BABS1201-2 - Google Docs PDF

Preview

Summary

all the notes are good and thorough includes PowerPoint slides info (black) + the extra info the lecture says that aren't written (pink)...

Description

Lecture 15: DNA replication Overview of Lectures -

How is genetic information passed from one generation to the next? How is the genetic information used to direct cellular processes? Textbook reference: Campbell Biology (11th edition) provides an extensive overview and is relevant for this and the following lectures.

LO1: Explain the semi-conservative model of DNA replication. DNA replication -

There is hydrogen bonding between the bases which directs base pairing There are two hydrogen bonds between A and T and three hydrogen bonding between G and C A always pairs with T and G always pairs with C Parental DNA molecule is the DNA that gets copied and replicated, the daughter DNA molecule is the copy/replicate

-

The first step in DNA replication is the separation of the two DNA strands You can still see the parental DNA molecule The missing ones on the right-hand side are known as A always pairs with T and G always pairs with C

-

Each parental strand serves as a template for the order of nucleotides along the new complementary strand. The nucleotides are connected to form the sugar-phosphate backbones of the new strand We now have 2 DNA molecules that are identical in sequence to the original parental molecule but what we have is one parental strand (dark blue) and one daughter strand (light blue) This process is called ‘semi conservative’ as we haven’t lost the parental strands but they’re not joined which means they’re semi-conserved

-

-

Origins of replication -

-

What we know is that in DNA there are specific sequences of AGCT that are recognised by the replication machinery and those are regions that we call origins which is where replication will begin So this is where the two strands will be pulled apart and the whole process will commence We also know that it can proceed in both directions from the same origin of replication There are also multiple origins of replication along the DNA and replication starts at the same time in all of them until eventually, they all meet and this marks the end of the process

-

Multiple origins are needed so that all the DNA in a cell can be replicated in a short time Human cells contain 6 billion bases which are all copied within a few hours In eukaryotes, DNA begins at many sites along the giant DNA molecule of each chromosome Structure of double-stranded DNA - The sugar in DNA is deoxyribose - This sugar has 5 carbons (blue pentagon) - Hanging of the first carbon (1 prime carbon) is where the base attaches - The 3 prime carbon has the OH group hanging from it - 5 prime carbon has the phosphate group hanging from it which connects to the 3 prime of the next carbon sugar - The right-hand side however does that in the opposite direction

-

-

-

-

The two DNA strands are antiparallelthey run in opposite directions with respect to a sugar-phosphate backbone We need to know whether it goes from 5 prime to 3 prime or vice versa due to the DNA polymerase DNA polymerase catalyses the addition of new nucleotides When we’re synthesising the DNA we start from 5 prime to 3 prime because the DNA polymerase is going to take the nucleoside triphosphate and it’s going to join it to the 3 prime hydroxide end We only need 1 phosphate for the DNA backbone however the reason there is 3 phosphates is that we need the energy to join all of the bonds and breaking phosphodiester bonds releases a large amount of energy which is used up in the catalyses of new nucleotides

Requirement of DNA polymerase Must have a 3’OH group to add on to Consequently, - Will only elongate DNA in the 5’-> 3’ direction - Cannot initiate DNA synthesis unless there is a primer (a short piece of RNA) that contains a 3’OH DNA is replicated from 5’ to 3’ end

Leading strand synthesis

LO2 Describe the basic steps involved in the process of DNA replication. -

In the parental DNA, all the bases are hydrogen bonded together and they have to be broken apart by an enzyme called helicase which unwinds the DNA helix - This causes the two strands to move apart - As it moves along, right behind it the strands will happily rejoin as bases will rejoin - This is where the single-strand binding protein arrives and it will only bind to single-stranded DNA, this ensures that the two strands are held apart and don’t just reform - After that, you need to layout an RNA primer and the enzyme primase will lay down the primer - Once the short DNA section RNA primer is laid down the DNA polymerase does not need the 3’ OH group to add on to - Right at the origin of replication where the strands are separated, it can just lay that down - It then moves away and along comes the DNA polymerase which adds the DNA nucleotides based on the sequence that’s on the template strand - It also has a feature called a ‘sliding clamp’ that wraps around the DNA which stops the polymerase from moving away and keeps it synthesising the DNA - The polymerase then continues to keep laying nucleotides on the new daughter DNA strand, along with the template of the parental strands while the helicase moves ahead of it unwinding the helix, the single-stranded binding protein prevents it from rejoining together DNA Replication (leading vs lagging strand) - YouTube An issue -

The two new strands are formed in different ways We know that the DNA synthesises along 3’ to 5’ and that DNA strands are antiparallel We also know that DNA polymerase will only synthesise DNA in a 5’ to 3’ direction So what happens to the 5’ to 3’ section? What happens is that once we unzip enough of the strand we also create a 3’ end in between which synthesize backwards This bottom strand is called the lagging strand The fragments are called Okazaki fragment We cannot unzip the whole thing because the DNA is too long and twisted

Lagging strand synthesis

Summary video: DNA replication: the basics - Bing video

LO3 Describe the function of the major enzymes involved in DNA replication.

DNA polymerase 3 elongate the fragment

to

DNA polymerase 1 for replacement of RNA primer by DNA

Replicating the ends of chromosomes - We know how the whole process starts, however, the ending is a bit of a problem when replicating the ends of the chromosomes. - The problem starts when you need to remove the RNA primer as near the end there is no 3’ OH group - What ends up happening is that the piece just gets shorter - This is a problem as each time you replicate the DNA strand the overall DNA will get shorter and shorter

The solution: Telomeres and telomerase -

-

-

At the end of our chromosomes, there are repeated sequences called telomeres The ends of all chromosomes have telomeres which can be stained with fleuron dye and seen (yellow) The enzyme telomerase can link the ends of a chromosome Telomerase is a special enzyme as it also carries within it some RNA nucleic-acid which it uses as a template to lengthen the end of a chromosome It adds it from 5’ to 3’ This is good because when it gets shortened through the process of replication, it won’t matter anymore because it was already lengthened in the first place However, there is still the issue regarding that telomerase is inactivated in post-embryonic cells

-

Telomerase is inactivated in post-embryonic cells As we get older the telomeres get shorter and shorter This is the reason why cells age and why we end up eventually dying Theoretically, we can reverse aging if we reactivated the telomeres However immortal cells are also cancerous cells because one of the traits of cancerous cells is that they’ve reactivated the telomeres

Nucleotide analogues can be used to treat disease

Lecture 16: Cell Division and Reproduction Lecture 15 recaps: - Genetic material gets replicated and once it’s been copied it then goes from single-stranded molecules to two double-stranded molecules which get moved to the daughter cells to ensure that a cell has the right amount of DNA. Cell division in bacteria- binary fission -

-

Binary 2 fission divide DNA replication commences at the origin of replication Shown in the first e-coli cell The problem that scientists faced was how the cell ensured that the replicated daughter cells each went to the cell Replication of the chromosome continues and each origin becomes attached separately to the plasma membrane. It gets attached at different spots Once replication is complete, the plasma membrane grows inward to separate the 2 new daughter cells and a new cell wall is deposited The origin is still attached Finally, they divide completely

Cell division -

The number of cells in the human is -1 billion per gram of tissue- all derived from a single cell- the fertilised egg So, large numbers of cells must replicate often to produce the adult human. It is essential that this process is precisely regulated

The cell cycle 2 Phases: ● Interphase: growth and replication of cellular components. When the cell needs to divide, it goes into different sub-phases. ○ Growth phase 1 ○ DNA synthesis phase (Lecture 15) ■ When DNA replicates ○ Growth phase 2 ● Mitotic phase: nucleus divides and chromosomes are distributed to daughter cells (mitosis) and the cytoplasm divides into 2 daughter cells (cytokinesis)

Cell Cycle Times -

Cell cycle times vary for different cell types but for most replicating human cells it is between 10 and 30 hours (for E.coli it is 20 minutes)

-

DNA IS REPLICATED, PACKAGED WITH PROTEINS TO FORM CHROMATIN AND IS THEN CONDENSED FOR CELL DIVISION DNA gets replicated Goes from 1 double-stranded helix to two strands of DNA as a helix Each double helix is packaged with proteins into a structure called chromatin. When the 2 parts of the chromatin are held together, they are called sister chromatids. The sister chromatids are held together at the centromere. During cell division, the sister chromatids separate at the centromere to become a chromosome.

MITOSIS CELL DIVISION HAS DISTINCT STAGES IN ANIMAL CELLS (SOMATIC CELLS -non sexual cells)

-

Fluorescently stained cell DNA = blue Fibres that form the spindle fibres = green Cytoskeleton = red

G2 of Interphase: -

The DNA has been replicated (and looks like spaghetti, the DNA is tangled around itself) however it is still in the nucleus within the single cell The DNA needs to be split evenly into the 2 new daughter cells. It is hard to split the DNA in its current form. ANALOGY: If you condense the spaghetti into bowtie pasta, it is easier to split it evenly. This is what is happening when the DNA is being condensed.

Prophase: -

DNA is starting to condense, still inside the nucleus. Individual chromosomes are starting to form. The spindle fibres are starting to extend from the centrosomes. Each centriole is moving farther away from each other to the opposite sides of the cell, further extending the spindle fibres.

Prometaphase: -

Just before metaphase The nuclear membrane is degraded to free the chromosomes The spindle fibres are attaching to the centromere region of the chromosome. Both centrioles (at this stage called spindle poles) are at the opposite ends of the cell.

Metaphase: -

The chromosomes are lining up at the equator of the cell still attached to the spindle fibres are the centrosome. ⇒ They are lining up as each chromosome is attached to fibre from the north pole and fibre from the south pole.

-

Each spindle fibre has equal pressure, so the chromosomes line up in the middle. The cell won’t go to the next step until all the chromosomes are lined up in the middle. This is to prevent a cell from having an extra chromosome and another cell missing one chromosome.

Anaphase: -

Once the chromosomes are lined up, a special protease chews through the protein that is holding the chromosomes together at their centromeres. The pulling pressure from the north and south spindle fibres pulls each half of the chromosome to the north or south side of the cell. They get pulled apart

Telophase: -

The cytoskeleton is reconfiguring to allow the cell to separate. A nucleus then starts to form in each separated cell along with the nuclear envelope. The chromosomes then start to decondense within the newly formed nucleus and nuclear envelope. The two new daughter cells are starting to form

Cytokinesis -

Cyto means cell, kinesis means movement There is a contractile ring of microfilament fibres which start to move into the centre of the separating cell in order to separate the cell to create 2 new daughter cells. In plant cells, it is more complicated as they have solid cell walls which give their structures. Once DNA has divided, cell wall material begins to form between the 2 nuclei to form a cell plate. This then fuses with the outer part of the cell wall, thus creating two new daughter plant cells.

Apoptosis- programmed cell death -

Apoptosis removes unwanted cells during development It also removes damaged cells throughout life If these damaged cells weren’t removed they would continue to replicate leading to cancer cells

Meiosis -

-

Somatic cells (all cells except sperm and ova) have 2 x 23 = 46 chromosomes (2n) For us the n is 23 for other species it differs Gametes (perm and ova) have half this number (n) so that when they fuse at fertilisation, the correct chromosome number will be maintained The process for producing cells with half the number of chromosomes (2n ⇒ n) is called meiosis.

Interphase 1 of Meiosis: -

-

2n chromosomes (homologous pair of chromosomes, 1 chromosome from mum, 1 chromosome from dad) within the parent cell are replicated to create 4n chromosomes (homologous pair of replicated chromosomes. The chromosomes from mum and dad are replicated to have sister chromatids)

Meiosis 1: -

The cell separates which halves the chromosomes back to 2n. One chromosome from dad in 1 daughter cell, 1 chromosome from mum in the other daughter cell.

Meiosis 2: -

The separated cells separate again to halve the chromosomes to 1n = 23 chromosomes.

Homologous: -

2 copies of chromosome 1, one copy inherited from mum, one copy inherited from dad. We say that they are homologous which means that the sequence of genetic material information lined up along that chromosome is the same on both. The genetic material is coding for the same information on that particular gene.

Diagram summary - During the meiosis 1 process we need to make sure that we get 2 copies of chromosome 1 and 2 copies of chromosome 2. And we need to make sure we get a copy of each in each gamete. - Prophase 1: chromosome 1 needs to find the other chromosome 1. Same with chromosome 2. The homologous chromosomes pair up and line up. - Anaphase 1: The homologous chromosomes are separated - Telophase + Cytokinesis: the homologous chromosomes are separated into daughter cells - The next step is interkinesis: between cell divisions. The DNA is not replicated here. - Meiosis II: once again they line up in the middle - Prophase II: The chromosomes don’t need to line up as there is only 1 copy of chromosome 1 with the sister chromatids attached. The same for chromosome 2. - Metaphase II & Anaphase: The chromosomes line up and the sister chromatids are separated. - Telophase II & Cytokinesis: 4 new gametes created with 1 chromatid from chromosome 1 and 1 chromatid from chromosome 2 in each gamete. Genetic variation arises during the process of meiosis

-

-

The reason the separation of the chromosomes happen into 4 gametes is due to genetic variation Humans have this process which engenders genetic variation so that we can respond to environment changes There are 4 different combinations of chromosomes due to meiosis. This is why we don’t look exactly like our siblings. Random assortment E.g. if there were 2 chromosomes from the dad and 2 chromosomes from the mum, there can either be 2 dad chromosomes in a gamete, 2 mum chromosomes in a gamete, 1 dad and 1 mum chromosome, 1 mum and 1 dad chromosome. This gives genetic variability. If a person looks more like their dad, then they have gametes with just the chromosomes from the dad. If a person had their dad’s eyes but mum's hair, they have gametes with a chromosome that programs the dads eye colour and a chromosome that programs the mums hair colour.

Crossing over increases the variability - When homologous chromosomes pair up, what can happen is that the ends of the chromosomes that are near each other can swap genetic information. - It's still the same trait, but they have swapped genetic information. - When they separate during meiosis ⇒ there is 1 gamete with the light orange with the dark yellow, 1 gamete with the dark orange dark yellow, light orange light yellow, dark orange light yellow. - More genetic variability is produced as the ends of the chromosomes can cross over when they pair up. - This creates gametes with more genetic variability. - Process is called crossing over -

-

-

Crossing over happens mostly with gene traits that are located at the very ends of chromosomes because they are the easiest to cross over. The closer it is to the centromere region, the harder it is for the process to occur as it is anchored very tightly. The chiasma is the site where the crossing over occurs. Crossing over between the ends of the chromosomes produces recombinant chromosomes which now combine information inherited from each parent.

SUMMARY: Mitosis occurs in somatic cells Meiosis is only in special process germ cells ⇒ increasing genetic diversity

Lecture 17: Gene expression I Gene expression - DNA contains the instructions for how a cell will function - Proteins perform these functions - How are the instructions translated from the nucleotide language of DNA into the amino acid language of proteins? - Via an intermediary - RNA - Central dogma: DNA ⇒ RNA ⇒ Protein - A dogma is a universal concept that we can rely on - DNa can be expressed into a protein via an RNA - However there are some exceptions bacteria carry DNA in the form of RNA - DNA encodes RNA, RNA encodes protein DNA vs RNA

DNA -

RNA Double stranded Deoxyribose (without the oxygen Thymine instead of uracil (Thymine has a CH3 group instead of H) DNA more stable than RNA so it can function in our cells through multiple rounds of replication

-

Single stranded Ribose sugar (with oxygen) Uracil instead of thymine (Uracil has H group instead of CH3) RNA is used transiently so it is less stable

Transcription/Translation -

DNA is transcribed into messenger RNA (mRNA), using base pairing. Same nucleic language but in a different form. This mRNA is translated into a protein. Nucleic acid language is changing to an amino acid language. Transcription ⇒ same language, different form. 3-letters on the mRNA makes a codon which encodes 1 amino acid. The code is a triplet code - 3 nucleotides code for each amino acid.

The genetic code Why a triplet code? - 4 bases can’t code for 20 amino acids. - Duplex code: 2 bases would code for 1 amino acid. There would be 16 possible combinations which is still not enough.

-

-

Therefore a triplet code is correct. 4^3 = 64 possible combinations. The code is redundant as it has more coding power than is needed. The third base is redundant, e.g. CCC = proline but CCA, CCG, and CCU also equals proline. The code was decoded by using synthetic mRNAs and translating them in vitro. UAA, UAG, and UGA a...