Week 3

Question 1

Sequencing genomes

Answer 1

resulted in a shift from studying single or a few genes to studying all genes simultaneously

proteome and transcriptome

Question 2

Genome

Answer 2

all DNA and identification of all DNA elements (transcriprion units)

Question 3

Transcriptome

Answer 3

all transcripts expressed (list plus analysis of expression)

Question 4

Proteome

Answer 4

all proteins expressed (list plus analysis and modification)

Question 5

Large scale ORF finder

Answer 5

Looking for open reading frames in the bacteria

Simple for bacteria because of the fact that DNA contains the coding region that is not interrupted.

So you can go from DNA to the protein coding capacity of that DNA very simply.

We can’t do the same for the eukaryotic DNA.

Question 6

Eukaryotes and ORF finders

Answer 6

not for most eukaryotes, we can’t go from the eukaryotic genome to the eukaryotic proteome that simply

Splicing

We need the transciptome to get the proteome of the genome.

Question 7

transcriptome is

Answer 7

all expressed RNA:
mRNA
rRNA
tRNA
siRNA
miRNA
non coding RNA
snRNA
crRNA
snoRNA

Question 8

eukaryotic mRNA

Answer 8

exstensively processed

5’ prima cap

AUG first codon of ORF

Messenger RNAs are processed with the additon of a poly-A-tail that helps us annotate the proteosome

Question 9

reverse transcriptase

Answer 9

the DNA copy is made with reverse trancriptase which requires a DNA primer. A common approach is to use an oligo dT primer that hybridizes with the poly A tail. therefore the total transcriptome is not represented

Before nanopore only DNA could be sequenced so RNA always had to be turned into a complementary DNA copy.

Question 10

post translational processing

Answer 10

a barrier to annotating the genome

a primary transcript is processed, splicing, poly-a-tail and cap

Therefore anytime we make a complementary DNA copy we’re making a complementary copy of the mature mRNA after the intronic sequences are removed.

A large amount of the genome is not expressed: intragenic regions which are not trasncirbed, intronic regions that are transcribed but spliced out.

Question 10

post translational processing

Answer 10

a barrier to annotating the genome

a primary transcript is processed, splicing, poly-a-tail and cap

Question 11

Alternative Splicing

Answer 11

Genes undergo alternative splicing, when you align different cDNA sequences to the genome you find that some genes that these aligments are quite different from one cDNA to another

indicating that they came from transcripts that have undergone alternative splicing

This gene produces six distinct messenger rna transcripts.

That encode three distinct polypeptides.

When you align this sequence to drosophila DNA you ifnd six different patterns of alignments due to six different splicing patterns of the mRNA transcripts.

Alternaitve splicing increases the number of proteins that can be encoded by a single gene.

Question 12

Types of splicing

Answer 12

alternative poly-a-tail sites
alternative promoters
Exon included or excluded
Mutually exclusive inclusion.
Alternative 5’ splice sites.
Alternative 3’ splice sites.
Retained intron

In some messages splicing occurs such that the intron remains in the mature mRNA, in other the mature mRNA the intron is removed.

Question 13

RNA seq Two major goals

Answer 13

Count the relative number of transcripts in the sample.

Determine the structure of the transcripts in the sample.

Often done after they’ve converted the RNA to complementary DNA and sequenced the complementary DNA.

Question 14

How do we get distinct cell types

Answer 14

differential gene expression

Question 15

sc RNA seq goals

Answer 15

To determine the poly A+ transcriptome of individual cells

Useful in the study of development and human disease

Question 16

sc RNA seq function

Answer 16

1-In drop single cell seq, suspension of cells, microparticles and lysis buffer,

2-mixed in a microfluorodics apparatus and encased into droplets by using oil, oil droplet contains a cell and microparticle

3-lysis buffer in the droplet lyses the cell releasing rna/dna

4-the poly adenlyted RNA is hybridized to a primer on the microparticle that contains oligodT.

5-Barcoded primer beads contains a unique sequence barcode sequence between the PCR handle and an oligodt tail

6-break droplets, reverse transcription with template switching formation of STAMPs

7-STAMPs are amplified by PCR so these microparticles that have these individuals cell barcodes and transcriptome attached are amplified. the amplified fragments are synthesized.

8-Generation of paired end reads.

One read goes through the cell barcode the other read goes through the cDNA

Illumina

Because they’re paired end reads we can tell which cell this cDNA comes from by read one having the cell barcode

9-Even though we are sequencing a complex PCR product from a multidtude of different stamps, each one of those microparticles had a distinct cell barcode that we can use to identify which cell the paired end reads came from.

Organize the data and ask which trasncripts are expressed in cell one.

Determine what genes are expressed in the cell and to what level., count the number of observed trancripts.

Question 17

Changing pattern of gene expression through development

Answer 17

When you start off as a single cell you have one transcriptome, but as the cells specialize during development you start to get expression of different patterns of genes in the each cell.

Zebra fish development, single cell RNA seq during development on each cell and looking fro changes in gene expression (sequence transcriptome)

Each point is a cell, change in gene expression of the cells as they differentiate.

Question 18

Proteome

Answer 18

Catalogue of the proteins expressed by an organism?

What proteins are unique to an organism or shared?

what is the function of the protein?

Information encoded in the genome.

All of the proteins encoded within a genome.

Function can be determined by taking advantage of the relatedness of all organisms.

Question 19

What proteins are unique to an organism or shared?

Answer 19

all life is related; genes are shared

homologous genes can fall into two categories:

• orthologs
• paralogs

Question 20

orthologs/paralogs

Answer 20

homologous genes in different species. have the same common ancestor

homologous genes in the same species. result of a duplication of a gene

Question 21

What was the function of the protein?

Answer 21

if an orthologous protein is well characterized in one organism then it may be reasonable to proopose that all the orhtologous proteins share its function.

Complex proteins often contain conserved protein domains of known function like DNA binding for example. Therefore, conserved domains can suggest the biochemical function of the protein in the proteome.

Question 22

Interactome

Answer 22

proteins interact with one another either in stabke complexes like RNA polymerase, or via transient interactions like initiation factors for translation.

An interactome is the result of a systematic analysis of the proteome.

Question 23

Systematic analysis of interactomes

Answer 23

1-Yeast two hybrid screen

2-affinity purification and mass spectrometry

Question 24

Yeast two hybrid screen

Answer 24

Gal4 binds to a uasgal4 and can drive gene expresion, we drive the expression of the reporter gene, beta galactosidase.

when yeast expresses b.galactosidas in the presence of chromogenic reagent the yeast cells will go blue.

yeast gal4 transcription factor is made up of two seperable domains, one is the DNA binding domain, the other is the activation domain

to have transcription AD must bind to DBD

Question 25

Yeast two hybrid screen steps

Answer 25

• seperate the DNA binding domain from the DNA activation domain, if these domains are expressed independently of one another so not fused to one another there will be no expression of beta galactosidase
• fuse the dbd to the bait protein and fusw the prey protein to the ad
• if these proteins interact you will get blue colonies

Question 26

Affinity purification

Answer 26

-add an ap tag to a protein and pass the protein mixture through a column, the protein will bind to the column containing the ligand that binds the ap tag and bind the protein along with it, binding them and the protein they are attached to the column

Question 27

How can genomes vary?

Answer 27

Genome size
Genome content/number
Genome structure/shape
Genome type

Question 28

Genome type/shape/peices

Answer 28

RNA or DNA

Circular or linear

of peices

double stranded or single stranded

Question 29

genomes can be complex structures

Answer 29

a mixture of linear and circular or linked circles

Question 30

Advantages of genome structure/shapes

Answer 30

circular dna is easy replicate; go around the circle

linear is hard to replicate at the ends (telomeres), the 3’ ends are difficult to replicate, they get shorter

Question 30

Advantages of genome structure/shapes

Answer 30

circular dna is easy replicate; go around the circle

linear is hard to replicate at the ends (telomeres), the 3’ ends are difficult to replicate, they get shorter

Question 31

Telomerase

Answer 31

reverse transcriptase

telomerase has an RNA template embedded in it that it can use to elongate the 3’ end creating a set of repeats

some chromosomes have circular telomeres

Question 32

What is a genome? What is genome size?

Answer 32

a set of genetic instructions within a biological compartment

the length of those instructions

Question 33

Units of size of the genome

Answer 33

nucleotide (single stranded)

base (both single and double stranded)

base pair (double stranded)

Question 34

bp Units

Answer 34

1 bp

1000 bp = kilobase (kb)

1,000,000 bp = megabase (Mb)

1,000,000,000 bp = gigabase (Gb)

Question 35

What is genome size

Answer 35

One full haploid set, don’t measure duplicated information

Count the length of one chromosome for identical chromsomes, add up the length for the different chromosomes.

Question 36

Complexity

Answer 36

does complexity increase with genome size or gene number?

Question 37

Bacteria and archae

Answer 37

genome size is linearly related to gene number.

the larger size of the genome the more genes

Question 38

Who has the biggest genome?

Answer 38

plants have an immense variation in genome size

single cell amoeba have a very large genome

genome size is not associated with complexity

Question 39

What do big genomes have that little genomes don’t have?

Answer 39

non-coding DNA

Differences in gene to base ratio

As genome size increases the precent of non ciding genome increases

The percent non coding varies within the genome.

Gene deserts.

Variation between organisms in the terms of the amount of noncoding DNA they have is due to the race to replication, some organisms that need tro replicate more quickly have lost noncoding regions of their DNA

Question 40

Is gene number related to complexity?

Answer 40

nope

we have this notion of implicit phylogeny that somehow because we possess specific qualities that some species are less evolved than we are.