Evolution and Development - Biologie UvA

Regulation of Gene Expression

How to recognize/detect an mRNA with a specific sequence?
 Rna-gel blot
 Rt-pcr
 In situ hybridisation
 Rna sequencing

RT-pcr: isolate mrna, convert to cdna, amplify by pcr, run products on a gel

RNA-seq: isolate mrna, convert to cdna, sequence individual cdna

Possibilities to regulate the expression of a gene




Gene expression can be regulated at multiple levels
 regulation of transcription initiation
 transacting TFs specifically bind to sis-regulatory gene elements
o Sis regulatory elements often cluster in regions called enhancers
o enhancers come be very far away from the transcribed region
 many TFs are part of families

Methods
 Measuring RNA levels is most versatile and commonly used
o Few genes -> RNA gel blot, RT PCR, Q PCR, in situ hybridization
o Many genes -> microarray, RNA-seq
 protein DNA interactions
o EMSA, footprinting, chromatin IP (ChiP-seq)
 Promoter-reporter genes
o trans genes that mimic expression of endogenous genes

Gene regulatory networks (GRNs)
 transcription of TF's genes is activated/repressed by TF’s
 All genes operate within complex regulatory networks
 combinatorial control
o multiple TF's needed

, o one TF may have multiple roles
 Hierarchical relations
o example -> MyoD, Eyeless

mutant phenotypes reveals roles of a gene in development
 Lose it -> gene necessary for process X?
 Move it -> gene sufficient for process X?

Mechanisms that create cell memory

DNA methylation

chromatin -> metalation
 histone modification
 recruitment of de novo DNA methylase

where a C is placed next to a G methylation is possible -> CpG
Half methylation -> methylase recognizes it
no metalation -> methylase doesn't recognize it

auto regulation of TRX factors genes -> widely
used
 positive feedback loops -> stable gene
expression
 negative feedback loops -> oscillators

DNA rearrangements -> specific cases

Antibodies: immunoglobin's (Ig), excreted or
membrane bound. Two heavy chains and two
light chains.

how can you recognize so many different
antigens?
 multiple combinations are possible by distinct IG domains on the DNA. This comes
for the heavy chain but with the light chain there's VJ joining.
 The intervening DNA has TIRs (Terminal inverted repeat)

RAG1 and RAG2 resemble transposases
 Enzymes with similar structures and sequence
 RAG1 and RAG2 genes are linked

cloning IPS cells

stem cell: not fully differentiated and self-renewing

zygote: fully totipotent

, ES cells
 isolated from inner cell mass
 undifferentiated
 pluripotent

adult stem cells
 limited capacity, not toti- or pluripotent
 they live in stem cell niches (bone marrow)
o there are cells around them that stimulate them to act as stem cells

organoids:
 organ like structures
 they are made in vitro from adult stem cells
 they are living representation of the organ they originated from
 Between in vivo and in vitro
 clinical relevance

Embryogenesis

Gastrulation: massive cell movement to the inside

zygote -> blastula -> gastrula

ectoderm:
 epidermal cells of skin
 the nervous system
 pigment cell

endoderm:
 digestive tube
 pharynx
 respiratory tube

mesoderm:
 noto chord
 bone tissue
 kidney
 blood cells
 facial muscle

germ cells
 sperm
 egg

neurulation: invagination in embryos where the neural plate closes itself to form the neural
tube, the embryo is now called the neurula