Chapter 20 - Genomics and Proteomics

Question 1

What is structural genomics?

Answer 1

The study of the organization and sequence of the genetic information contained within a genome

Helps generate genetic and physical maps

Question 2

What are genetic maps used for and their limitations?

Answer 2

Genetic maps: provide the location of known genes relative to other genes (measured in map units)

Limitations of genetic mapping were overcome with the advances of molecular genetics
- Limitations: low resolution with regard to actual physical structure of chromosome/genes, doesn’t always accurately correspond to the actual physical distance between genes

Question 3

What are physical maps?

Answer 3

Place genes in relation to distances measured in bases

Tend to have higher resolution than genetic maps

Question 4

How are physical maps created?

Answer 4

Created through restriction site mapping
- Determines positions of restriction sites that are cut by the enzymes – gives physical landmark of where sites are in genome

Cut sample of DNA up and run it on a gel
- Number and positions of bands that you get can tell how many restriction sites are in DNA for enzyme you used - Not going to tell order of fragments

Take sample and cut it with a different restriction enzyme that cuts at different sites and run it on a gel

Then take another sample and use both restriction enzymes – called a double digest
- Run this sample on a gel and compare the bands from the single and double digests to find where overlaps occur to determine order

Question 5

What are the 2 obstacles and 2 approaches of whole genome sequencing?

Answer 5

Obstacles:
- Large genome sizes
- Short reads and assembly can be tedious

Approaches:
- Map-based sequencing
- Whole-genome shotgun

Question 6

How does map-based sequencing occur?

Answer 6

Take short sequenced fragments that are assembled into correct sequence where restriction sites or markers overlap

Provide known location of genes within genome at regularly spaced intervals along chromosome

Assembled into a contig – continuous stretch of DNA

Question 7

How does whole-genome shotgun working?

Answer 7

Sequence small fragments that are arranged into correct sequence using overlap in sequence

Assembled in pretty powerful bioinformatic program

One requirement in finding overlaps is to increase coverage – average number of times a nucleotide in the genome is sequenced
- As it increases, increased confidence in assembly of genome

Can compare the genomes between species or organisms

Question 8

What is metagenomics?

Answer 8

Genome sequences of entire group of organisms that inhabit a common environment are sampled and determined

Typically, heard of within microbial communities
- Useful for microbes that can’t be cultured
- Study communities of microbes

Question 9

What is amplicon sequencing?

Answer 9

Sequence multiple copies of fragments from one target gene (the amplicon)

Question 10

What is metagenomic sequencing?

Answer 10

Short sequence fragments from all DNA in population

Help see what organisms live in a studied environment

Question 11

What is functional genomics?

Answer 11

Studies the functions and interactions of the genetic information contained within genomes
- Characterizes what genes do (especially in coding genes)

Identifies transcriptome (coding region of DNA) and proteome (proteins encoded by region)

Focuses on dynamic interaction, aspects such as gene transcription, translation, regulation of expression, and protein-protein interactions

Focuses:
- Predicting function from sequence
- Gene expression - what ways or to what degree are genes expressed

Question 12

What are the 2 ways that functional genomics predicts function from sequence?

Answer 12

Homology searches

Protein domains

Question 13

What are homology searches?

Answer 13

Comparing DNA or protein sequences from the same or similar species as the one you’re getting sequences from

Genes you’re looking at between individuals called homologs – genes found in different species that have evolved from the same gene in a common ancestor
- Ortholog – found in different species
- Paralog – found in the same species

Paralogs typically arise from gene duplications

Looks at different functions of genes between species

Question 14

What are protein domains?

Answer 14

Regions of the protein that have specific shapes and functions

Common domains between species have similar arrangements of amino acids

Domains and their functions tend to be conserved over time

Can infer the functionality of proteins

Question 15

What are the 2 ways that functional genomics determines gene expression?

Answer 15

Microarrays

RNAseq

Question 16

What are microarrays?

Answer 16

An ordered array of known DNA sequences that serve as probes to detect the presence of complementary sequences through a process called nucleic acid hybridization

Can use this to assess the expression of genes in various tissues or under various conditions

Question 17

What are the steps for microarrays?

Answer 17

Extract RNA from tissue sample and convert it to cDNA
- Converted through reverse transcription using reverse transcriptase

cDNA labeled with fluorescent nucleotides

cDNA is run over microarray chip

cDNA will bind to complement probes that are on chip

If they hybridize, they will emit fluorescence

Question 18

What are the 3 limitations of microarrays?

Answer 18

Genes under investigation have to be known

Sometimes sequences will hybridize to the same probe to create artifacts

Ability to quantify degree of expression is limited

Question 19

What is RNAseq used for?

Answer 19

Can provide detailed information about gene expression including types and number of RNA molecules produced by transcription, presence of alternatively processed RNAs, differential expression or two alleles

Question 20

What are the steps of RNAseq?

Answer 20

Isolate RNA

Convert to cDNA

Fragment the cDNA and prepare libraries

Sequence cDNA

Assemble sequencing reads into RNA transcripts
- Can map the reads to known genes/genomes
- Can be done de novo – creating it from itself

Question 21

What is comparative genomics?

Answer 21

Studies similarities and differences in gene content, function, and organization among genomes of different organisms

Gives us an idea of the evolution of genomes within and across species

Question 22

What is single cell RNAseq?

Answer 22

RNAseq done in single cells

Looks at differences between two cells to see how gene expression differs

Question 23

What are the 4 characteristics of prokaryotic genomes?

Answer 23

Single, circular chromosome

Typically small

Size of genome is variable within and between species
- Tend to see small prokaryotic genomes in very stressful or extreme environments

High association between number of genes and genome size

Question 24

What is horizontal gene transfer and the 3 types?

Answer 24

Transfer of genetic material between organisms

Types:
- Conjugation
- Transformation
- Transduction

Question 25

What is conjugation?

Answer 25

Genetic material is passed directly from one bacterium to another

Question 26

What is transformation?

Answer 26

Bacterium takes up DNA from the medium in which it is growing

Question 27

What is transduction?

Answer 27

Bacterial viruses (bacteriophage) carry DNA from one bacterium to another

Question 28

What has been the main effect of horizontal gene transfer?

Answer 28

Because it is so widespread, many bacterial chromosomes are a mixture of genes
- Has become difficult to define species of bacteria because bacterial species are not isolated from the genes of other species
- Also makes reconstruction of ancestral relationships difficult because relationships are based on similarities and differences between species

Question 29

What are draft genomes?

Answer 29

Unfinished or unannotated genome sequences

Question 30

What are the 9 characteristics of eukaryotic genomes?

Answer 30

Genome size is larger than prokaryotes

No correlation between genome size and complexity or number of genes

Segmental duplications are common

Vast amount of noncoding DNA

Moderately to highly repetitive sequences that come from replicative transposable elements

Vertebrates tend to have more protein diversity than invertebrates
- Vertebrate genomes don’t necessarily encode more protein domains than invertebrates, but the existing code is formed into more protein types (through alternative splicing)

Many genes are present in the same order in the same genome – called collinearity
- Genes that are descended from a common ancestor genome and evolutionary forces tend to conserve the same order of genome between species, even though the genes themselves have changed

Homologous genes

Multigene families

Question 31

What are segmental duplications?

Answer 31

Duplicated chromosome segments that are over 1000 base pairs

Can found intrachromosomally or interchromosomally and result from unequal crossing over

Can promote or permit further duplication, which furthers misalignment of duplicated region

Can give rise to new genes as they occur over time

Question 32

What is the role of noncoding DNA?

Answer 32

Can play a role in gene expression or can code for other RNA molecules and not proteins

A lot is unknown

Gene deserts – large areas of noncoding DNA

Question 33

What is proteomics?

Answer 33

Aims to determine the complete set of proteins in a given cell (proteome)

Proteins undergo many posttranslational modifications that are not encoded in DNA, so looking at gene that encodes a protein doesn’t necessarily give all of the information on the structure and function of a protein

Question 34

What are the 2 methods for determining cellular proteins?

Answer 34

2D PAGE/Liquid chromatography

Mass spectrometry

Question 35

What are the steps of 2D PAGE/Liquid chromatography?

Answer 35

2D PAGE:
- Proteins are separated in one dimension in polyacrylamide gel by charge
- Also get separated by mass in a second dimension
- Then they are stained – so you can see size and charge of protein
- Size of each dot shows how much of each protein is present

Liquid chromatography (automates process):
- Take a mixture of protein molecules and dissolve it in a liquid
- Passed through a column and proteins stick at different parts
- Run another liquid through and a computer reads each of the molecules that passes through based on their charges or affinity as the liquid pulls the molecules out at different times

Question 36

What is mass spectrometry and the steps?

Answer 36

Molecular mass of protein is determined by determining the migration rate of an ionized molecule in an electric field

Small molecules will migrate rapidly whereas large will take longer

Steps:
- Digest proteins – cleave large protein into smaller peptides
- Then use mass spectrometry to separate peptides in a way that determines their mass-to-charge ratio
- A profile of peaks is produced
- Computer program compares profile with those of known and predicted proteins and a match identifies the protein