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Summary Molecular Genetics

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This is a summary of the lectures of Molecular Genetics, a mandatory course for biomedical science students that are in the second year of the study Biology at the RUG. With this summary (and the shorter version I made) I was able to get a 9.0 for this exam. Especially understanding the pictures he...

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  • 5 november 2022
  • 59
  • 2022/2023
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Molecular genetics

Contents
Recap: Genomes, Transcriptomes, Proteomes (3 pages) ....................................................................... 2
Recap: Studying DNA (2 pages) ............................................................................................................... 5
Lecture 1 Genome replication (5 pages) ................................................................................................. 7
Lecture 2 Mutations and repair (6 pages) ............................................................................................ 12
Lecture 3 Homologous recombination (3 pages).................................................................................. 18
Lecture 4 SSR and Transposition (5 pages) ........................................................................................... 21
Lecture 5 Transcription and splicing (6 pages) ..................................................................................... 26
Lecture 6 Transcription factors and their study (3 pages) .................................................................... 32
Lecture 7 Genomes of prokaryotes and eukaryotic organelles (5 pages) ............................................ 35
Lecture 8 Architecture of the genome in eukaryotes (4 pages) ........................................................... 40
Lecture 9 Genome expression in response to the external environment (3 pages) ............................ 44
Lecture 10 Understanding the function of genes (3 pages) ................................................................. 47
Lecture 11 Proteomes (7 pages) ........................................................................................................... 50
Lecture 12 DNA sequencing (3 pages) .................................................................................................. 57




1

,Recap: Genomes, Transcriptomes, Proteomes (3 pages)
The genome of humans (like that of all other animals) consists of two distinct parts
• Human nuclear genome:
- 3.2 billion bp
- 22 autosomes, 2 sex chromosomes
- ~45.000 genes
• Mitochondrial genome:
- 16,569 bp; 37 genes
- ~10 copies/mitochondrion
Genome = biological information to construct/maintain ‘encoded’ organism
Most genomes = DNA
Some genomes (e.g. that of Sars-Cov-2 virus) = RNA
An experiment suggesting that genes are made of DNA

The ends of a polynucleotide are different
➢ DNA has polarity (‘a chemical DIRECTION’)
DNA synthesis occurs in 5’ to 3’ direction: new
Nucleotide is added to 3’ carbon at end of existing polynucleotide
All DNA polymerases can only perform 5’ to 3’ synthesis > problem in
replication!
The DNA double helix is stabilized by base pairing and base stacking…
- Base stacking: attracting forces between adjacent base pairs
… and are flexible as well

Also important are the minor and major grooves, these are important for
protein interactions

RNA and the transcriptome
First product of genome expression is the transcriptome (ALL RNA molecules of a cell
(organisms) at a given time point) > transcription
➢ Total RNA in bacterial cell ~6% of total weight
➢ Total RNA in mammalian cell ~1%

Template-dependent RNA synthesis
RNA is synthesized in 5’ tot 3’ direction by DNA-dependent
RNA polymerase reading the DNA in 3’ to 5’ direction
Sequence of transcript is determined by base pairing to DNA
template

Cells contain different types of RNA
Heterogeneous nuclear RNA and long noncoding RNA only in
eukaryotes




2

,Splicing of eukaryotic pre-mRNA molecule
Many RNAs in eukaryotes initially synthesized as
precursors (pre-mRNA)
Splicing occurs in nucleus
Most eukaryotic mRNAs are modified at their 3’- and
5’- ends
End modifications occur during synthesis of eukaryotic
mRNAs, most have a cap at 5’ end and a poly (A) tail at
3’ end

Some examples of chemically modified bases occurring in rRNA and tRNA molecules




Reasons for these modifications are mostly unknown
Some modifications do have a known function:
- In tRNA: provide specificity for attachment of proper amino acid
‘RNA editing’: chemical modification on mRNA (in eukaryotes)
- Alters biological information in the modified mRNA
- Leads to altered structure of encoded protein

Proteins and the proteome
Second product of genome expression is proteome (the cell’s repertoire
of proteins at a given moment) > translation
Protein: linear unbranched polymer of amino acid residues
Amino acids ‘stitched together’ via peptide bonds
- Primary structure: amino acids in a chain
- Secondary structure: a helix/beta sheet etc.
- Tertiary structure: 3D structure
- Quaternary structure:
• Association of two or more separate polypeptides
• Each folded in its proper tertiary structure
➢ “multi-subunit protein”
Side (R) groups of the 20 proteinogenic amino acids
Amino acid diversity (R groups) underlies (function) diversity

Relation between ‘punctuation codons’ and gene product (protein)
The genetic code is (not entirely) universal
- At least 2 additional amino acids can be inserted during translation
➢ Selenocysteine - Sec (U) and pyrrolysine - Pyl (O)
Insertion is directed by modified reading of the genetic code
Selenocysteine: in proteins in all kingdoms of life, over 50 selenoproteins in humans




3

,Context-dependent reassignment of a 5’-UGA-3’ codon
UGA normally functions as a translation STOP (termination) codon)
UGA-3’ codon specifying selenocysteine is distinguished by stem-loop structure:
- Positioned in mRNA downstream of the codon in prokaryotes
- In the 3’-untranslated region of a eukaryotic mRNA
- Serves as a signal to insert selenocysteine in growing polypeptide chain
Something similarly takes place for insertion of pyrrolysine into protein




4

,Recap: Studying DNA (2 pages)
Engineering nature
Humans have always ‘manipulated’ the world around them
They either ‘designed’ new techniques or stole ideas from examples
from nature
Scientists are no different
Examples of manipulations (only
a few!) that can be carried out
with DNA




Many of these manipulations use (altered) enzymes from nature
- Nucleases are restriction enzymes
- A bacterial defence mechanism against bacteriophages: to cut and get rid of
incoming phage DNA
Restriction enzymes allow cutting DNA at specific positions
- Fragments cut by BamHI and Sau3A can be ligated by ligase (same sticky ends)
Agarose gel electrophoresis: to visualise/examine DNA/RNA fragments
- Agarose concentration determines range of fragment sizes that can be resolved

Mode of action of DNA-dependent DNA polymerases
Used in e.g. PCR, DNA sequencing and more
NB. Synthesis is always 5’ to 3’ direction!
Enzyme requires a starting point (primer)

DNA polymerase can ‘make a break’ DNA
- Primer first anneals to template
- DNA polymerase adds nucleotides to 3’- end of primer
to remove wrong nucleotide (self-correction)
➢ Proofreading
Important for genome stability

DNA ligase: a ‘molecular glue’ used in vivo and in
the lab to stitch DNA fragments together




5

,Linkers can be used to increase efficiency of ‘blunt-end’ ligation
- High concentration of linker DNA
- More linkers can be ligated in a ‘train’ (resolved by cutting with
BamHI)
- Efficient ligation of sticky BamHI ends

A 1980’s revolution in (molecular) biology: PCR

Exponential
amplification



DNA and RNA can be amplified by PCR
Specific primers allow cloning any DNA or RNA fragment
- Without requiring restriction enzymes
- In only a few hours
- Sequence information is required
cDNA clones are intron-free

DNA cloning
Or use PCR when sequence is known

Cloning vectors
- Some are based on plasmids with low or high copy
number
- (antibiotic) selection marker
- ‘multiple cloning site’ (MCS) for easy insertion of
DNA fragments
- DNA can also be cloned in other organisms, often
initial cloning is still done in E.coli


Repurposing a natural plant engineer




6

, Lecture 1 Genome replication (5 pages)
Flow of genetic information

DNA replication is semiconservative
Watson and Crick (1953)
• Semiconservative replication
• Each ‘parent’ DNA strand produces a new
‘daughter’ strand
➢ Topological problem:
- How to unwind DNA
- Semiconservative, conservative and dispersive replication
mechanisms
Matthew Meselson and Franklin Stahl
• Meselson-Stahl experiment
N15: heavier and thus this DNA strand
will have a higher density when
incorporated. It was indeed
incorporated in the DNA strand
Then this DNA strand was put in
another medium with N14, these
mixed and created an ‘intermediate’
density gradient.

Semiconservative Conservative Dispersive
First generation Intermediate 1 light Intermediate
1 heavy
Second generation ¾ Light ¼ heavy Intermediate
¼ Intermediate ¾ light


The topological problem
The topology of DNA is defined by how the two DNA strands are intertwined. It plays an
important role in processes as replication, recombination, transcription, etc.
Chromosomes contain both negative (underwound) and positive (overwound) supercoils in
different regions.
Topoisomerases: enzymes that catalyse the state of DNA by cutting
DNA strands
• Type I topoisomerase: single-stranded
break
• Type II topoisomerase: double-
stranded break
➢ Gyrase (type II topoisomerase):
enzyme that introduces negative
supercoils. It relaxes and prevents
overwinding during DNA replication (positive to negative
supercoils necessary for DNA replication, needs ATP)



7

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