OBM1 Cell och Molekylärbiologi PDF

**OBM1** **Cell och Molekylärbiologi** ============================= **Mål/Goals of the course** - - - - - **Content of the course** Cell Biology, Molecular biology and Laboratory methods. - - - - - - - **Cell Structure and microscopy** **What is life** Cells are...

**OBM1** **Cell och Molekylärbiologi** ============================= **Mål/Goals of the course** - - - - - **Content of the course** Cell Biology, Molecular biology and Laboratory methods. - - - - - - - **Cell Structure and microscopy** **What is life** Cells are the fundamental unit of life, they are different in shape and size and chemical requirements. All cells have similar basic chemistry. The central dogma, DNA is made from 4 different nucleotides (A,G,C,T) RNA: Made from 4 different nucleotides. Similar to DNA except U instead of T. Protein is built from amino acids (20 different) From DNA TO RNA TO Protein. Life is an autocatalytic process. DNA and RNA provide the sequence information that leads to proteins later being made. Thereafter catalytic activity needs to synthesize nucleic acids and themselves. **Cell theory** states that all organisms consist of one or more cells, The cell is the basic unit of structure for all organisms. All cells also arise only from preexisting cells. **Cells under the microscope.** Z. Janssen/H. Janssen was the first to make the first useful light microscope. M.Knoll/E.Ruska Invented the electron microscope which made it possible to view detailed structure of the cells, The light microscope uses visible light to illuminate specimens and glass lenses to form an image. Electron microscopes use beams of electrons as a source of illumination and electromagnetic lenses to form an image. So for light microscope we can see beyond cells but more specifically organelles With an electron microscope we can start seeing molecules. **The cell structure.** There are 30 trillion human cells in an adult human being. There are 200 major cell types. More than 20 cell types are involved in tooth development/tooth formation. The cell has main parts: A membrane that defies the boundary of the cell and a chemical processing unit. There is a division of cellular organization into 2 broad groups. 1. "Organisms without a cell nucleus or any other membrane bound organelles" Bacteria Archaea (survive +65oc) 2. "Cells organized into complex structure by internal membranes and a cytoskeleton" Eukaryon = "True nucleus" Animal, plants and fungi **Comparison between prokaryote and eukaryote cells** Eukaryote and larger than prokaryotic Eukaryote has membrane bound organelles which prokaryote does not Eukaryote has microtubules which prokaryote does not This phenomenon is the same with intermediate filaments and exocytosis and endocytosis, eukaryotes have and prokaryotes do not. The cell division is fission for prokaryote and for eukaryotic mitosis and meiosis. For genetic information Prokaryote has dna bound with few proteins while eukaryote has dna bound with histones to give chromosomes. The RNA processing is little in prokaryote while eukaryote has a lot. They differ in ribosomes. Looking at function, prokaryote has single identical entities while eukaryote specialize for certain functions. **Prokaryotic cells** - - - **Eukaryotic cells** - **Plasma membranes functions** - - - - - - **Nucleus** Nucleus is the information center, where most genetic material is stored. Not all because some of it is in the mitochondria. The nucleus is the most prominent organelle. The nuclear envelope separates the nucleus from the rest of the cellular organelles. It has an inner and outer membrane that is divided by perinuclear space. Nucleus is where ribosomal RNAs are synthesized and combined with proteins to form ribosomes. **Mitochondria** Site where most of the energy is composed. Has an outer membrane, intermembrane and an inner membrane. It is a large organelle. - - - Many hundreds per cell and can indicate function, high demand for ATP as energy can be expected to have large numbers of mitochondria. **Endoplasmic reticulum** Has only one membrane. It consists of interconnected tubular membranes and flattened sacs. There are 2, Rough ER and the Smooth ER. The rough ER has ribosomes on the membrane that faces the cytosol. Ribosomes actively secrete proteins , which are transported into or across the membrane they form. They are involved in the synthesis of proteins. Since the smooth er does not have ribosomes, they are not involved in the synthesis of proteins. Instead they are involved in biosynthesis of steroids, such as cholesterol and the steroid hormones derived from it. Biosynthesis of membranes, particularly lipids. Detoxification of a variety of organic molecules including alcohol. The function of it depends on the location of the cell. If it's in the liver then it's involved in carbohydrate metabolism. If it's in the muscle then it's involved in storage and release of ca ions that trigger contraction. **Golgia apparatus** Consists of a stack of flattened sacs called cisternae. It has a role in processing and packaging of secretory proteins and synthesis of complex polysaccharides. Whatever goes in is being modified and shipped out. Vesicles that bud off from ER are accepted by golgi complex. The process of coming in is endocytosis, the opposite is exocytosis to export out of the cell. Contents passed onto other components of the cell by vesicles budding from the golgicomplext **Lysosome** Responsible for intracellular digestion. It contains acid hydrolases. The lysosome releases nutrients from ingested food. It's also involved in degradation of unwanted molecules for recycling or excretion. **Relevance to dentistry lysosome** Chediak-higashi syndrome: Mutation in the lysosomal trafficking regulator leading to abnormally large intracytoplasmic granules. This leads to early onset for severe gingival inflammation, Tooth exfoliation or deep probing affecting dentition. **Peroxisome** Contained environment where oxidative reactions are used to degrade lipids and destroy toxins. **Ribosome** An organelle not surrounded by a membrane. It has a large complex made from dozens of ribosomal proteins and several ribosomal RNA:S. It is composed of a large subunit and a small subunit. Units come together to synthesize RNA to make a protein. More than 100k ribosomes in eukaryotic cells. They are also present in mitochondria where they produce organelle-specific proteins. They have a central role in protein synthesis. Orientation of the mRNA and amino acids carrying tRNAs in proper relation to each other. Catalyzing the formation of peptide bonds to link the amino acids into a polypeptide. **Cytoplasm** Portion of the interior of the cell that is not occupied by the nucleus. It includes all organelles and the cytosol **Cytoskeleton** Important in cell movement, division and migration. It has an internal framework of filaments and tubules to give distinctive shape and a high level of organization. There are 3 major structural elements. Microtubules, Microfilaments, Intermediate filaments. **What is the minimal requirement of a living cell** **An example of relevance to dentistry - pemphigus** Autoimmune disease in which autoantibodies target desmogleins (found in desmosomes) Desmosomes are important for maintaining intercellular adhesion in stratified squamous epithelia. **Different microscopes and what we use them for** To analyze cells or tissue, there needs to be a stain with dyes to visualize the tissue or cell structure. You can identify proteins or structures of interest by staining tissues with various dyes for different structures. Then you can use antibodies to detect different proteins through enzyme reactions or fluorescent probes (molecules) **Light microscopy** Magnification of cells up to 1000 times Three requirements: A bright light must be focused onto the specimen by lenses in the condenser The specimen must be prepared to allow light pass through it an appropriate set of lenses must be arranged to focus an image of the specimen in the eye. **Fluorescence microscopy** Use of fluorescent dyes to stain cells Illuminating light passes through two sets of filters 1. 2. **Online lecture 1.2 Genes, Chromosomes & Chromatin & 1.3 DNA & replication** **The structure of DNA** - - - - - - - - - - - - - - - - **DNA Packaging** - - - - - - - - - - - - - - **DNA & Replication** **Seminar 1 - Genes, Chromosomes and DNA** **Chapter 5: DNA and Chromosomes** **1)What is a gene, the genome, genetic code, an allele, a chromosome, a chromatid, sister** **chromatids, double-helix, nucleosome?** A gene is a sequence or a segment of a DNA that contains instructions for making a particular protein or RNA molecule A genome is the entire set of DNA instructions found in a cell An allele is one of two or more versions of DNA sequence (a single base or a segment of bases) at a given genomic location. An individual inherits two alleles, one from each parent, for any given genomic location where such variation exists. If the two alleles are the same, the individual is homozygous for that allele. If the alleles are different, the individual is heterozygous. A chromosome is a thread-like structure made up of DNA. It's in the nucleus and is a package of DNA information A chromatid is one of two identical halves of a chromosome that has replicated to be ready for cell division A sister chromatids it's the other half of the identical chromatid. Nucleosome **2)How is the structure of the DNA (which molecules, how are they held together)?** A DNA molecule consists of two long polynucleotide chains. Each chain is composed of four nucleotide subunits. The two chains are held together by hydrogen bonds between the base portions of nucleotides. The nucleotides are composed of a nitrogen containing base and five-carbon sugar, to which a phosphate group is attached. The sugar for the nucleotide in the DNA is deoxyribose. The base pairs are adenine to cytosine and guanine to thymine. **3)Which bases pair together? How are they paired?** A pairs with T and G paired with C. They are paired together through a hydrogen bond. Depending on pair it's either 2 or 3 hydrogenbonds. **4)Define anti-parallel, complementarity, chemical polarity** Each DNA strand has a chemical polarity because the ester linkages to the sugar molecules of either side of the bond are different. The nucleotide subunits within a DNA strand are held together by phosphodiester bonds that link the 5ʹ end of one sugar with the 3ʹ end of the next The DNA strands, the nucleotides are antiparallel because they go in the opposite direction. The're parallel and do not overlap but go towards different direction When you look at a base-pair, A always pairs with T and G with C. In each case, a bulkier two ring base is paired with a single ring base (purine with pyrimidine). Each pair is a base pair. Adenine and guanine are purine while cytosine and thymine are pyrimidine. Because each base-pairing has a specific pair, it's complementary because it enables the base pairs to be packed in the energetically most favorable arrangement along the interior of the double helix. In this arrangement each base pair has the same width, holding the sugarphosphate backbones an equal distance apart. the complementarity is crucial when it comes to copying and maintaining the DNA structure. **5)How many gene products (mRNA, protein) are usually encoded in a eukaryotic gene? / in a** **prokaryotic gene?** **6)What is chromatin? What is the difference between euchromatin and heterochromatin? How** **is the DNA packed in these two states?** Chromatin is a mixture of DNA and proteins that form the chromosomes found in the cells of humans or higher organisms. DNA is formed into proteins that is divided into two classes called histones and nonhistones. The highly condensed form of interphase is called heterochromatin. It makes up to 10% of an interphase chromosome. The rest of the interphase contains euchromatin. Both chromatines are composed of mixtures of different chromatin structures. **7)What is karyotyping? Which chromosome aberrations can be detected by karyotyping?** karyotyping is the order full set of the 46 human chromosomes. If parts of chromosomes are lost or switched between chromosomes, they can be detected. **8)What are histones? How many different types of histones are there and what do they do?** Histones are proteins that bind to DNA to form eukaryotic chromosomes. **9)What is the replication of origin? The centromere? The telomeres?** **10)How many chromosomes does a somatic human cell have in total? How many different chromosomes does a somatic human cell have?** **Chapter 6: DNA Replication, Repair, and Recombination** **1)What is replication?** For each cell division, a cell has to copy its genome with accuracy. So replication is the process in which the genome DNA is copied in cells. Before the cell divides it must copy or replicate the entire genome. **2)Explain conservative versus semi-conservative replication?** **3)What is a replication origin? What are replication forks?** The replication origin is the process begun by initiator proteins that bind to specific DNA. In this phase, the initiator proteins pry the two DNA strands apart, breaking the hydrogen bonds between the bases. DNA molecules in the process of being replicated contain Y shaped junctions called replication forks. These forks are formed at each replication origin. AT each fork, the replication machine moves along the DNA opening up the two strands of the double helix and using each strand as a template to make a new daughter strand. The two forks move away from the origin in opposite directions, unzipping the DNA double helix and copying DNA as they go. The replication fork is asymmetrical. The reason why is because the 5' to 3' direction poses a problem as the sugar-phosphate backbone of each strand has a unique chemical polarity determined by the way each sugar residue is linked to the next, and the two strands in the double helix are antiparallel as they run in opposite direction. Because of this at each replication fork, one new dna strand is being made on a template that runs in one direction (3' to 5') while the other strand is being made on a template that runs in the opposite direction (5' to 3') **4)Which enzymes are needed for replication? What do they do?** The enzyme that is needed for replications is called DNA polymerase. The reason is because the replication fork is driven by the action of the replication machine. This enzyme catalyze the addition of nucleotides to the 3 primer end of a growing dna strand. **5)What is meant by "Direction of replication"? "Continuous versus discontinuous replication"?** **"Leading versus lagging strand"?** **6)What are Okazaki fragments? How are they joined together?** **7)What enzymatic functions does the DNA-Polymerase II have?** **8)Which molecule is the primer comprised of? Which enzyme produces the primer?** **9)What is telomerase? Why is it important? Which cells express telomerase?** **10)Most common types of base errors? How do they occur?** **11)Explain mismatch repair** **12)What is the consequence of a double-strand break? Non-homologous end-joining versus** **Homologous Recombination? In which instances is one better than the other?** **Seminar notes** Nonhomologous repairing. This repair happens when the two strands of the double helix are broken at the same time. This causes a problem because normally if a one strand breaks, leading to the nucleotides on that strand being broken, they can be repaired by using the information of the complementary strand. Reasons why the two strands of double helix being broken can be because of mishaps at the replication fork, radiation and chemical attack. Because this is a serious problem, cells have developed strategy to repair. One way is by quickly sticking the broken end of the strands together, this is called nonhomologous repairing. It is carried out by a special group of enzymes that clean the broken strands and rejoin them by DNA ligation. This repair is called quick and dirty because it seals the brake and repairs it fast. However, it poses a problem as when the brake is being cleaned, nucleotides could be lost at the site of the repair which leads to the cell potentially suffering some bad consequences. This strategy is quick but risky for fixing broken chromosomes. So when the strands are damaged in the double helix, the strands are quickly repaired by a nuclease that chews back the broken ends to produce flush ends. Thereafter a Ligase joins the two strands together. Problem: Some nucleotides could be lost in the reparation process. Protein called KU binds in both sides of the strandbreak, 1. 2. 3. 4. **[Molecularbiology]** ================================== **Structure of DNA** ==================== - - - - - - - - - - - Chromatin can be condensed into 2 forms 1. 2. Whenever cells undergo lots of replication, they want to allow the chromatin to be passed on to the daughter cells. Therefore chromatin during cell replication condenses to form chromosomes. They condense to chromosomes to prevent the DNA tangling and damage during cell division. DNA gets so condensed because histones which are octamere are positively charged are made of lysine and arginine while DNA is negatively charged because of the phosphate groups. They attract and make it compact structure. A chromatid is one of the two identical halves of a chromosome that has been replicated in preparation for cell division. The two "sister" chromatids are joined at a constricted region of the chromosome called the centromere. So what is DNA? - - **Nitrogenous bases** - - **Pentose sugars** - - - - **Phosphate group** - - - SO A Nucleotide is all of this together.In the pentose sugar the 1st carbon connects to a nitrogenous base, on the 5th carbon there is a phosphate group. Nucleotides together make up a DNA structure. **Complementarity** - - These interact with each other through hydrogen bonds that link the bases together. For A and T there are 2 hydrogen bonds and G with C 3 hydrogen bonds. The hydrogen bond between A and T is easier to break because it consists of 2 hydrogen bonds opposed to the other with 3. This comes to play in DNA replication. **Antiparallel** DNA has an ANTIparallel arrangement. This means that on one end, it's arranged from 5' to 3', the other has to be arranged the other way from 3' to 5'. They are oriented in the opposite way because of complementarity. We know adenine wants to react with thymine and guanine with cytosine. If one side isn't oriented the opposite way then purines will be together and the pyrimidines together which is not optimal for bonding. There is a sugarphosphate backbone. This backbone is made up of a bond called phosphordiesterbond, a strong covalent bond. This bond is formed between the 5' (carbon) end of one and the 3'(carbon) end of the other nucleotide. On the 3' end of the carbon in the sugar there is an OH group and 5' end is a phosphate. So the 3' end and the 5' end together makes a strong phosphodiester bond combining all nucleotides together. These ends are in every nucleotide. Combining the ends makes a whole nucleotidebond. **B) DNA replication** **What is the point of replication?** In order for cells to be replicated and make more cells, we need the DNA within the cells to replicate. DNA is what makes the cell what it is. So the whole purpose is to allow for cell replication (cell cycle). DNA replication happens in the S phase of the cell cycle. To simplify it, you're taking old parental strands, separating them and making new dna strands that are complementary to them in a semi-conservative process. Semi-conservative process means taking and making a mixed old and new double stranded DNA molecules. DNA replication has to occur in the specific direction. It always has to occur from the 5' end to the 3' end. When adding nucleotides, you're adding phosphategroup onto the 3' prime group of the preceding nucleotide. DNA replication is bi-directional. When we separate the two parental strands to create DNA, we create ends called replication forks from both directions. There are going to be enzymes called helicases to unwind the DNA on both sides moving in opposite directions. Then Enzymes called.DNA polymerases are going to move into these areas and follow the helicase to synthesize new DNA off the parental strand in a bi-directional fashion. **3 STAGES OF REPLICATION** [Stage 1. Initiation of DNA replication] - - - - - - - [Stage 2. Elongation of DNA] - - - - - - - - - - - - - [Stage 3. Termination of DNA replication] - - - - - **Extra about DNA polymerase,** it proofreads from 3' to 5' to see if it has made any mistakes and corrects it. **RNA TRANSCRIPTION and TRANSLATION** **TRANSCRIPTION** DNA transcription is a simple thing. It's taking DNA, double stranded, and converting to RNA. This happens in prokaryotic and eukaryotic, however the process is different. [Prokaryotic cells] - - - - - - [Eukaryotic cells] - - - - - - - - - - - [Gene regulation in Eukaryotic cells] - - - Steps of transcription 1. +-----------------------------------+-----------------------------------+ | [Prokaryotic ] | [Eukaryotic] | +===================================+===================================+ | - | - | | | | | | | | | | | - | | | | | | | | | | | | - | | +-----------------------------------+-----------------------------------+ | RNA holopolymerase | RNA polymerase (II) to make mRNA | | | that will be used to translate | | | into proteins | +-----------------------------------+-----------------------------------+ | | | +-----------------------------------+-----------------------------------+ | | | +-----------------------------------+-----------------------------------+ +-----------------------------------+-----------------------------------+ | [Prokaryotic] | [Eukaryotic] | +===================================+===================================+ | - | | | | | | | | | | | | - | | | | | | | | | | | | - - - - | | +-----------------------------------+-----------------------------------+ [3, Termination of transcription process] It's where we have made the RNA transcript and basically have to detach it away from the DNA and prevent the RNA polymerase from reading. [Prokaryotic] [Eukaryotic] -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- In prokaryotic cells there is an RHO dependent mechanism. RHO-protein that causes the RNA polymerase to break away from the DNA As the polymerase makes RNA it hits a sequence where when it reads DNA it makes a sequence of AAUAAA. This is called a polyadenylation signal. It is being synthesized by the RNA polymerase. It activates a particular enzymes that will cleave the RNA from the RNA polymerase and the DNA. The next mechanism is RHO-independent termination. There is no RHO protein. RNA polymerase bind to template strand, it encounters an RNA sequence called inverted repeats that makes a hydrogen bond reaction between them causing them to loop. The loop signal brings particular enzymes to break the RNA from the RNA polymerase. [4. Post Transcriptional modification] - - - - - - - - - - - - - - - - - - - - - - - [How the SNRP work] - - - - - - - - - - [Alternative RNA splicing.] This give variants of a protein [RNA editing] **[RNA TRANSLATION/Protein synthesis]** Translation is taking mRNA from transcription to make proteins. [Genetic code] [mRNA] mRNA or messenger RNA. It has a specific sequence of nucleotides that are in triplet forms. In one end there is a 5' end in the and a 3' end. The triplet is called codons, they are triplets of nucleotides. Nucleotides of RNA are different from the nucleotides of DNA. Why? because of the nucleotides that contain different nitrogenous bases or to specific 1 different.The nitrogenous bases are Adenine, guanine, cytosine and Uracil. There are 4 different types of nucleotides. There are only triplet sequences. 4 to the power 3 means that there are 64 different types of codons. There are 64 codons , 61 of them read. The codons consist of nucleotides with triplet nitrogenous bases. For example, AUG codes for an amino acid called methionine. So 61 codons code for an amino acid. The 3 other codons don't code for an amino acid. They code for terminating the translation process. They are called stop codons. UGA,UAA and UAG will not give a particular amino acid and instead stop the process. mRNA contains codons, codons are made of nucleotides, nucleotides contain 4 different nitrogenous bases A,G,C,U. 64 codons exist, 61 code for an amino acid the rest stop the translation process. [tRNA (transferRNA)] - - - [Characteristics of genetic code] For most part you go from 5' to 3' continuously. The translation process is called commaless. It is commaless because if there are a couple of nucleotides between a codon, it doesn\'t skip the nucleotides, it still reads it. The only exception to it is viruses. The genetic code is non overlapping. The only exception is that it can occur in viruses. Non overlapping means that when it reads the codon, it doesn't start again from the 2nd nucleotide on the codon. It just goes from a codon triplet to another codon triplet. The genetic code is redundant and degenerate. Some amino acids have different codons. You can't track the amino acid back to the specific codon. The only exceptions are methionine and tryptophan. In methionine there is only one codon, and tryptophan there is only one codon. All other amino acids have multiple codons that code for it. There is something called the wobble effect, that is particular for tRNA. The tRNA has a 5' end that goes to the 3' end. On the first position of the 5' end there is something called inosine in the anticodon. Inosine is complementary to adenine, uracil and cytosine and gives the same amino acid. The wobble effect decreases the risk of mutation, because if there is any mutation of the DNA, there will be mutation in the mRNA which then leads to changes or substitution in the codons. If you switch up the codons there will be changes in amino acids. If there is an Inosine the risk of mutations decreases as it's complementary to all other nitrogenous bases which leads to it coding the same amino acid. [tRNA structure charging] T-arm is closest to the 3' it tethers the tRNA to the ribosome. The D-arm allows for identification of the tRNA by the synthetase enzyme.  [Ribosomes] **Eukaryotes** - - - **Prokaryotes** - - - - - Ribosome = rRNA + proteins **Phases of translation** 1. +-----------------------------------+-----------------------------------+ | Prokaryotes | Eukaryotes | +===================================+===================================+ | - | - | | | | | | | | | | | - - | - | | | | | | | | | | | - | - | | | | | | | | | | | - | - | | | | | | | | | | | - | - | | | | | | | | | | | - | - | | | | | | | | | | | - | - | +-----------------------------------+-----------------------------------+ 2. This is the same for Eukaryotic cells and prokaryotic cells. This example is for Eukaryotic. There is an A, P and an E site. A is the arrival site, p synthesis site and E, exit site. - - **Elongation Factors\ **In elongation, elongation factors (EFs) participate, proteins that associate with the ribosome and facilitate the correct binding of tRNA to the different binding sites of the ribosome. The elongation factors also contribute to proper codon reading and help keep the error rate low. Without these, approximately every thousandth codon would be misread, leading to protein defects. **Codon Reading\ **Codon reading occurs at the A-site (aminoacyl site) of the 40S subunit. Here, a codon on the mRNA molecule pairs with the corresponding anticodon on an amino acid-carrying tRNA molecule. There are a total of 64 codons, 61 of which code for amino acids, and three function as stop codons. 1. 2. 3. 4. 5. 6. 7. 8. To terminate translation, three stop codons are used: UAG, UAA, and UGA. These do not code for an amino acid but trigger a signal that causes the binding of a release factor to the A-site. In eukaryotes, this release factor is called eRF1. eRF1 prevents further tRNA molecules from binding to the A-site. Subsequently, other release factors bind to the ribosome, leading to the dissociation of the two subunits from the mRNA molecule. Once the ribosome breaks apart, the newly synthesized polypeptide chain and the mRNA molecule are released. The mRNA strand will then be degraded. For a functional protein to be produced, the newly formed polypeptide chain usually needs to undergo post-translational modifications **Posttranslation modification** Many polypeptides are modified before they become functional proteins.\ This can occur while they are still being synthesized by the ribosome (cotranslationally) or after the synthesis is complete (posttranslationally). Modifications can involve the removal of parts of the translated protein or the attachment of molecules to the polypeptide. These changes affect the protein\'s structure, stability, localization, and function. Trimming\ Some proteins are synthesized as large, inactive precursor molecules. One example is proinsulin, which must be cleaved into smaller components to become the active hormone insulin. The cleavage is performed by enzymes called endoproteases. The location of this process varies between proteins. Some undergo trimming in the rough endoplasmic reticulum (RER) or Golgi apparatus, while others are cleaved after being secreted from the cell. Addition of Molecules\ A polypeptide can be modified by covalent attachment of different molecules. These processes can occur in various places in the cell and at different times. Common modifications include: - - - - - - Protein Folding\ After synthesis, the polypeptide must fold into its correct structure. This can occur spontaneously, mainly due to the hydrophobic effect and the formation of various intramolecular bonds. However, some proteins need the help of other proteins, called chaperones, to fold correctly. A chaperone is a protein shaped like a barrel. The unfolded polypeptide is placed inside the chaperone, where it can assume its proper structure without interference from external factors. Protein Degradation\ The path from DNA to protein involves many steps, and despite strong regulation, errors can occur. Proteins that are faulty for any reason, such as improper folding, must be degraded. A cell filled with non-functional proteins cannot survive. For proteins to be broken down, they must first undergo ubiquitination. The protein is tagged with small proteins called ubiquitins via covalent bonding. The ubiquitins are recognized by a protein complex called the proteasome. The proteasome is an ATP-driven proteolytic system responsible for protein degradation in the cell. In the proteasome, peptide bonds are broken down, and the amino acids can be reused for the synthesis of new proteins. 1. 2. - - - When a protein is meant for the ER or for secretion, its translation begins in the cytoplasm, but as the ribosome synthesizes the protein, it recognizes a signal sequence, and the ribosome is directed to the rough ER. The ribosome then attaches to the ER membrane and continues translation, with the newly made protein either inserted into the ER membrane or translocated into the ER lumen. So, **translation** happens in both the **cytoplasm** and the **rough ER**, depending on where the protein is destined to go. **Seminar** The central dogma of molecular biology is a theory stating that genetic information flows only in one direction, from DNA, to RNA, to protein, or RNA directly to protein. Operons are short sequences of regulatory gene segments found only in the genetic material (DNA) of prokaryotic cells (bacteria and archaea). Operons function to activate or deactivate gene expression in response to environmental triggers, called inducers/promotors or repressors **DNA repair** There are 5 major DNA repair pathways 1. 2. 3. 4. 5. **Basic excision repair** Base excision repair (BER) is the most common DNA repair mechanism in the cell. BER involves removing an incorrect base and replacing it with the correct one. This method is used, among other things, to replace bases that have been altered due to deamination. When only the incorrect base is replaced, it is called short-patch BER. If instead, a longer nucleotide sequence (up to ten nucleotides) is replaced, it is referred to as long-patch BER. The BER process proceeds as follows: 1. 2. 3. 4. 5. ##### Nucleotide excision repair (NER) can recognize more types of DNA damage than BER. NER is often applied when damage has affected multiple nucleotides simultaneously. NER is primarily activated by UV-induced pyrimidine dimers or when large side groups have bound to nitrogenous bases. The damage is repaired by cleaving away an approximately 30 base pair-long oligonucleotide and replacing it with undamaged bases. NER can occur independently of, or be coupled with, transcription. **Transcription coupled repair (TCR)** occurs in the following steps: 1. 2. 3. 4. 5. **Global genomic repair (GGR)** can occur at any time. The process is largely similar to TCR. The difference is that the process does not take place during transcription, and other proteins, XPA and XPC, recognize the damage. XPA and XPC are proteins that constantly move along the DNA molecule to detect damage. Afterward, the same procedure follows as in steps 3-5 in TCR. **Mismatch repair** Mismatch repair (MMR) is used to remove bases that have been incorrectly inserted during DNA replication. Although DNA polymerase has a built-in proofreading function, some errors still occur in the process. These bases are recognized by the proteins in the MMR complex. MMR has been primarily studied in the bacterium *E. coli*, so this process will be described here. The principles are the same for eukaryotic cells, but the exact enzymes involved are not fully understood. After replication, the newly synthesized DNA strands are scanned by specific MMR enzymes. When a mismatch is detected, the following occurs: 1. 2. 3. 4. **Homologous recombination** Homologous recombination is usually only used during meiosis. Its purpose is to facilitate the exchange of genetic material between two sister chromatids. The system is generally inactivated in other cells. However, it can be activated in response to double-strand breaks. Double-strand breaks can be caused by free radicals, ionizing radiation, or chemicals. A cell with a double-strand break is at risk of undergoing apoptosis. Since both strands are damaged, the previously mentioned methods, where one strand is used as a template to repair a removed faulty segment, do not work. Instead, homologous recombination or non-homologous end joining is used to repair the damage. In homologous recombination, one of the sister chromatid\'s DNA is used as a template for repair. In eukaryotes, this method can only be utilized after DNA replication, during the time when the two sister chromatids are still connected. The repair mechanism proceeds as follows: 1. 2. 3. 4. **Nonhomogolous repair** Non-homologous recombination (NHEJ) involves repairing a double-strand break by simply gluing the two ends back together. This is the most common repair mechanism for double-strand breaks, as it does not require access to an undamaged sister chromatid. The downside of non-homologous recombination is that it often leads to deletion mutations. This is because the nucleotides that are lost at the break site and during repair are not replaced. Non-homologous recombination occurs as follows: 1. 2. 3. 4. **Gene regulation** What is an Operon? An **operon** is a cluster of genes that are transcribed together as a single mRNA from one promoter. These genes are usually related in function and are co-regulated, meaning that they are either turned on or off together. Operons allow bacteria to efficiently control the expression of multiple genes that are involved in the same biological process. A typical operon consists of: 1. 2. 3. 4. Types of Operons 1. 2. Let\'s discuss two classic bacterial operons: 1\. The Lac Operon (Inducible Operon) The **lac operon** in *E. coli* is responsible for metabolizing lactose. The structural genes in the lac operon code for enzymes needed to break down lactose into simpler sugars (glucose and galactose). Key components of the **lac operon**: - - - Regulation of the Lac Operon: - - Additional regulation: - 2\. The Trp Operon (Repressible Operon) The **trp operon** in *E. coli* controls the production of tryptophan, an essential amino acid. It is a **repressible operon**, meaning it is normally **on** and producing tryptophan, but it can be turned **off** when tryptophan is abundant. Key components of the **trp operon**: - Regulation of the Trp Operon: - - Summary of Operon Regulation: - - Advantages of Operon Systems: - - This operon model illustrates how bacteria tightly regulate gene expression to conserve energy and resources, only producing proteins when they are needed. **[Cell biology]** ============================== **Cell Junctions** - - - - - There are a couple of different types of cell to cell junctions. 1. 2. 3. 4. 5. **Tight Junctions** [Structure, significance and fuction] There are two particular proteins that come out of the cell membrane and interact with each other. One is called claudins and the other is called occludoins. These proteins span through the extracellular space and are anchored into the cell membrane. In the inner cytosol or the cytosolic side there are black circular proteins called zona occludins that are bound to the claudins and cludin proteins. The last protein on the inner cytosolic that are bound to the zona occluddins are called actin filaments. Tight junctions connect cell to cell at the apical surface. The significance of the thigh junction is to tightly hold the cells together, they're designed to act as a diffusion barrier to block transport of ions and large molecules between cells. **Adherens Junctions** Adherens junctions more specific for resisting shearing and abrasive forces, allowing for stretch. prevent cells from separating from each other. There are proteins called E-cadherins that anchor into the cell membrane and then there is a component of it that comes out in the extracellular space. There is a special molecule that helps the 2 cadherins to bind together. The molecule that helps is calcium, it acts as a bridge to help them bind together. The next protein is vinculin on the inner cytosolic side. There is another protein called the catenin proteins in the cytosolic side and then finally actin filaments in the most inner part of the cytosolic. **Desmosomes** Good for shearing forces and abrasive forces, keeping cells together. They are stronger than tight junctions and adherens junctions. There are proteins called cadherins that come out from the cell membrane into the extracellular space. There are two different types of cadherins that interlock each other, Desmoglein and desmocollin. Because they are cadherins they are calcium dependent for bonding. There is another protein called desmoplakin (plaque), which anchors the desmoglein and the desmocollin. It comes out of the cytosolic area into the membrane. On the inner side of the cytosolic there is a protein called intermediate filaments, the main component is keratin. **Hemidesosome** It is actually not a true cell to cell junction, more of a cell to extracellular matrix junction/basal lamina that consists of fibronectin, collagen, laminin. Hemidesmosome is the connection between the basal lamina to the cell membrane. Hemidesmosome consists of integrins, it is a protein that spans through the cell membrane and connects it to the extracellular matrix. On the cytosolic side there are intermediate filaments that consist of keratin. The basic function is to form what is called the basement membrane. **Gap junctions** Made of 2 proteins called connexons. 2 connexons make up a gap junction. A connexon is made up of 6 connexins. Allows cell to cell communication. If you for example have an iron like sodium and calcium, it can move onto the next cell through this cell communication. Gap junctions (GJs) consist of clusters of double-membrane spanning hydrophilic channels that provide direct cell-to-cell communication by allowing the passage of signaling molecules, ions, and electrical currents. **Extracellular Matrix** A large network of proteins and other molecules that surround, support, and give structure to cells and tissues in the body. The cells in the body are connected to their surroundings, the extracellular matrix ( ECM ). \[ The ECM consists of a complex network of proteoglycans (eg, aggrecan and decorin), collagen and other fibrous proteins (eg, elastin ), and adhesive glycoproteins (eg, fibronectin, nidogen, and laminin ). \] ECM is produced and secreted by surrounding cells, in most connective tissue types it is fibroblasts that manufacture ECM \'s macromolecules. Collagen is the body\'s most commonly occurring protein and makes up the majority of the extracellular matrix. Collagen has the form of fiber-like strands. The strands organize in different ways depending on the type of collagen. Collagen\'s main task is to give structure to the ECM. Collagen is particularly abundant in bone and cartilage tissue. Elastin forms stretchable fibers and contributes elasticity to tissues. Elastin is found e.g. in skin, blood vessels and lung tissue and is the most commonly occurring protein in arterial walls. Collagen has a triple helix formation. The individual strands come together to make a triple helix. The collagen contains glycine + 2 other amino acids. Glycine is always on the 3rd amino acid spot. It is important for the formation of the triple helix as it is small. The ECM helps to attach the cells and structure into tissues, and informs the cells when to grow, die, produce etc. The ECM is connected to integrins which together makes the hemidesmosome. Proteoglycans are protein molecules found in the space between our cells. They are found in the ECM. They consist of polysaccharides linked to a protein chain. They attract water and form a gel that makes the tissue better able to withstand shocks and pressure. An example of a proteoglycan is aggrecan in cartilage.Proteoglycans can also bind various growth factors and thus affect cell growth and cell division. Decorin is one such example. **Cell signalling** [Paracrine signalling] Paracrine communication involves short-distance communication between adjacent cells. \] \[ Cells secrete factors such as proteins and peptides into their local environment to which surrounding cells react [Autocrine signalling] Cells can produce and release signaling molecules that they themselves react to. This type of signaling is called autocrine signaling. \] \[ Autocrine signaling occurs, for example, in the immune system and in cancer cells. [Endocrine signalling] Endocrine signaling means that endocrine cells release hormones into the bloodstream which are then distributed throughout the body and thus reach their target organs. \] Endocrine cells are specialized in hormone secretion. These cells are usually part of a gland , such as the thyroid or pituitary gland. Hormones are chemical substances that have a specific regulatory effect on one or more target cells. \[ The body\'s hormone system operates in different so-called hormone axes whereby hormone release is regulated by superior signal systems and feedback loops. \] Different cell signaling systems are integrated into these hormone axes where both chemical synaptic transmission with neuropeptides and endocrine signaling are relevant. \[ Negative feedback means inhibition of the various cells that are part of the hormone axes. \] \[ All of the body\'s hormone systems are controlled by feedback systems whose purpose is to coordinate the body\'s various hormonal functions. - [Juxtacrine signaling] Juxtacrine signaling involves contact-dependent signaling between cells where a membrane-bound signaling molecule is in direct contact with a receptor on a target cell. \] Sometimes gap junctions and contact between proteins in the extracellular matrix ( ECM ) and receptors on cells are also counted as juxtacrine signaling. Integrins are a group of transmembrane proteins whose main task is to connect cells to each other or to the ECM. Integrins participate in many important processes, such as embryonic development, hemostasis and wound healing **Mitosis and Cell cycle** **Organelles in the cells** [Nucleus ] - Outer layer is where you have lots of ribosomes. Inner membrane has a protein structure that binds to the DNA and histone proteins. These are Inner membrane is called lamins. On the nuclear envelope there are pores called nuclear pores. If you want to move things out and in and out, that is the function of the nuclear pores. Whole purpose is transport. - - - - - -  [Rough endoplasmic reticulum (ER)] - - - - - - [Smooth ER] - - - - - - - - [Golgi apparatus] It takes the vesicles from the rough and smooth ER. The area that takes it in is called the cis golgi. Through systematic steps the proteins or fatty acids will go through the golgi and bud off some type of molecule and leave the area of the golgi called trans golgi. - - - - - [Cell membrane] - - - - [Lysosomes] Contains enzymes called hydraulic enzymes. Enzymes - - - - - - - [Peroxisomen] It Contains a lot of enzymes. The important ones are catalase, oxidase and metabolic enzymes. One of the main functions of peroxisomes is to detoxify the cell by splitting hydrogen peroxide - - - [Mitochondria] - Oxidative phosphorylation - - - - - - There is mitochondrial DNA. [Ribosomes] 2 components, large subunit (60s) and the small subunit (40s) for eukaryotic cells. Ribosomes made up of rRNA and proteins. - - - [Cytoskeleton] There are 3 different structures. 1. - - - - 2. Primarily tough, not much movement. - - - 3. Made of alpha tubulin and beta tubulin. These two come together and form filaments, then 13 filaments come together and make microtubules. - 1. 2. 3. **[Cell cycle]** - - - **Interphase** [G1 PHASE (GAP 1)] - - - - - - **G1/S phase checkpoint to check that everything is okay** [S - PHASE (Synthetic phase)] - - - [G2 PHASE (GAP 2 PHASE)] - **Mitosis (M PHASE)** [Prophase] - - - - [Metaphase] - - [Anaphase] - - [Telophase] - - - - - - Chromatin refers to a substance found in the cell nucleus that\'s composed primarily of DNA and proteins. When cells divide, chromatin condenses to form chromosomes which split into two identical strands called chromatids. Each chromatid then becomes a chromosome in each new cell that is formed. **[Microscopy]** ============================ [Lightmicroscopy] A typical eukaryotic cell has a diameter of 10 - 20 μm, which is about a fifth of the smallest object that the human eye can perceive. The job of a microscope is to magnify an image so that the human retina can discern information that would have been missed by the naked eye. In the light microscope, visible light directed through optical lenses is used to magnify the image. For light microscopy, the specimen/cells to be studied should be fixed and stained. The wavelength of visible light (390 - 770 nm) is a limiting factor for the light microscope\'s maximum magnification The light microscope is used to study histological preparations (tissue samples) at a microscopic level. It is very common and is used in the diagnostics of e.g. tumors , anemias and other blood diseases, inflammations, infections, in diagnostics of autoimmune diseases and in karyotyping (mapping of a cell\'s chromosome set). Fluorescence microscope is a type of light microscope with high-frequency radiation (eg UV), which causes a specimen stained with a fluorescent dye to emit light. This allows one to look at only the structures that have been stained, while the rest of the specimen remains dark. The wavelength of visible light (390 - 770 nm, violet - red) is a limiting factor for the light microscope\'s maximum magnification. It is not possible to study objects much smaller than the wavelength of light. In practice, mitochondria (about 500 nm in diameter) and bacteria are the smallest objects that can be studied with a light microscope. With a light microscope, you can e.g. not directly see plasma membrane , ribosomes, cytoskeleton and small intracellular vesicles. - - - - - - Electron microscopy refers to microscopy using electron radiation. This method provides a significantly more powerful magnification compared to light microscopy as the wavelength of the electrons is less than a thousandth of the wavelength of visible light. Electron microscopes are mainly used in research but also in the diagnosis of certain diseases. The electron microscope has up to 200 times better resolution than the light microscope - - - - bright field microscope (fluorescent) and phase-contrast microscopy. With live cells, phase-contrast is the choice. **ANALYSIS GENE EXPRESSION** For tissue analysis we want to amplify the DNA or gene expressions. Therefore we use DNA -polymerase chain reaction Components are - - - - 3 step procedure - - - **To determine if a gene is expressed?** Old methods such as northern and southern blot are used. Northern blot for RNA, southern for DNA. New methods have come such as In situ hybridization = **Specific gene expression in a tissue sample** Next generation sequencing or RNA sequencing = **Global gene expression** Quantitative - polymerase chain reaction = **Analysis of the extent to which a specific gene is expressed or inhibited** Sanger sequencing = **Sequence of the genetic code** Microarrays reverse transcriptase CRISPR CAS-9 is used to study gene function in a variety of species. **PROTEIN ANALYSIS** Even though we have a gene expression does not necessarily mean that the protein is transcribed. Therefore protein analysis is needed to accompany gene analysis. [Whole tissue] Immunohistochemistry or immunofluorescence [Isolated tissue/cells] Western blot ELISA Extraction or isolation of proteins Common for all these methods is that they use antibodies to detect proteins. The antibody is akin(similar or corresponding) to the gene primers in DNA tech. They are specific for the protein of interest. The antibody consists of a primary and a secondary one. The primary one attaches to the protein of interest. The secondary one targets the primary to form a complex. Attached to the 2nd could be an enzyme or a fluorescence tag where a detection signal can be assessed. The best way is the ELISA method. - - - 1. 2. 3. 4. 5. 6.