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The Learning Library
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Principles of GeneticsGenetic Structure Both DNA and RNA are made up of the basic component of nucleotides. Each nucleotide is made up of 3 smaller parts. The fist is the nucleic acid often called the base, the ribose sugar and then the phosphate group. The nucleic acids are of 5 types with Adenine, Guanine, Cytosine, Thymine and Uracil. They are dividend in purines with Adenine and Guanine. The purines have 2 rings. The pyrimidines are made up of Uracil, Thymine and Cytosine and have 1 ring. Four of them are used in DNA with Adenine (A), Guanine (G), Cytosine (C), and Thymine (T). The are base paired together into A-T and G-C. The A is always with a T and the G is always with a C. The A-T pair shares 2 hydrogen bonds while the G-C pair shares 3 hydrogen bonds. The RNA uses four of the nucleic acids with Adenine, Uracil, Guanine and Cytosine. The only difference between RNA and DNA is the T is swapped for a U. The ribose sugar is the second part of a nucleotide. The ribose is a 5 carbon sugar with the 1 carbon attached to the base and the 5 carbon attached to the Phosphate group. The difference between the ribose of DNA and RNA is the second carbon lacks the OH (hydroxyl) group. That is how we know the difference between Deoyxribonucleic Acid (DNA) and Robonucleic Acid (RNA). The second carbon has 1 less Oxygen group making it deoxyribose. The phosphate group is the exact same in both RNA and DNA. The main one attaches to the 5 prime (5') carbon while the next nucleic acid in the chain attaches to the 3 prime (3') carbon. This makes DNA and RNA run from a 5' to a 3' direction. The DNA is double stranded with both strands running in opposite directions called antiparallel. This means one strand runs 5' to 3' and the other runs the exact opposite of 3' to 5'. RNA is single stranded since that extra Oxygen on that 2nd carbon would cause static hinderance when trying to bind into double strands. To a certain extent, you can get double stranded RNA as static charges will bind together, but it ends up in a stem and loop structure. The last structural concept of DNA is it is wrapped into a helical structure. Since its double stranded, we call it the double helix. The double helix structure tends to have one grove that is larger than the other. The larger group is called the major grove and the smaller is called the minor grove. Each twist of the helix is about 10 base pairs in length. This structure puts the bases to the inside as they are electrostatically bound to each other and puts the phosphates to the outside as they are hydrophilic. This protects and makes the DNA slightly negative in electrical charge which is important for packaging on histones. Histones are DNA packaging proteins. They are made up of an octamer (8) total proteins with 2 H2a, 2 H2b, 2 H3 and 2 H4 proteins in what looks like a spool for thread. The histone has a slight positive charge. The DNA wraps twice around each histone complex. The histones, the DNA wrapped around the histones and the small span of DNA linked to the next Histone is called 1 nucleosome. That is the smallest structure of DNA packaging. The nucleosomes are packaged in a string of beads formation with many histones wrapped twice with the DNA. When DNA is tightly packaged for mitosis, we call it a Chromosome, but when its loosely packaged during normal cell function, we call it Chromatin. If the chromatin is unwrapped on the histones, we call it euchromatin. If its more densely packed, we call it heterochromatin. There are multiple levels of packaging for which DNA will go through to fit 6 feet of DNA into the tiny nucleus of a cell. They start at the 30 nanometer and go all the way up to the 1400 nanometer which is the chromosome we see in mitosis. At the first stage, the nucleosome are bunched up like in a six pack of beer. The empty space it take out. This is the 30 nm packaging. The next level is the DNA is bound in loops onto structural scaffold. This gets you to the 300 nm. Then it supercoils into the 700 nm form. There are over 3.2 billion nucleotides in the human genome which encodes over 22,000 genes. Its broken down into 23 pairs of chromosomes which you get 1 set from your mom and 1 set from your dad for 46 total chromosome. The first 22 chromosomes are called autosomes. The last 2 are the sex chromosomes. Two X chromosome is female and XY is male. Chromosomes The term ploidy is used in genetics do represent how many copies of each chromosome each species has. If you have only 1 copy of each like our germline cells, that is haploid. Often shown as (N). If you have 2 copies of each chromosome like our somatic cells its called diploid (2N). Some species have more chromosomes called ploidy. The strawberry is octoploid with 8 sets of each chromosome. This becomes important as we study genetics in human disease. There can be abnormal amounts of chromosomes in some diseases. Chromosomal abnormalities account for 60% of all spontaneous abortions. The term monosomy refers to having only 1 of a specific chromosome. The only known monosomy disease to yield a viable fetus is single X chromosome or Turner syndrome. There are several genetic disorders that lead from trisomy like trisomy 13, trisomy 18 or trisomy 21 which is Down Syndrome. This starts to get important in cancer genetics where genetic instability can lead to chromosomal abnormalities. The next important component of Chromosomes is the location of their centromere. This is a key feature to each chromosome. The centromere is critical to the chromosome. Its an anchor point during DNA replication for the sister chromatids. Its also is used in mitosis. There are 4 types of centromeres and they are named by their location in the chromosome. If the centromere is right in the center, its called metacentric. If its slightly off center, it is called sub metacentric. If the centromere is closer to the end of the chromosome, then its acrocentric. If its at the very end of the centromere, then its telocentric. There are no natural telocentric human chromosomes. The last part of the chromosome is the Telomere. This is the cap on the ends of each chromosome. It plays a key role in cell division. The telomere is there to protect the ends of the DNA strands. Each time a cell copies its DNA for mitosis, the telomere gets a little shorter. This is called the hayflick limit and averages 40 to 60 times a cell can go through mitosis before it hits this limit. Once any cell hits this limit it goes into senescence which it will no longer divide which plays a role in many age related disorders. Stem cells have and enzyme that is called telomerase. This extends the telomere each time the cell divides making stem cells immortal. One of the hallmarks of cancer is when a cancer cell mutates to turn on telomerase and become immortal like stem cells. Polymerases This is typically something you won't see covered in genetics. You usually get into this in Virology, but I think it is important to understand now. This is about the different types of polymerases and what they do. The polymerase functions by building chains of nucleotides to form RNA or DNA chains. There are 4 basic kinds of polymerase based on what type of genetic code they read and what type of genetic polymerase they create. The first is the DNA dependent DNA polymerase. It reads DNA and it creates DNA. Our human DNA polymerase that synthesizes our DNA or does DNA repair comes from this family. You will often see the DNA polymerase referred to by its Greek alphabet name such as DNA pol Theta. There are several of these DNA polymerases working in human cells. They all have different functions. The second kind of polymerase is the DNA dependent RNA polymerase. This is what we typically call the RNA polymerase in human cells. It copies the DNA into RNA. The DNA to RNA polymerases come in 3 kinds in humans with RNA polymerase I making ribosomal RNA, polymerase II makes the messenger RNA, and polymerase III makes the transfer RNA. The third kind of polymerase is RNA dependent DNA polymerase. This is typically called the Reverse Transcriptase. It takes RNA and can write it into DNA to inset into the genome. You see this in retro viruses like HIV. There are several of these reverse transcriptase used in biotech right now with Lentivirus and Gamma Retroviruses. They use them to do basic cell editing in the lab. The last polymerase is the RNA dependent RNA polymerase. This is not natural to human cells. Its used by viruses to copy their RNA genome into more viral genome or messenger RNA in human cells. The viral RNA to RNA polymerases are also targets of anti viral therapies. DNA Synthesis There are about 3 billion nucleotides in the human genome. It would take a very long time to copy all of them with just one set of replication enzymes. This means the DNA will synthesize using many points of replication. The site where the replication starts is called the origin of replication. There is one of these replication start points every several thousand nucleotides in the human genome. These are at sites rich with A and T pairs as they have less bonds. The first enzyme will be Helicase which opens the DNA at the origin of replication. It unzips the DNA along the hydrogen bonds that hold the DNA together. The spot at which the Helicase unzips the DNA is called the replication fork. Since the DNA has an electrostatic charge, it will want to snap back closed. There is a set of proteins called Single Strand Binding (SSB) proteins that bind to each strand of the DNA and stabilizes the charges. The DNA polymerase that is responsible for copying the DNA can only bind to double stranded DNA, but in this case the DNA is separated. There is a RNA primase that comes in and lays in a few RNA primers into the starting spot so the DNA polymerase can attach. Both strands are copied at the same time. Each strand acts as a template while a new strand is synthesized from each of the original strands. This process leaves each new DNA with 1 original strand bound to 1 new strand. This is called a semi conserved replication process as each parent strand is paired with to the new synthesized strand. The DNA polymerase has to read the DNA going from the 3' to the 5' direction because it creates DNA from 5' to 3'. This makes one strand of the DNA easy to copy. The polymerase just binds to the 3' end and goes copying the DNA along the way as it is always moving forward. The other strand runs in the opposite direction of this leading strand. That mean the DNA polymerase has to work backward as that strand is going in the 5' to 3' direction. It does this by jumping several thousand nucleotides ahead and working backwards. These sections of DNA, it copies by jumping ahead and working backwards, are called Okazaki fragments. Initially, they are not connected together. There is another enzyme that comes along and connects these Okazaki fragment together. This enzyme is called DNA ligase. The leading strand is the one that goes easily following from the 3' end toward the 5' end. The lagging strand is the stand that goes from 5' to 3' and requires the DNA primase to jump around. The DNA is wound together into a double helix. As the helicase moves along the DNA and opens it up, it will create a twisting action on the DNA. There is another enzyme that binds to the double strands of DNA ahead of the helicase enzyme. As the tension on the DNA gets too high, the topoisomerase enzyme will break the bonds of the DNA and allow it to unwind the tension before binding it back together. The topoisomerase is a critical enzyme that is necessary for DNA synthesis to release the tension. There are cancer drugs that target this topoisomerase enzyme to prevent DNA synthesis in rapidly replicating cells. Transcription The DNA of the cell serves as a master blue print. Every cell of our body has a complete copy of this DNA blueprint. Depending on the cell and its functions, it will use specific parts of the blueprint found in the DNA to produce the proteins and enzymes needed by that cell. The rule is that each gene creates one fully functional protein or enzyme. The gene itself has a few key regions that are important to be familiar with. The very first nucleotide to be copied is the start of the actual gene. The part that in copied is called the open reading frame. The gene includes all the nucleotides that make up the actual messenger RNA. About 50 to 100 nucleotides before the start site will be the promoter region. The most basic part of the promoter is called the TATA box. Its a sequence of Thymine and Adenine nucleotides. This is the most basic promoter region and allows for a low level of gene expression. The transcription factors will bind to the promoter region and increase the level of gene expression. That means more copies will be made of that gene. When transcription begins, the RNA polymerase will bind to the promoter and begin copying the gene. There is actually a big complex of proteins that must bind to the promoter region before the RNA polymerase. This is called the initiation complex. It starts with the TATA binding protein (TBP) and builds this complex that recruits RNA polymerase. There are other regulatory segments in the DNA that can influence gene expression. They are called regulatory elements and are located thousands of nucleotides before the promoter. They can bind transcription factors called Repressors or Enhancers. The DNA will fold in a loop which allows the regulatory sequence to come in contact with the promoter region. This will allow the Repressor to block binding of the RNA polymerase. The Enhancer can also make contact with the RNA polymerase, but it will increase the activity of the RNA polymerase leading to increased gene expression. Once the transcription factors have bound to the promoter region, the RNA polymerase can bind to the transcription factors and begin copying the gene. The RNA polymerase opens up the DNA itself. It doesn't need any assistance like the DNA polymerase did. It will begin copying the DNA using the 3' to 5' strand of DNA as a template and create a RNA strand in the 5' to 3' direction. It begins with the first start nucleotide and continues until it reaches the end point. Then the polymerase will fall off and the RNA strand will be free to move into post transcriptional modification. The initial strand of the RNA is called the primary transcript. It will undergo three modifications before it can leave the nucleus of the cell. The first is it will undergo splicing by the spliceosome complex. The vast majority of the gene is made up of non coding information called introns. The actual coding part of the gene is called the exons. The spliceosome will follow splicing marks on the primary RNA and remove all the introns and paste the exons back together. This also leads to the ability of alternative splicing by cells to use a gene differently. One cell might use Exons 1, 2, 4, and 5 while another might use Exons 1, 3, 4 and 5. That is the concept of alternative splicing. Two different proteins can be made from the same gene, but by different splicing of the exons. After the splicing of the RNA is complete, there are enzymes that will add a Guanine cap to the 5' end of the RNA. This serves to allow the RNA to exit the nucleus and assists in loading the RNA into the Ribosome. The last process is the addition of the poly A tail. This just adds about 250 or more Adenine nucleotides to the end of the RNA. This signifies the end of the RNA when its read by the Ribosome. The final RNA product is now called a messenger RNA (mRNA). Its built to take the DNA information and deliver it to the Ribosome for production of a protein. Translation When the messenger mRNA exits the nucleus, it will be loaded into a organelle called the ribosome. These little factories take the RNA blueprint and use it to build a protein. Here we introduce the concept of the Codon. The DNA is made up of 4 bases with Adenine, Guanine, Cytosine and Thymine. They have to encode 20 different amino acids. To do so, the DNA uses a codon. Its a combination of 3 nucleotides. The start codon is always AUG (Adenine, Uracil and Guanine) and codes for the amino acid Methionine. Each 3 bases of nucleotides is one codon and encodes one amino acid until it reaches the stop codon. There are actually 3 different stop codons with UAA, UAG and UGA. The Ribosome takes the RNA and begins with coding the start codon with Methionine and continues to read each and every codon until it reaches the stop codon. Inside the ribosome there are Transfer RNA (tRNA) that have an anticodon on one end and the amino acid on the other end. The tRNA will match the mRNA codon with its equal opposite anticodon. When the ribosome makes the match, the amino acid on the end of that tRNA is added to the chain of amino acids it is building. This process continues reading each codon of the messenger RNA and matching it to a amino acid to build a peptide chain of amino acids. Mutations Mutations in the DNA can cause some very serious damage to the final product which is the protein. Since 3 nucleotides come together to make one codon and produce one amino acid, you can get some dramatic changes from even a single nucleotide mutation. The first mutation is called the missense mutation. Its when the change in a single nucleotide changes the final amino acid to be coded. This is exactly what drives the genetic disease of Sickle Cell. One single nucleotide is changed. That leads to another amino acid being coded. The two different amino acids have dramatically different behaviors. The one is hydrophobic while the other is hydrophilic. That simple change will change the entire shape of the protein. In proteins, shape determines function. The second mutation is the nonsense mutation. This is where a mutation changes the codon from encoding an amino acid to a stop codon. This terminates the production of the protein early. A nonsense mutation will make a truncated version of the protein. In some cases, the shorter proteins are still functional or partially functional. In many cases, they lose complete function of that protein. The last mutation is called the frame shift mutation. That is when a single nucleotide gets inserted or deleted. That causes every codon in that gene to get changed. None of the codons will be right when they all get shifted for one extra or one less nucleotide. Protein Folding After the ribosome creates initial peptide of amino acids, the peptide will undergo folding. There are various proteins and enzymes that assist in protein folding. The proteins will have many electrostatic, hydrogen, hydrophilic and hydrophobic forces. These will all come into play as the protein folds. The hydrophobic amino acids will be pulled into the center of the protein while the hydrophilic amino acids will turn outward. When proteins fold, they create some basic structures that play a key role in the protein's function. They are the Primary, Secondary, Tertiary and Quaternary structures. Not every protein will have all these structures, but you will see at least the first few basic structures in all proteins. The Primary structure is very simple as it is the actual long peptide of amino acids. The primary structure is just the sequence of amino acids and their place in the overall chain. The Secondary structures will form into alpha helix and beta pleated sheets. The alpha helix is just a coil shape and these play a huge role in many protein structures. The part of a protein that spans across the membrane of a cell will be an alpha helix in many cases. Even the points on a T cell receptor that bind the antigen are the tips of the alpha helix. The beta pleated sheet looks like a radiator structure where the strands alternate back and forth to make a sheet like structure. These are not the only structures, but they are the most common. Tertiary Structure is the final 3 dimensional shape of the protein. You will hear them called globulins which means protein. The final structure of the protein will determine its function. If there is even the smallest mutation in any of the sequences of the protein, it can vastly change the function of the protein. There are many genetic diseases where the body produces the protein, but its not shaped correctly and therefore fails to function properly. The Quaternary structure is when multiple proteins come together to form a larger structure like in Immunoglobulins or Hemoglobin. In these structures several proteins are coming together to build a larger protein that functions as one. The Hemoglobin of the Red Blood Cells is a great example. This is made up of four proteins in the shape of a 4 leaf clover. Mitosis Most cells spend their lives in G0 phase of growth. This means they are adults and they go about their normal function. Some cells are created frequently like red blood cells and neutrophils. You make millions of these cells every day. Cells are not created from nothing. They use a process of mitosis. That is where one cell splits into two cells. When a cell gets the signal to replicate, it will go from G0 phase into G1 phase. This is called the growth or gap phase. In G1 phase of cell growth, the cell will build up resources and grow so that it is ready to split in two. Then it moves into S phase for Synthesis. That is where the DNA gets synthesized so that every chromosome is copied. Once the cell has 2 copies of every gene called sister chromatids, it will advance into G2 phase of growth. In G2 phase it will check all the DNA and verify it is ready to divide. This whole growing up part of the cell cycle is called interphase. When its all complete, the cell advances into the final M phase which stand for Mitosis. This is where the DNA gets split up between the two new cells. The first step in mitosis is called Prophase. In this phase, the centrosomes will move to opposite sides of the cell and begin to produce protein strand. Then the membrane of the nucleus will dissolve in the stage called Prometaphase. The long protein strands of the centrosome will attach to each and every chromosome of DNA. This then begins Metaphase which is where every chromosome has a strand attached to it from each Centrosome. There is an enzyme that will cut the sister chromatids apart and each new chromosome will be pulled to opposite sides of the cell. This will be Anaphase where each centrosome gets one of each and every chromosome. Then a new nucleus will be formed around each set of chromosomes in the Telophase of mitosis. The last part is cytokinesis which is the actual splitting of the two cells Epigenetics Epigenetics means on top of genetics. The study of Genetics is all about the DNA. Its about how its structured and packaged. Epigenetics is about how Genes are expressed and regulated. We are born with our Genetics, but our Epigenetics is acquired by experiences and environmental exposure. I don't want to get too deep into epigenetics, but I think there are a few concepts that are important to understand. The first one is the process of gene methylation and gene silencing. Each gene has a promoter in front of the gene where the transcription factors bind and activate transcription of that gene. The promoter of the gene will often have Cytosine and Guanine rich regions called CpG islands. That stands for Cytosine, Phosphate, and Guanine. These CpG rich regions can become methylated. Methylation of these CpG regions of the promoter can come from environmental factors like UV exposure, chemicals, radiation, smoking and so many other things. This exposure can cause the methylation of the promoter and eventual silencing of the gene. The silencing of a gene plays a big role in understanding tumor genesis in cancer. The loss of tumor suppressor genes don't always come from a mutation of the DNA which renders them ineffective. It will often come from gene silencing by epigenetic forces. The second concept of epigenetics is the Acetylation and Deacetylation of the Histones which package the DNA. We learned in genetic that the DNA gets wrapped twice around each histone. While the DNA is packaged like this, it is transcriptionally inactive. The proteins and enzymes that do transcription can not access packaged genes. For a gene to be transcribed, it has to be exposed to the transcription machinery. This is controlled by acetylation or deacetylation of the histones. The DNA normally has a slight negative charge. The histone has a slight positive charge. They like to electrostatically bond to each other. By adding or removing a acetyl group to the tails of the histone, the charge can change allowing the DNA to be unwound. This is an important concept to understand as a gene needs to be exposed to be active. Some areas of the DNA are always inactive and densely packaged like round the centromere and the telomeres. There is no genes encoded in this region. One cell might have a gene active as it uses it all the time while another cell will keep that gene packaged as it never uses that gene. Each cell only uses genes specific to that cells role and functions. Some oncology drugs will target the acetylation or deacetylation of histones to suppress the transcription of genes in cancer. |
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