Summaries

Contents

DNA Structure and Replication:

DNA: Basic concepts

  • The genetic code of life (all cellular organisms and most viruses)
  • Contains the genetic material of the cell
  • Found in nucleus in the cell

Genetic code is almost universal

Approximately 22-25000 genes in the human genome

Structure of DNA:

Monomer of DNA has 3 parts:

  1. Pentose sugar (2-Deoxyribose)       Pentose sugar + phosphate group form the sugar           
  2. Phosphate group                          phosphate backbone via Phosphodiester bonds
  3. Nitrogenous base

A-adenine,T-thymine,G-guanine,C-cytosine – Bases in DNA

Purine=A,G (Double-ring)

Pyrimidine=C,T (Single-ring)

DNA is a double stranded molecule. The 2 strands are complementary to each other and anti-parallel. Double strand twisted to form right-handed double helix. 

Double helix contains major and minor grooves. It is easier for proteins to form complexes on major grooves as they are more exposed 

Complimentary base pairing:

A goes to T (2 hydrogen bonds)

C goes to G (3 hydrogen bonds) 

Central Dogma Of Biology

DNA → RNA – Protein

DNA 

DNA →  DNA = Replication

DNA →  RNA = Transcription

RNA → DNA = Reverse-transcription

RNA →  Protein = Translation

The 3 models of DNA Replication:

Conservative replication- The original DNA molecule is used as a template and the double helix stays intact

Results in: One original parent molecule + one completely new daughter double helix

Semi-conservative replication- The original DNA double helix is broken up into 2 single strands which are then used as templates for new complementary bases to form the complementary strand

Results in: 2 DNA strands produced each strand conserving half the DNA from the first strand

Dispersive model-Parental double helix is broken into double helix segments which acts as templates for new DNA synthesis. Segments then join together.

Results in: 2 DNA strands that are mixtures of parental and daughter DNA. 

DNA Polymerase

DNAPs in E.Coli

DNAP I: Functions in repair and replication

DNAP II: Functions in repair

DNAP II: Principle DNA replication enzyme 

DNAP IV: Functions in DNA repair

DNAP V: Functions in DNA repair

3 actions of DNA polymerase:-

  1. 3’ to 5’ exonuclease activity
  2. 5’ to 3’ exonuclease activity 
  3. 5’ to 3’ polymerising activity

Exonucleases:

Exonucleases are enzymes that removes successive nucleotide bases from the end of a polynucleotide strand

3’ to 5’ exonuclease activity- 

  1. Addition of an incorrect base stalls the action of DNA polymerases-it can’t add the next nucleotide in the sequence 
  2. Exonucleases removes 1 nucleotide base at a time
  3. Polymerase reversing direction (moving from synthesising 5’ to 3’ to 3’ to 5’ and correcting a single base mistake) 
  4. Proof-reading is quite a slow process                                                                        

5’ to 3’ exonuclease activity-

  1. Removes upto 10 nucleotide bases at a time
  2. Removes the RNA primer at the end of replication

DNA POL1 has exonuclease activity (and replication)

  • Exonuclease activity used as a means of proofreading
  • Removes incorrect bases and replaces them with correct ones
  • Also degrades the RNA primer and replaces it with a newly synthesised strand of DNA at the end of replication
  • Also has some replication capabilities but is too slow (600 dNTPs added/minute) and moderately processive (refers to the amount of dNTPs that can be added until the enzyme falls off) to be the principal replication enzyme
  • Functions in multiple processes that require short lengths of DNA segments 

DNA POL2

  • Also participates in exonuclease activity
  • Similar to POL1

DNA POL3 has synthesis activity

  • Main synthesis molecule in DNA replication
  • Synthesises 5’ to 3’ direction 
  • Fast (1000 dNTPs added/sec), highly accurate plus highly processive (can add a lot of nucleotides at a time without dissociating) 

DNA Replication:

Split into 3 parts:

  1. Initiation
  2. Elongation
  3. Termination

Initiation:

– DNA Helicase breaks the hydrogen bonds between the 2 strands to unwind the double helix at replication origins which are rich with A-T pairs

-RNA Primase catalyses the synthesis of a short RNA segment upfield of the DNA polymerase. These short segments are called primers.

-Primers help to expose the 3’ end of DNA so DNA polymerase can bind to the strand and begin synthesising

Elongation:

-DNA polymerase III binds to the 3’ end of the primer. Makes a continuous strand of DNA on the 3’ to 5’ strand (synthesising 5’ to 3’)

-5’ to 3’ strand is referred to as “lagging strand” as helicase unwinds the DNA strand, polymerase has to join on the 3’ end leading to multiple polymerases synthesizing the new strand resulting in okazaki fragments

-Before polymerase can bind to the DNA strand RNA primase must synthesise more primers allowing the binding of polymerases

-Okazaki fragments have to be adjoined by DNA ligase

-DNA Topoisomerase helps keep the original DNA strands unwound so the DNA polymerase can continue to synthesise

Termination:

-The primers (segments of RNA)  present in the newly synthesised strand are stripped away by exonucleases which polymerases replace with DNA 

-More than one helicase acts on DNA when replication takes place, so there are multiple replication forks 

-The 2 okazaki fragments at the place of two forks meeting are all joined by DNA ligase to form 2 continuous DNA molecules (Semi-conservative)

DNA Packaging:

DNA is wrapped twice around histone octomer proteins forming nucleosomes in a ‘beads on a string’ formation. Nucleosomes further foiled into solenoids. Solenoids can further loop and coil into chromosomes.

Total of 10 Histone isoforms:

7 somatic

3 germline

Chromatin:

Chromatin-complex of DNA and histones

2 types of chromatin-Heterochromatin and Euchromatin 

  • Heterochromatin– Highly condensed therefore appears darker 

                               Lacks RNA synthesis

  • Euchromatin– Less condensed therefore appears lighter

                                    Has RNA synthesis

Telomeres:

-Telomeres are the end regions of chromosomes

-Clusters of TTAGGG- can be thousands of base pairs long

-Help protect the integrity of chromosomes + allows replication of extreme ends

-Telomeres slowly eaten away after every division

-Makes the replicated strands shorter as the telomere sequence signals DNA polymerase to unbind and leave the strand. Therefore as replication occurs strands get shorter

-Related to aging

-Telomerase: extends the telomeres of chromosomes 

-Enzymes bind to a RNA molecule with complementary sequence to telomeric repeat

-Extends the overhanging strand of DNA of telomeres using the RNA as a template

-Complementary strand made by normal replication machinery 

Cell cycle:

  • Cell cycle is the sequence of events leading up to and during the process of cell division. 
  • Somatic cells- all the cells in the body that aren’t haploid / gamete cells. Diploid with the full set of 46 chromosomes. 
  • Two main components of the cell cycle: 
  1. Interphase: Cell prepares to divide. 
  2. Mitosis: Actual process of cell division. 
  • Interphase: 

3 main subphases. 

  1. G1: This is when the cell continues to grow and replicate organelles, synthesising proteins ready for DNA replication. 
  2. S: DNA replication. 2 complete copies of DNA are available. 
  3. G2: cell increases in size and produces more proteins. Checks if cell is ready for mitosis
  • M phase: 

2 main components: 

  1. Cell Division
  2. Cytokinesis 
  • After the mitosis phase cells enter G1 and continue to divide once again. 
  • Some cells can stay in a “rest phase” : G0. This is where the cell does not divide or prepare to divide. This is common in skeletal cells and neurons. 

Chromosomes

  • Not visible at interphase. They condense at the mitotic phase – during prophase. 
  • Post replication they contain 2x the normal amount of chromatids. 
  • P arm → short arm
  • Q arm → long arm 
  • Healthy somatic contain 46 chromosomes – 23 pairs. 1 maternal set and the other is a paternal set. 
  • Gametes are haploid – they contain a single set of chromosomes (23 chromosomes). 
  • Homologous chromosomes: have the same gene loci (same region on the chromosome)  but different alleles 

Karyotyping

  • Method of showing all chromosomes under a light microscope. 
  • Looks for number of chromosomes, position of centromeres on chromosomes, sex determining, lebgth of chromosomes, 
  • Chromosomes are taken during the metaphase stage of mitosis. Condensed chromosomes all line up on the metaphase plate. All chromosomes are visible. 

Mitosis: 

  • Single cell divides into two daughter cells, identical to the parent cell and includes the complete genome identical.
  • Important for: 
  • development of embryos
  • Growth
  • Healing
  • Repair of damage 
  • Replacement of damaged cells.
  • Stages: 
  1. Prophase: 
  • Centromeres holding the chromatids together are surrounded by a kinetochore.  
  • Chromatin fibres condense and shorten to form chromosomes.
  • Centrioles move towards the poles (Responsible for spindle fibre production of cells) 
  • Centrosomes consist of 2 centrioles perpendicular towards each other. 
  1. Prometaphase
  • Spindle fibres of centrioles will extend and connect to the kinetochore of the chromosomes. 
  • Nuclear membrane disintegrates, causing the chromosomes to lie free within the cell. 
  1. Metaphase
  • Chromatids are led by mitotic spindle fibres 
  • Chromatids line up on the metaphase equatorial plate 
  1. Anaphase 
  • Centromere and kinetochore split. Allows chromosomes to separate into individual sister chromatids.
  • Microfibres shorten and centrosomes will pull with equal force at opposite poles, allowing full separation of chromatids. 
  1. Telophase 
  • Nuclear envelope reforms 
  • Nucleolus reappears 
  • Chromosomes uncoil into chromatin (no longer so condensed) 
  • Microtubules de-attach from kinetochore
  1. Cytokinesis
  • Begins halfway through anaphase 
  • Results in the splitting of parent cell into 2 identical daughter cells.
  • Late anaphase: contractile ring develops along line previously occupied by the metaphase plate. 
  • Allows the formation of the “cleavage furrow”, stimulates the pinching of the cytoplasm
  • As cleavage furrow deepens the cells will eventually separate with own nucleus, own chromosomes and cytoplasm

Meiosis: 

  • Occurs in germ line cells – produces gametes. 
  • End product is 4 non-identical cells with half the chromosome content of a somatic cell (haploid, 23 chromosomes) 
  • Cells don’t go through G2 phase – stop at S phase of the interphase
  • 2 steps
  1. Meiosis I
  2. Meiosis II
  • Stages
  1. Prophase I:
  • Chromatin Fibres condense and shorten
  • Microtubules extend from poles to the centre of the cell.  
  • Spindle microtubules attach to the kinetochores of each chromosome from the centrosomes
  • Nuclear envelope breaks up and nucleolus diminishes
  1. Crossing Over
  • Exchange of genetic material between homologous chromosomes. Results in recombinant chromosomes during sexual reproduction 
  • Occurs between prophase I and metaphase I
  • Allows genetic variation to occur. Chromatids are held together by the centromere. No longer identical hence after separation daughter cells will continue a combination of both maternal and paternal alleles. 

2. Metaphase I:

– Homologous pairs will line up along the metaphase plate. 

3. Anaphase I:

– Microtubules attached to each kinetochore shorten.  

-Centrioles pull with equal force from opposite poles

-Allow 1 complete chromosome from each homologous pair to be pulled towards each pole. 

4. Telophase I:

– Nuclear Envelope reforms

– Microtubules of the mitotic spindle break up

-Chromatin uncoils and becomes looser as the nuclear membrane is formed 

5. Cytokinesis I:

-Cleavage furrow and causes the separation of the cytoplasm

6. Meiosis II: Exact same steps as mitosis. 

  • Interphase of Meiosis I: 46 x 2 chromosomes = 92 Chromatids
  • End of Meiosis I: 46 Chromatids (genetically variated) 

2 cells

  • End of Meiosis: 23 Chromosomes

4 cells

  • 1 diploid germ cell divided into 4 gametes in males, all ae functional. Female ovum: ¼ of the gametes are functional cells. Other 3 are resulting daughter cells degenerate polar bodies

Errors in Mitosis and Meiosis 

  • Male Gamete Formation
  • Sperm are produced after puberty
  • Production of sperm is continuous and prolific. Each ejaculation contains 100-650 million sperm cells.
  • Occurs within the testes 
  • Spermatogonia are the stem cells that give rise to sperm. Located in the periphery of the seminiferous tubule 
  • Developing sperm move towards the central opening as they undergo meiosis and differentiation
  • 1 spermatogonia = 4 spermatozoa. Spermatogonia is the diploid stem cell. Sperm cells are haploid. 
  • Female gamete formation
  • Development of ova (unfertilised, mature egg) occurs within the ovary. 
  • Oogonia are the stem cells that give rise to ova. Multiply and begin meiosis. 
  • Stops at prophase 1 – here ova are primary oocytes.
  • Puberty causes stimulation of FSH and LH 
  • FSH and LH regulate development, growth, pubertal maturation, reproductive processes of the female body. 
  • FSH and LH is synthesised and secreted by gonadotropic cells of the anterior pituitary gland
  • FSH periodically stimulates the growth of a follicle and induces the primary oocyte to complete meiosis I and start meiosis II. This is a secondary oocyte
  • Halt of meiosis II in oocytes. Continues after the oocyte has been penetrated by a sperm cell , where LH stimulates completion of meiosis II. 
  • Birth of a female: no more oocytogonium, only primary oocytes. 
  • 14th day of the menstrual cycle: primary oocyte turns into a secondary oocyte. Stops at metaphase II till sperm penetration. If fertilisation occurs a zygote is formed, if not degeneration occurs. 
  • 1 reproductive cell and 3 polar bodies. Egg needs nutrients to support life so excess chromosomes are discarded as one section takes everything it needs. 
  • Nutrients are distributed to only one cell. This helps to support ovulation

Mechanisms for mutations in the sperm and egg

  • Women don’t have many germ-line cells. They start with a pool of egg cells that degenerate from puberty. 1 degenerates each month if no fertilisation occurs. 
  • Older eggs are more susceptible to mutations and destruction. This is because they are exposed to more risk factors caused by life-style and environment that can potentially mutate the egg.
  • Spindle assembly mitotic checkpoint occurs just after the metaphase before the anaphase. This checkpoint ensures all chromosomes are lined up by spindles on the metaphase plate. 
  • Inhibits progression of mitosis to anaphase until all homologous chromosomes are aligned to the spindle apparatus. 
  • Unequal separation of chromosomes can lead to trisomy and other disorders 
  • Lifestyle and environmental factors can increase the risk of aneuploidy – presence of an abnormal number of cells. 
  • Most aneuploidy are maternally derived. This is because of prolonged arrest  of oocytes causing reduced activity of the SAC, leading to maternal age linked errors in segregation control. 

Laws of segregation: 

  • Allele pairs separate during gamete formation but unite during fertilization
  • Aneuploidy = an abnormal amount of chromosomes due to improper segregatio post anaphase
  • Trisomies = additional chromosome leading to 3 homologous chromosomes at a particular chromosome instead of 2. 

(2n+1)

  • Monosomies is 1 missing chromosomes. This means there is 1 chromosome instead of a homologous pair. 

(2n-1)

  • Polypody = the total number of chromosomes is a multiple other than 2 (e.g. 3n, 4n, 5n where n=23 chromosomes) 
  • Nondisjunction is the failure of chromosome separation in anaphase of mitosis or meiosis. 
  • Results in a 1:1 ratio of daughter cells with an extra chromosome (2n+1) to those with a loss of a chromosome (2n-1) 
  • If occur during meiosis it is a germ-line mutation and therefore transmissible to the next generation
  1. In Meiosis I : failure of homologous chromosomes to separate 
  2. In Meiosis II: Failure of sister chromatids to split 
  • Nondisjunction occurs due to spindle fibres not attaching properly leading to unequal splitting of chromosomes 

Disorders: 

  • Polyploidy – an uneven gain of a whole haploid set of chromosomes. 
  • Caused by 2 sperms fertilising the same egg. Zona pellucida fails to form a hard protective layer 
  • Incorrect meiotic genesis leading to diploid copies of chromosomes within single oocyte.  Diploid oocyte + haploid sperm = triploidy 
  • Trisomy 21: Down’s Syndrome

– Autosomal, 3 copies of chromosome 21

– Results in varying degrees of mental disability, decreased immunity to diseases and organ defects. Distinguishable facial features. Increased prevalence of leukaemia. Increased incidence of early Alzheimer’s disease 

  • Trisomy 18: Edward’s Syndrome 

– Autosomal: 3 copies of chromosome 18

-Failure of all organ systems, death in a few months, small lower aw, low set ears, overlapping fingers 

  • Trisomy 13: Patau’s Syndrome 

– Caused by 3 copies of chromosome 13

-Multiple congenital abnormalities: polydactyly (more than 5 fingers/toes), holoprosencephaly (prosencephalon fails to develop within the two hemispheres), die within a month. 

  • Monosomy 45: Turner’s Syndrome

– Only 1 X chromosome being inherited 

-Results in a sterile female, short in stature, heart and kidney defects, reduced effects of puberty

  • Trisomy 47: Superfemale 

– 3 X chromosomes inherited. 

– Results in healthy fertile female, delayed motor development, low IQ, delayed speech, abdominal pain 

  • Trisomy 47: Klienfelter’s Syndrome 

– Extra X chromosome in male (XXY)

-Sterile male: taller and less muscular, broader hips, longer legs, larger breasts, weaker bones, small penis and testicles, delay in puberty and less facial and body hair post puberty. 

  • Trisomy 47: Klienfelter’s Syndrome 

-Sex chromosome inherits 2 Ys (XYY)

-Results in a sterile male, violent, decreased intelligence and taller male. 

Non-disjunction of sister chromatids 

  • If nondisjunction occurs during mitosis, after ovum and sperm fuse (post-zygotic), individual will exhibit mosaicism. 
  • Mosaicism: the state of being composed of cells of two genetically different types. 
  • Earlier the mutation that occurs during embryogenesis → greater the number of aneuploid fetal cells 
  • Degree of mosaicism depends on when the mitotic error has occurred. 
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