathogen extermination
• Habitat destruction
• Natural events, i.e Elm yellow, white noise disease, transmissible cancer
• Climate change
• Species translocations
• Environmental pollution
• Indirect effects due to species interdependence.

Question 10

The mechanisms of CRISPR/Cas

Answer 10

Clustered regularly inter-spaced short palindromic repeats.
- Prokaryotic genome regions used as defense against viral infection/ similar to vertebrae immune systems in that they are responsive and heritable
- When a phage infect the bacterium cell, regions of the phage DNA are clipped out, replicated and integrated into a new CRISPR locus with a spacer in between
- Transcription of that region produces CRISPR RNAa that bind to Cas proteins
- Using the bound RNAs as a probe, the Cas proteins will clip viral DNA when a match against a region of DNA from an invading virus is detected, thus defense has been effected

Question 11

Potential application of CRISPR/Cas

Answer 11

• Gene knockout and editing
  > Gene knockout - silencing gene expression
  > Gene editing - replace/insert DNA sequences into endogenous gene
• Gene drive
  > Using CRISPR/Cas to accelerate the dispersal of a chosen gene throughout a population
  > A possible weapon against a vector that carries the Zika virus.

Question 12

Distinguish between Highthroughput sequencing projects

Answer 12

-De novo sequencing - Determining the full-genome sequence without using a known reference sequence from an individual of the same species
- Resequencing - Determining the sequence of an individual of a species for which a reference genome sequence is already know
- Exome sequencing - resequencing project that sequences only exons/coding regions, identify trait-linked mutations in protein-coding region.

Question 13

Describe the contents of the human genome and functional roles (protein coding regions and other main regions)

Answer 13

• Protein coding regions - Areas in the genome that produce different kinds of proteins, these proteins will perform various cellular activities either as enzymes or being structural components in different entities.
• Non protein coding regions - Areas in the genome that produce different kinds of RNA molecules (e.g tRNA,siRNA, miRNA, rRNA), RNAs will then perform various structural and regulatory roles, most cintrol gene expression e.g miRNA and siRNA
• Pseudogenes - Degenerate genes that have acquires many deleterious mutations over a period of time, these genes encode non-functional proteins
  >Processes pseudogenes - picked up by virus from mRNA and reverse transcribed and they tent to lack introns and promoters.
• Binding sites for ligands - Regions (i.s promoters) that are targeted by various DNA interacting proteins such as enzymes or regulatory proteins, ligands will tend tend to perform regulatory roles as in the case of transcription factors activating/repressing gene expression.
• Repetitive elements - Repeated DNA sequences that tend to constitute the largest component of any genome, some are functional such as rRNA genes, while the majority are non-functional such as minisatellites or microsatellites (15%), Lines (21%), SINES (13%)

Question 14

Characteristics of protein coding genes and specific examples

Answer 14

• There are 23000 protein-coding genes in the human genome
• They occupy a small fraction of the genome 2-3% of the overall sequence
• They are distributed unevenly across chromosomes and appear on both strands
  -Many appear in multiple copies, either identical or diverged into families due to duplication or divergence (e.g over 900 related olfactory-receptor genes). Closely related genes may co-localize (e.g hemoglobin genes) and some genes may occur on different chromosomes (e.g ubiquitin)
  -Some chromosomes are gene-rich (e.g chromosome 19 and 22) and some are gene-poor (e.g subtelomeres, chromosome 18 and X)
  -Protein-coding genes - in humans typically contain coding (exons) and non-coding (introns) regions. Splice-signal sites delineate borders between the coding and non-coding regions. On average exons are roughly 200bp in length and introns vary in sizes leading to diversity in protein coding lengths

Question 15

Characteristics of protein-coding genes in the human genome: Gene structure

Answer 15

• Protein-coding genes in the human genome typically contain coding (exons) and non-coding (introns) regions
  -Splice signals sites delineate borders between the coding exon and non-coding intron regions
  Exons are roughly 200bp in length, introns vary in sizes leading to diversity in protein-coding gene lengths.

Question 16

Characteristics of protein-coding genes in the human genome: Gene transcription

Answer 16

• Gene expression is regulated by cis or trans regulatory elements that may be located upstream or downstream of candidate genes

Question 17

Characteristics of protein-coding genes in the human genome: Gene Copies

Answer 17

-Protein-coding genes occupy a small fraction of the human genome 2-3%
-many appear in multiple copies either identical or diverged into families due to duplication and divergence (e.g over 400 related olfactory-receptor genes)
-Closely related genes may co-localize (e.g hemoglobin genes)
-Some genes occur in different chromosomes (e.g ubiquitin)
-Protein-coding genes are distributed unevenly across all chromosomes and appear on both strands

Question 18

Describe mechanisms that may interfere with the genome-proteome relationship

Answer 18

• Alternative splicing, mature mRNA molecules are formed from different choices of exons of a gene but always in the order in which they appear in the genome
  >this means more than one protein will be encoded from a single gene that is spliced alternatively
  > this occurs in 95% of the human genome
  >Genes that have multiple promoters, if the reading frames are out of phase then different proteins occur.
• RNA editing, produces one or more proteins with different amino acids that differ from what is predicted in the genome
  > Vitis vinifera, mitochondrial protein-coding genes are subjected to multiple C to U editing events.
  > In humans some genes experience A to I (=G) changes tat are tissue specific
• Post-translational modifications, complexes of polypeptide chains e.g hemoglobin
• Special combinatorial DNA splicing, certain protein-coding DNA regions recombine to create novel gene combinations that will result in extensive protein diversity, e.g antibodies

Question 19

Distinguish between moderate and highly repetitive DNA regions

Answer 19

• Moderately repetitive DNA
  
  • Functional: Dispersed gene families created by gene duplication and divergence (e.g actin globin. Tandem gene repeats e.g 250 rRNA genes of histone gene
    
    • Without known function: SINES (100,000 of Alu copies 200 - 300 bp long, LINES 10 - 10,000 copies of 1-5kb long, pseudogenes
• Highly repetitive DNA
  - Minisatellites: repeat arrays of 14 - 500bp, 1 - 5kb long
  - Microsatellites: repeat arrays of up to 13bp, 100s pf kb long, occur mainly as heterochromatin around centromeres
  - Telomeres: typically comprise of 6bp repeats that occurs 250 - 1000 times per chromosome end

Question 20

Distinguish between two classes of TEs in terms of their methods of transposition

Answer 20

Transposable elements are skittish segments of DNA, found in all organisms that move around the genome.
- Retrotransposons (Class 1): Replicate via an RNA intermediate (RTase) and uses a copy and paste mode e.g LINES(L1) and SINE (Alu)
- Transposons (Class 2): Produce DNA copies without an intermediate RNA stage, encode transposase that recognizes sequences within the transposon itself, cuts it out and inserts it elsewhere thus using cut and paste mode

Question 21

Discuss the biological effects of TE transposition in genomes

Answer 21

• Sequence broadcasting: multiple copies distributed to various locations in the genome
• Alter gene properties: Non-functional gene product (knockout effect), affect gene regulation ( TEs in promoters) even when in introns (slow down RNA polymerase) or alter its splicing pattern
• Serve as an important engine of evolution: Gene evolution by fusion or exon shuffling, generate species-specific alternative splicing patterns leading to protein isoforms, change reading frame leading to truncated proteins and thus diseases.
• Cause chromosome rearrangement: mispairing of chromosomes during cell division ( e.g inversions, translocation, transpositions and duplications), deletions (e.g mutation nearby TE sequence e.g Prader-Willi and Angelman syndrome)
• Leakage of epigenetic modification: Natural defense against TEs is methylation or regulation of TEs using siRNAs, TE silencing can also affect neighboring genes, hypomethylation lead to transcriptional reactivation of TEs due to cancers and other diseases.

Question 22

The concept of developmental toolkit remaining the same in similar or different body plans

Answer 22

• HOX genes: responsible for anterior-posterior patterning in the body plans of flies, humans and nematodes
• PAX6 gene in humans is required for proper eye development but if expresses in Drosophila can transform embryonic wing tissue to an ectopic eye
  >Despite huge differences in gross eye anatomy in vertebrates, insects and octopus, visual systems arose from a common ancestor, this can be seen by the structure of rhodopsin and the architecture of neural pathways.
• HOX genes illustrate the conservation of the developmental toolkit in species with very different body plans
• DNA methylases: signals for transcription control in vertebrate development and tissue differentiation, the distribution of DNA methylases illustrates the diversification of developmental toolkit even in organisms with similar body plans.

Question 23

How do proteins evolve, What insights can be gained from evolution of globin genes?

Answer 23

Mechanisms of protein evolution
- Mutations: at the DNA level (substitutions or indels), substitutions can have no change, conservative or severe change, indels can vary in length
- Domain mix and match: Safe way to generate diversity, often domains don’t retain their functions

How do essential proteins evolve
- Gene duplication followed by divergence e.g globins
- Multi-function proteins: depending on the cell’s requirements, evolution would the optimize the protein for one or other functions if not retain both, prokaryotes import plasmids with desired function

Globins:
- Within each cluster are more closely relates than between clusters
- Others (myo-,neuro-,cytoglobin) are distantly related
- Divergence at molecular level correlates with species level divergence

Question 24

Regulatory mechanisms affecting protein activity

Answer 24

Transcription:
- Gene copy number
- Promoter activity
- Repression/attenuation
- Induction
- DNA methylation/ chromatin remodelling

Translation:
- mRNA lifetime
- Codon usage, tRNA levels
- Ribosome binding
- Alternative splicing
- RNA interference

Post-translational modification:
- Inhibition
- Allosteric change

Question 25

Control mechanisms that take place at the level of transcription and translation

Answer 25

Mechanisms at the level of transcription:
- Antisense RNA: forms a double helix with mRNA and block transcription, Flavr Savr tomato - artificitial gene transcribes into antisense RNA that reduces translation of a gene involved in ethylene synthesis delaying ripening
- RNA interference: A short stretch of double stranded RNA elicits degradation by ribonucleoprotein complex of mRNA complementary to either of the RNA strands, gene knockouts in studies aimed at deducing gene functions
- Attachments of ligands to the Shine-Dalgarno sequence in mRNA, prevents the mRNA from binding to the ribosome in E.coli

Mechanisms at the level of translation
-Regulating the translation of slice variants
> Degradation of specific mRNA variants by miRNA and siRNA which may repress translation of specific splice variants
> Splicing factors may interact with transcripts of specific exons and splicing machinery to direct maturaturion of the mRNA
> At transcriptional level, chromatin remodelling may render certain exons inaccessible and affect expressed splice variant