Notes for Test 3.doc

OCTOBER 17, 2008 *DNA Sequencing: This is the determination of the precise sequence of nucleotides in a sample of DNA. The common method is chain termination, where you can stop where you want to stop the sequencing. If you modify the 3? OH, it changes the sequencing. This method is also called di-deoxynucleotide sequencing. DNA synthesis is 3? to 5?: Once you attach a dideoxynucleotide, there is a H instead of an OH of the 3? carbon. This terminates the process of complementary matching of nucleotides. *DNA REPLICATION: You can replicate DNA in a bucket (test tube). The following is what you need for DNA replication: 1) Single stranded DNA template. We need lots of copies of these. Polymerase chain reaction helps to amplify amount of DNA. 2) Primer: This is for matching a small part of the template. 3) DNA Polymerase 4) dNTP?s: This include dATP, dGTP, dTTP, and dCTP. 5) Appropriate deoxnucleotides (one per tube). You start with a template and a primer. The primer floats around and finds the right spot on the DNA strands and sticks to it. If you want to find all the Ts in the sequence, after putting in the usual dATP, dGTP, dTTP, and dCTP, you then put in ddTTP. This will cause DNA to start priming. You get compliments until it reaches an A. The sequence either grabs a dTTP or a ddTTP as the compliment. If it chooses a dTTP, that?s fine and the process keeps going. If it chooses a ddTTP though, the process will stop. This keeps repeating until every A has been stopped at. You get different fragments by continuing to repeat this. To find the other letters, set up 4 buckets. Set up one for A, one for T, one for C, and one for G. You end up with a whole pile of fragments that need to be separated. Use electrophoresis next. This uses an electrical field to separate fragments based on size and length. You use a siv or a sponge, and you use polymerase. Gel has regular holes in them. You first load up the sample and then you put a charge on it. DNA is negatively charged therefore it will run towards the positive charge. Fragments will be separated based on size. The shorter ones are at the bottom (where the + charge is) and the longer ones are up top (where the ? charge is). You then take all the fragments from all the buckets and it will separate in separate lanes. With one row at a time, you keep reading and get the base sequences. The United States not has automated sequencing which is laser driven This is done with putting a fluorescing tag on DNA. *OTHER APPLICATIONS: This could have a medical application where stopping replication could be good for cancer. This hasn?t been figured out yet, but it could also be good for viruses. The drug called AZT (Azidodideoythymidine) that is modified as a way that?s attractive to virusus. This stops virus replication. OCTOBER 22, 2008 *CHROMATIN STRUCTURE AND DNA CONDENSATION: DNA has semi-conservative replication. Two strands of the parental double helix unwind. Each strand specifies a new daughter strand. *CONDENSATION OF DNA TO FORM CHROMOSOMES: The size of haploid is 10^11 and the size of diploid is 2 x 10^11. This extends to 2 meters in a linear form (per cell). This is packaged into the nucleus a diameter of 5 um (up to 1000x contraction or a 500:1 ratio by the time you get through to a metaphase chromosome). *BASIC STEPS IN DNA CONDENSATION: The Folded Fiber Model consists of nucleotide pairs, then the DNA forms into a helical double stranded form (20A wide). Then the DNA and protein interact to form chromatin (100A particle). You see this in the G1 phase (20-100A wide or 2-11 nm, but some go out to 30 nm). The centromeres and telomeres are condensed so they?re going to be wider. So you can tell how active the areas are by the thickness. If being used, it will be much thicker and packed down. Once we go through replication, it goes to about 300A wide in size. It keeps packing until G2/M boundary, where this is the final packing into chromosomes for mitosis. *HOW DOES CONDENSATION OCCUR? Double stranded DNA + protein (histone) = a nucleosome particle (including H1 histone, etc.). A chromatin fiber is like beads on a string where nucleosomes are arranged down the ?string.? A chromatin fiber of 3 nucleosome has around 600 base pairs (200 per nucleosome). *NUCLEOSOME: Histone octomers are the circles in the structure. There are 8 octomers in each nucleosome, not including the Histone H1. The ones in the middle are hydrophobic and the ones on the outside are hydrophilic. The nucleosome is 100A tall. Linker DNA goes in between the nucleosomes and connects them. They are ? charged. The Histone H1 nucleosome acts like a putty at the bottom to hold it together. It pulls the nucleosome together to make another structure. The double stranded DNA has around 150 base pairs. Histones are tightly conserved proteins found only in the nucleus of numerous organisms. This tells you that they?ve been there for a longtime and that they?re really important and critical to the operation of a system. There is electrostatic attraction with DNA (-) and histones (+) The association of histone octamers and DNA creates a beads on a string arrangement The DNA between nucleosomes (linker DNA) is associated with the histone H1. *THE SOLENOID: The helical winding of the nucleosome strands result in the formation of a solenoid. It has 5+ nucleosomes per wrap usually. You get a 300A fiber and this is as compact as DNA gets in G1. A solenoid: Solenoids start to form up in a scaffold. This happens during G2. This is getting closer to mitosis or meiosis. It keeps condensing and solenoids coil around each other The fully condensed chromatin fiber is up to ~6000A in diameter. This is the metaphase chromosome: The scaffold is a protein that acts as a stuff sac called lamin. The final stage of condensation take place at G2/M. Why? This is because of the presence of nuclear scaffold make of lamin (protein). This allows for attachment of the highly condensed material to form a chromosome. The scaffold is only present at the G2/M boundary. Chromosomes are only visible at this time! The metaphase chromosome: SEPTEMBE 24, 2008 *HISTONES: Histones have regulatory functions. It only used maybe 10% of the genes you have. You have to have genes turned on and tuned off. This occurs through gene packaging. The non-needed DNA is also packaged. G1 is the normal cell life 99% of the time. DNA is beads on a string, and is 20-100 A wide. Synthesis and replication make and condense sister chromatids. They are packaged down to around 300A. As we move through G2, there is more packaging and shortening and widening of DNA. DNA here is 2000A. In M phase, there is lamin formation with condensation. The metaphase chromosome is here, and this is the final shortening and thickening. *CHROMOSOME BANDING: Around 1970, techniques were developed that allowed greater visualizations of chromosomes than had previously been possibly. These techniques took advantage of the fact that heterochromatin and euchromatin stained differently. In fact, chromosomes can be identified by their unique staining patterns. This is a quick way we could look for deletions, changes, etc *KARYOTYPE: This is an alignment of chromosomes based on size and shape. Idiogram is another name for this, and is the actual arrangement. The dark areas are more dense and includes the centromeres and telomeres. Each chromosome has a different banding pattern. Banding (or staining) is a high degree of condensation and is tightly coiled. Each chromosome has a different number of nucleotides. The number of nucleotides equals the length of chromosomes. The type of nucleotides equals the banding properties *TWO TYPES OF CHROMATIN: Heterochromatin and euchromatin. *HETEROCHROMATIN: This is tightly coiled, dark staining. This is noncoding and contains no genes. This determines chromosomes structure. This primarily occurs at centromeres and telomeres (ends of chromosomes that shorten after replication). There is not as much crossover and recombination especially around the centromere because it?s so dense. It remains densely coiled throughout the cell cycle. Two types of heterochromatin include 1) constituitive and 2) facultative. *CONSTITUITIVE: ?Always? This is always consitutitive. This includes the centromeres and telomeres (these are always centromeres and telomeres). These are the noncoding regions of chromosomes. This contains HR/MR type nucleotides. *FACULTATIVE: ?Potential to become? This includes whole chromosomes. This includes X inactivation (Barr bodies). An example is where whole chromosomes are noncoding. *EUCHROMATIN: This is the less densely packed, light staining regions and are the coding sequences. They are tightly coiled only during metaphase. *SUMMARY: Large DNA molecules must be highly condensed to fit within the cell nuclei. Condensation occurs in stages: Nucleosomes (beads-on-a-string) Solenoids: supercoiling of supercoils Intense condensation: supercoiling of supercoils (rosettes) Folded around protein scaffold in prophase Chromatin exists in more than one form: heterochromatin and euchromatin. Staining patterns of chromatin are replaced to the underlying structure (coiling) and sequence organization. This led us to the understanding of Genomic Complexity. *GENOMIC COMPLEXITY: The evolutionary progression is going up in complexity with a higher among of DNA. You go up by orders of magnitude. The flowering plants have the most DNA. Relationship of genome size to genome species: The C-Value is the amount of DNA contained in the haploid genome of a species. The C-value paradox is that excess DNA is present that does not seem to be essential to the development or evolutionary divergence of eukaryokes. Also, closely related organisms with the same degree of complexity in body form, tissue, and organ types often vary by tenfold or more in DNA content. In an experiment, scientists heated DNA (from G1) up and saw how fast it came together. Short DNA pops apart but then come back together quickly. HR are highly repetatives. They are simple and small and come together very quickly. MR are moderately repetatives. They are a little longer and more complex. U are the genes. It comes together very slow and is very complex. *CLASSES OF NUCLEOTIDE SEQUENCES: (From least complex to most complex) 1. Highly repetitive (HR): These are non-coding areas with rapid reassociation (simple sequences). They are 5-10 bp in length and comprises 5-10% of the genome. It is clustered around centromeres and telomeres. They play a role in maintaining chromosome morphology. 2. Moderately repetitive (MR): This is the non-coding region of 150-500 bp in length. This is 5-10% of the genome. One sequence can be repeated 700-900,000 times. 3. Unique (U). This is the coding region where the genes are. It is 1000-15,000 bp in length on average. There are approximately 23,000 genes in humans. This is 1-5% of total nucleotides. The coding region is the genotype (so 95% of the genotype doesn?t make phenotype). So what about the other 70%? This includes ?trash,? and pseudogenes (fake genes). OCTOBER 27, 2008 *WHAT IS A GENE? A gene includes a locus = globin, alleles (A or a), and genotype is Aa A gene is a sequence of nucleotides (genotype) and carries genetic information which is to be expressed (phenotype). The human genome has approximately 22,000-28,000 ?genes? with an average size of 1,000-15,000 bp. It has a huge letter sequence in a chunk of DNA, and you have to find the start and end of genes. What defines a gene boundary? When do genes start and stop? This can be found because lots of genes and highly conserved and universal. *CONSENSUS STRUCTURE OF ANY EUKARYOTIC GENE: A gene is a transcriptional unit, a sequence of DNA that actually gets transcribed. Exons are actual pieces of the code that will be used to make phenotype. Introns are ?intervening sequences? or areas of genes that do not generally code for phenotype. ?Gene graffiti? Why would you have an intron? No one really knows but there is an exon shuffling theory. If we need to evolve some stretch or region, we evolve it and shuffle exons around to make it what we want. The beginning and ending sequences of introns are highly conserved (same across different organisms). There are also introns that protect exons ?buffers.? They don?t pile critical supplies in one spot. There is safety with spreading things around. ?Generally? is mentioned in the intron defition because we have more proteins then we have genes. We have alternate splicing in which a particular intron will be an intron sometimes and an exon sometimes. So one gene can make 2 different genomes. TATA box is a highly conserved sequence which serves as the binding site for RNA Pol (start site of transcription)?This is called the promoter region. ?On-off? ?START Some proteins for transcriptional unit are ?on? and some proteins sit there and block it ?off? therefore DNA Pol cannot bind. CCATT box is the site at which DNA binding proteins attach and modulate the rate and copies of a gene made ?accelerator? ?by proteins binding to it. An enhancer specifies DNA sequences shared by genes to which proteins bind to coordinate gene activity ?coordinator? *ORGANIZATION OF GENES: EUCHROMATIN 1. Solitary (Unique): This is a single copy of a gene (haploid). There are 2 copies of a gene in a diploid situation. This compromises the bulk of euchromatin. 2. Duplicated: These are duplicated gene sequences in the genome. It consists of very similar nucleotides. Each codes for a similar polypeptide but having a distinctly different function It arises from unequal crossover during meiosis. 3. Multigene Families: These are genes or identical or closely related DNA sequences which share similar functions and chromosomal location. They are most often used or synthesized at different times. Example includes globin genes?fetal, embryonic, and adult in mammals. 4. Pseudogenes: These are nonfunctional sequences due to significant substitutions or deletions in the nucleotide sequences. 5. Repeated Genes: These are multiple copies of small genes clustered throughout the genome at specific chromosome sites. They are present in a high copy number and in a tandem configuration. OCTOBER 29, 2008 *TRANSCRIPTION: The central dogma (major set of ideas or rules): There is actually no such thing as phenotype. It is actually proteins that cause physical characteristics. mRNA is messenger RNA. This is the message. It is usually from the unique region and will make 1-2 copies. rRNA is transcribed ribosomal RNA. tRNA is transfer RNA. They are assembled together into rough ribosomes (little factories). Translation is converting information into proteins. Amino acids are the base units of proteins. So the arrow at the end of the central dogma actually goes from translation to amino acids, which then make proteins. It takes 3 nucleotides to make an amino acid. *TRANSCIPTION: This occurs in the nucleus (if it?s present). It has nothing to do with mitosis and meiosis. It occurs in either G1 or G2 (period when genes coding for cellular organelle proteins are synthesized). Gene structures (TATA) defines which nucleotides will be transcribed. The traditional model: 1 gene (genotype) yields 1 RNA (interim) yields 1 ?protein? (phenotype) But actually this correspondence is not correct. The ratio is not 1:1:1 and sometimes RNA can make different numbers of proteins. You also do not make proteins but rather a string of amino acids. So one gene does not necessarily equal 1 RNA. Proteins are actually amino acids that come together to make proteins. *TRANSCRIPTION: All nucleotides of transcriptional unit are converted from DNA ro RNA. Recognition: TATA (finding and attaching to TATA or other promoter). Initiation: Binding of RNA Pol Elongation: Movement of RNA Pol (down the DNA strand). Termination: GC rich region?GC rich regions like to fold back on each other and crossbonding helps to stop the process. Processing: This is taking what we?ve got and modifying it. Cap and tail addition is adding ends (closing it up). Intron removal is splicing and removing unwanted parts. *RECOGNITION: RNA Pol recognizes and binds at the TATA box. There are protein helpers that come along and bind to the TATA to help signal binding site for RNA Pol. *INITIATION: RNA Pol uses one strand for transcription called the sense strand. You have to knock nucleosomes off- every 200 nucleosomes. Transcription begins at the initiation site. RNA Pol doesn?t do well hanging onto DNA. It falls off so it has to do multiple trips. RNA synthesis is 5? to 3? (new strand). The 3? OH is where new phosphodiester bonds can form. *ELONGATION: The growing RNA strand is complimentary to DNA. Introns and exons are both transcribed. RNA Pol moves down the DNA strand and synthesizes RNA. *TERMINATION: Toward the end there are GC rich regions. GC regions like to crossbond and this makes it hard for RNA pol to continue. There are also termination factors that help. It goes back to the origination formation usually, unless you want multiple copies of the strand. The termination site is reached causing release of both mRNA and RNA pol. REVERSE TRANSCRIPTASE: With reverse transcriptase, you start with mRNA and go in reverse. There is a library of mRNA with lots of copies of mRNA. We reverse transcriptase and go backwards and make DNA. You can stick a label on it, throw it into the genome, and it will stick to where it belongs. This shows which genes are active. *TYPES OF RNA PRODUCED FROM GENES: 1) mRNA is messenger RNA and is the template for translation. 2) tRNA is transfer RNA and carries amino acids. 3) rRNA is ribosomal RNA and combines with proteins (30-50) to form subunits of the ribosome, the site of translation. OCTOBER 31, 2008 *STEPS OF TRANSCRIPTION: All nucleotides of a transcriptional unit are converted from DNA to RNA (hrRNA). The steps are: 1. Recognition: This includes finding TATA. 2. Initiation: This includes the binding of RHA Pol. 3. Elongation: This involves the movement of RNA Pol (down the DNA strand). 4. Addition of cap and tail Processing: This is the post-transcriptional addition of the cap and tail, and is for intron removal. *POST-TRANSCRIPTIONAL MODIFICATION: The steps include 1. The 5? cap is added to the mRNA. The cap is made of guanine (serves as the ribosome recognition site) 2. The Poly A tail is added to the 3? end of mRNA (protects mRNA from degradation in the cytoplasm). This is added by poly A polymerase. *INTRONS: Introns are noncoding gene regions (in eukaryotes only). The number of introns in a gene vary (1 in insulin to 50 in collagen). There is no set intron size and the number of nucleotides vary. The intron/exon boundary is conserved. Introns are an evolved feature. Only genes in higher order organisms have introns (i.e. viral, mtDNA, and cpDNA lack intons). *ENDOSYMBIOTIC THEORY: Mitochondria invaded the nucleus to become a symbiont. The splicing mechanism of intron removal is by: Autocatylytic RNA Spliceosomes *AUTOCATYLYTIC RNA (Ribozymes): The splicing mechanism occurs most commonly in rRNA. The introns itself contains the enzymatic activity necessary for its removal. *SPLICEOSOME: This is a RNA and protein complex. It excises intones in the nucleus of cells. The RNA component is single stranded RNA (small 100-200 nucleotide RNA strands). The protein component is U (enzymes with excision activity). The two together make snRNPs (small nuclear ribonucleoproteins). This is sometimes called ?SNURPs.? *ALTERNATE SPLICING OF hnRNA: hnRNA stands for heterogeneous nuclear RNA. *REVIEW: Transcription is taking genes and going from DNA to RNA. mRNA, tRNa, and rRNA assist. In processing, only mRNA is involved and includes 1) cap addition 2) tail addition and 3) intron removal (splicing). NOVEMBER 3, 2008 *TRANSLATION: This takes place in the cytoplasm. It converts mRNA into amino acids which make proteins which cause phenotype. Genes (genotype) are create an mRNA (an intermediate) to yield a peptide (phenotype). There is a direct correspondence but this is not a 1:1:1 correspondence. Biostructure to where mRNA is used to make peptides. *RIBONUCLEOTIDE: This is the base unit of mRNA which make amino acids. How is this done? Through the genetic dictionary. The genetic dictionary allows us to understand how biological systems use the genetic message and convert it over). Nucleotides are read 3 nucleotides at a time, from 5? to 3?. Three nucleotides make up one codon. One codon make 1 amino acid. How? This is also by the genetic dictionary. *FEATURE OF THE GENETIC DICTIONARY: 1. It is written in linear form (stored in the mRNA). 2. It is coded as triplets (codons). 3. It is unambiguous (1 codon = 1 amino acid). This is an absolute 1:1 relationship. 4. It is also degenerate is another aspect. This is because one amino acid can be coded for by more than 1 codon. If you know the amino acid, you don?t know the exact codon. If you know the codon though, you know the amino acid. 5. There are specific start and stop codons. Start: AUG Stop: UAA, UGA, UAG 6. It is comma-less. There are no pauses in the code. Once you start, you start and keep it going until you stop. 7. It is non-overlapping. It reads right down the line. It doesn?t backtrack, etc. 8. It is universal. This means that it is generally the same across organisms. Sequoia trees and humans have the same genetic code. *GENETIC CODE: The genetic code can be found in the lab manual. For AUG, A is in the 1st position, U is in the 2nd position, and G is in the 3rd position. You read from the first position, then the 2nd, then the 3rd. AUG codes for met (methianine). The stop codons UAG, UGA, and UAA are TERM codes. There are 20 amino acids and 3 stop codons, therefore there are 23 we?re going to us, but there are 64 possible (4^3 = 64). The general rule is that the 3rd position can often change (different letter) and amino acids will be the same. This is called wobbling. This is the reason that it?s unambiguous going down and degenerate going up. *THREE MAIN PLAYERS IN TRANSLATION: The three main players include: 1. Ribosome (rRNA and proteins): This is the ?construction site.? 2. Transfer RNA (tRNA): This is the ?delivery system.? 3. Messenger RNA (mRNA): This is the message. *STEPS IN TRANSLATION: The steps include: 1. tRNA charging (This has to be done for translation, but is semi-independent. The first real step is initiation.) 2. Initiation 3. Elongation 4. Termination *TRANSFER RNA (tRNA): tRNA delivers amino acids one by one to the growing chain at the ribosome. The cloverleaf structure of tRNA: This is a mix of single stranded and double stranded regions. The anti-codon loop will have 3 bases sticking out on top that?s complementary to the message. It is important because it determines what will happen at the receptor site. *STEPS IN TRANSLATION: Here are the steps in more details: 1. tRNA charging: tRNA molecules are linked with amino acids known as being ?charged.? It recognizes the correct amino acid and puts it into place. Sticking the amino acid on there torques the top end. It goes from Written in short hand, the structure is: