Molecular Biology

When was DNA first isolated?

1871 (by Miescher)

What did Griffith mean by the term "transformation"?

Factor/process which converted non-virulent bacteria into virulent

What are nucleotides made of?

Pentose sugar, nitrogenous bases, phosphate group

In the famous experiment by Avery, MacLeod and McCarty, which treatment resulted in the destruction of the "transforming material"?

DNase

The Hershey and Chase experiment served as a powerful independent confirmation that DNA was indeed the genetic material. Which type of organism or methodology did they employ to make their discovery?

Two groups of bacteriophages (phosphorus + sulphur isotopes to radioactively label DNA + protein)

What is Chargaff's rule?

A = T and G = C

What did Watson and Crick unveil in 1953?

Structure of DNA

Is DNA hydrophobic or hydrophilic?

Both (backbone = hydrophilic; bases = hydrophobic)

What are the minor groove and major groove?

Regions of varying space between double helices

How many conformations of DNA exists?

3

Is DNA replication conservative, semi-conservative or dispersive?

semi-conservative

Where does the DNA replication process begin?

Sites of origin

In which direction does DNA synthesis proceed?

5’ to 3’

How can DNA replication be in relation to the direction of the replication forks with respect to the origin of replication?

mostly bidirectional (can be unidirectional)

What does it mean that DNA replication is semi-discontinuous?

Leading strand = continuously synthesised; lagging strand = discontinuously synthesised

What is the replication fork?

The separation zone where the synthesis of the two new strands takes place

Which proteins are involved in DNA duplication? (10)

Helicases, primases, DNA polymerase, DNA ligase, tethering protein (sliding clamp), SSB (single strand binding) proteins, topoisomerase, nuclease, nucleosomal histone, telomerase

What is the leading strand?

The strand which is continuously synthesised; DNA synthesis proceeds in direction of fork opening

What is the lagging strand?

The strand which is discontinuously synthesised; DNA synthesis proceeds in opposite direction of fork opening

For which analysis is bromodeoxyuridine (BrdU) commonly used?

Detection of proliferating cells (in vitro or in vivo)

What happens when live cells are exposed to bromodeoxyuridine (BrdU)?

Incorporated into DNA of S-phase cells

How long is it advisable to provide bromodeoxyuridine (BrdU) in cell proliferation assays?

Depends on proliferation rate [rapidly dividing cells = less time; slow division (primary cells) = up to 24 hours]

How can the incorporation of bromodeoxyuridine (BrdU) be detected in actively proliferating cells?

Fixing + denaturing DNA + adding antibodies that recognise BrdU and bind to other fluorescent antibodies + fluorescent microscope

What occurs in zebrafish after resection of the cardiac apex?

tissue regeneration; proliferation of cardiomyocytes

Does the mammalian heart possess regenerative capacity?

Yes, but only during prenatal development or immediate post-natal period

What is the technical limitation in using bromodeoxyuridine (BrdU) to measure cell proliferation in slowly proliferating tissues?

toxic effect

Which method was developed in humans to accurately measure the renewal of poorly proliferating tissues, such as cardiac muscle cells?

carbon-14

What is the rate of cardiomyocyte division in the adult human heart?

1% per year at age 25; 0.3% per year at age 75

Which percentage of cardiomyocytes is renewed during a normal lifetime?

≈50%

Where can totipotent stem cells be found?

Embryo; up to 4-8 cell stage

Where can pluripotent stem cells be found?

Inner cell mass of blastocyst-stage embryos + cellular reprogramming of somatic cells

Where can multipotent stem cells be found?

Most organs of cells

Which cell types can be generated from totipotent stem cells?

All (including extra embryonic cells)

Which cell types can be generated from pluripotent stem cells?

All body cells [derived from 3 germ layers (endoderm, ectoderm, mesoderm); no extraembryonic (e.g. placenta)]

Which cell types can be generated from multipotent stem cells?

Limited number -> depends which tissue

Which cells are generated by a stem cell undergoing symmetric cell division?

2 identical daughter stem cells

At which developmental stage is the division of embryonic stem cells exclusively symmetrical?

Beginning of embryonic development (4-8 cell stage)

At which developmental stage does asymmetric division of stem cells begin?

Formation of blastocyst

Which cells does a stem cell that undergoes asymmetric cell division give rise to?

1 daughter stem cell + 1 differentiated (usually progenitor)

Which types of stem cells are found in most adult tissues?

Multipotent (can be unipotent)

What does cell differentiation mean?

Process by which stem/progenitor cells become specialised + take on specific roles in organism

What does the term "embryonic stem cells" usually refer to?

Pluripotent stem cells (isolated from inner cell mass of blastocyst-stage embryos)

Where can adult stem cells be found?

Most body tissues

Which cell types can be generated from adult stem cells?

Limited number

Which cells enable cell renewal in highly renewing adult tissues?

adult stem cells

Where are hematopoietic stem cells mainly located in adult individuals?

Bone marrow

Which potency do hematopoietic stem cells have?

Multipotent

Where do adult intestinal stem cells reside?

base of crypts

Where do adult skin stem cells reside?

basal layer of skin

Which marker identifies adult skeletal muscle stem cells?

Pax7

In which state of activation are mainly found skeletal muscle stem cells?

Quiescent (out of cell cycle)

Which cell types can be generated from embryonic neural stem cells?

Neurons, glial cells

Do stem cells exist in the brain of adult mammals?

Yes. In hippocampus (sub-granules) + lateral ventricles (sub-ventricular)

Which stem populations contribute to the generation of cardiac cells during embryonic development?

Cardiogenic mesoderm, cardiac neural crest, proepicardial

Which stem populations give rise to cardiac muscle cells?

1st and 2nd heart field

Which signals can modulate stem cell differentiation and self-renewal?

Extracellular matrix, soluble fibres, other cells, mechanical stimuli

Which ligands are well known to regulate stem cells?

WNT, Notch, BMP, ERBB

How is cell differentiation controlled in response to different signals?

Transcription factors

Are the transcription factors that regulate cell differentiation specific or shared among different stem cell populations?

Usually specific; can be shared

What are recombinases?

Enzymes that facilitate DNA exchange reactions between short target sequences, specific to that recombinase

Which operations can recombinases perform?

Excision (insertion), inversion, translocation, exchange of DNA segments

What is the Cre enzyme?

A recombinase enzyme derived from P1 bacteriophage

Which events does the Cre enzyme promote?

Site-specific recombination of LoxP sites

Which sequences does the Cre recombinase recognize?

LoxP sites (34 bp, 2 inverted palindromic repeats, core sequence)

Which operations can Cre recombinase induce?

Deletion, inversion, translocation

Why is the Cre recombinase considered a revolutionary tool in genetic engineering?

Only needs 2 LoxP sites (no co-factors); can be used in any cells; fast, effective, precise

What is the Cre/Lox system used for?

Gene deletion in specific cell population + induce expression of transgenes

Is it possible to delete or induce a gene in a cell type-specific manner with the Cre/Lox system?

Yes. (Using cell-type specific promoter upstream of Cre gene)

What capabilities does the utilization of inducible Cre recombinases provide?

Temporal control of gene manipulation

How were inducible-Cre created?

Fusion between Cre recombinase + receptor which binds to a specific ligand and controls activation of Cre

What are the most well-known inducible Cre variants?

Tamoxifen, RU486, TMP (-inducible Cre)

What occurs in cells containing the following construct?

Overexpression of transgene (in cell-type specific population)

What occurs in cells containing the following construct?

Gene undergoes knockout in cells where Cre recombinase is actively expressed

Which analysis can be conducted using "lineage tracing"?

Contribution of cell populations to embryonic or regenerative organogenesis

How can the Cre/Lox system be adapted for lineage tracing analysis?

Tissue-specific promoter -> stop sequence excised -> reporter gene activated

What are the common reporter genes employed for lineage tracing investigations?

Fluorescent or LacZ protein

In zebrafish, which cell population mainly contributes to cardiomyocyte regeneration following cardiac resection?

Pre-existing/Differentiated cardiomyocytes

In neonatal mice, which cell population mainly contributes to cardiomyocyte regeneration following cardiac resection?

Pre-existing/Differentiated cardiomyocytes

Which therapies based on adult stem cells have been demonstrated to be effective in clinical protocols?

Cornea, bone marrow, skin regeneration

Which effect on heart function is usually observed following the injection of adult stem cells in patients with heart failure?

Most increase in heart function

Which cell types are primarily generated by cKit+ cardiac cells, proposed as cardiac stem cells?

Endothelial cells

Which cell population is responsible for generating new cardiomyocytes during the regeneration process following cardiac damage in neonatal mammals?

Pre-existing cardiomyocytes

Injection of adult stem cells into heart failure patients has been reported to induce modest and temporary beneficial effects on heart function. What are they attributed to?

Paracrine factors (that induce angiogenesis and cardiomyocyte activity)

Is it possible to use the Cre/Lox system to transiently induce the expression of a transgene?

No

Which molecular tool is suitable for transiently inducing the expression of a transgene?

Tet-ON/Tet-OFF

What is Tet Repressor (TetR) Protein?

A homodimer repressor of TetA

What is the Tet-OFF/Tet-ON system?

Tetracycline-controlled transcriptional activation

What is the Tet-OFF/Tet-ON system used for?

Transient gene expression control in eukaryotic cells

How was the tetracycline transactivation factor (tTA) generated?

TetR + VP16

What is the difference between Tet-OFF and Tet-ON system?

Tet-OFF: TetR (tTA) used + stops expression in presence of TET.

Tet-ON: rTetR (rtTA) used + enhances expression in the presence of TET.

What does cell dedifferentiation mean?

When a mature cells reverts back to a less-differentiated state

Which cellular process mainly contributes to limb regeneration in the salamander?

Depends on developmental stage:

before metamorphosis = expansion + stem cell differentiation

after metamorphosis = dedifferentiation

Which features characterize the partial cellular dedifferentiation observed in cardiomyocytes during the spontaneous process of heart regeneration in zebrafish or in neonatal mice?

• Disassembly of sarcomeres

• Reduced expression of sarcomeric genes

• Detachment of cardiomyocytes

• Expression of positive cell cycle regulators

What is ERBB2?

• Co-receptor for heterodimerisation for erbB4

• Responds to NRG1

• Regulates embryonic cardiac development

• Key player in cardiac regeneration

Which effects are induced by ERBB2 signalling in cardiomyocytes?

Cell dedifferentiation

(due to reduction in sarcomeric structures, increase in mature sarcomeric components and expression of dedifferentiation markers and proliferation markers)

Do ERBB2 expression levels change in the cardiac tissue during the early postnatal development?

Yes; decrease after birth

Which effects are induced by transient ERBB2 overexpression in cardiac muscle cells following myocardial infarction?

Increased heart regeneration (more proliferation + regeneration, reduced scarring)

Which processes are induced in the heart by the glucocorticoid receptor (GR) during postnatal development?

• Decreased cell proliferation

• Cytoarchitectural (myofibrils) maturation

• Energetic maturation

What are the primary hurdles hindering the therapeutic application of embryonic stem cells?

ethical dilemmas, immunological rejection, teratoma formation

Which strategy has been utilized to mitigate the tendency of embryonic stem cells to form teratomas when transplanted in vivo?

Differentiation before implanting