Lecture Notes: Meiosis, Genetics, and DNA Replication PDF

​ These chromosomes have already entered s phase and have already replicated ​ What is fundamentally different from meiosis than mitosis, is that the chromosomes find each other and come together ​ Centromere is AT rich ​ Sisterchromatids are held together by cohesin complex ​ Mitosis- cell divi...

​ These chromosomes have already entered s phase and have already replicated ​ What is fundamentally different from meiosis than mitosis, is that the chromosomes find each other and come together ​ Centromere is AT rich ​ Sisterchromatids are held together by cohesin complex ​ Mitosis- cell division in somatic cells ​ Meiosis- unique to the gonads ○​ 4 new haploid cells are genetically unique ○​ Happens through 2 mechanisms  ​ Indpendent assortment ​ Recombination ​ Meiosis 1- chromosomes come together and pair very tightly ​ Homologous chromosomes RANDOMLY pair together ○​ Either both will go to one pole, or one goes to one pole and the other to the opposite ​ Recombination also happens during Meiosis 1 ○​ Homologous chromosomes get very close together and exchange material between NON-sister chromatids ○​ Chromosomes go to opposite poles ○​ You have made new, unique chromosomes that are part mom, part dad ​ Statistically impossible to make 2 of the same gamete ​ Used Drosophila genetics ​ Identified large # of mutations ​ Wanted to identify the genes that made the flies different- tried to map the genes ​ Vestigial is in purple ​ They crossed the flies and expected all heterozygotes and to be all phenotypically wild-type ​ In the second progeny, expected ½ wild-type and ½ double mutant ​ Parentals are the expected ​ The 2 new classes are the recombinant classes, they are not expected ​ It was later determined that the percentage of recombinants can be used to determine location of the genes ​ If you have 2 genes that are closer together, then you will have way fewer recombinants than if they were farther apart ​ Another thing that led scientists to believe that there was an exchange of information was that when they looked at gametes (gonads), they could identify structures above in the cells ○​ Homologous chromosomes form loop-like structures ○​ They hypothesized that there must be an exchange of genetic material ​ Start in prophase l ​ Synaptonemal junction- made of many protein ​ At the end of prophase l after recombination has come to completion, you can see the crosses (chiasmata) ○​ Kinetochore microtuble form on top of microtuble ○​ Tetrad (last pic on left) - A four-part structure that forms during prophase I of meiosis and consists of two homologous chromosomes, each composed of two identical chromatids ​ In order for the chromosomes to pair, they undergo synapsis ​ Spo11 enzyme induces double stranded breaks ○​ Only expressed in cells undergoing meiosis, NOT mitosis. ​ First- it induces a lot of double stranded breaks ○​ When a cell has a break, it will try to repair that damage ○​ Exonucleus degrades 5’ ends, exposing 3’ stranded tails ○​ Now we have a free strand dna ​ Think of it as a nucleophilic attack ​ Free strand will invade, base complementary ○​ DMC1 will help one of the tail to invade, and open up the other chromatid’s double helix ○​ RPA will bind to the sequence and stabilize it because DNA doesn’t like to be single stranded ​ To join the 5’ end of one nucleotide to the other 3’ end of the other nucleotide, ligase is required to join ○​ Needs ATP to covalently bond the two ​ After this process has come to completion, you get a holiday junction ​ They then go through branch migration to lengthen the size of the heteroduplex ​ Holiday junctions is how you physically keep homologous chromosomes together ○​ Cohesin is how you keep sister chromatids together  ​ Non-disjunction- failure of chromosomes to separate during cell division, which can result in abnormal numbers of chromosomes in daughter cells. ​ Resolvase can cut in either one of 2 directions ​ In order to complete meiosis, holiday junctions need to resolve ○​ Can result good recombination (now made new chromosome) 1/29 Correct answer: Synaptonemal complex ​ During DNA replication, Cohesin complex keeps sister chromatids together ○​ Cells must enter S phase, whether they’re going through mitosis or meiosis ○​ ALL chromosomes have it ​ During Meiosis, in prophase 1, homologous chromosomes will come together and pair (synapsis) ○​ Structure that holds them together: Synaptonemal complex ​ You can get exchange of genetic material ○​ This is where recombination happens, where diversity stems from ○​ Cohesin keeps sisters together ​ Spo11 is an endonuclease that will induce double stranded breaks in a random manner on all homologous chromosomes ○​ When the break happens, the cells will want to fix themselves ○​ Starts the process of recombination ​ After you get the double-stranded break, you get another exonuclease, ​ What you get is the formation of the holiday junction hich resects the DNA ends to create single-stranded overhangs, facilitating the recruitment of recombination proteins like RAD51 for homologous recombination repair. ​ If the cell tries to divide, you can get breakage in DNA ​ How the holiday junctions get resolved is they get cut by an endonuclease ○​ Endonuclease= cuts within ○​ Exonuclease= have an open end and chew out a single strand ​ Asymmetric cut= you can result in a productive crossover effect ​ You can also get a holiday junction resolved in a manner that’s non-crossover ○​ You arent getting a chiasmata= a point at which paired chromosomes remain in contact during the first metaphase of meiosis, and at which crossing over and exchange of genetic material occur between the strands ​ Asymmetric cut- you can get a crossover ​ If you resolve the holiday junctions, you get a non-crossover ​ Dessolution= also non-crossover ​ Recombination happening during prophase ○​ Here it’s been further subdivided ​ When they were doing these experiments, they had the unexpected recombinant classes ○​ The only reason you got these is because there was an exchange of genetic material ​ ​ VG stands for vestigial ​ The key to doing these types of experiments is to look at the number of recombinants between each phenotype ○​ Count the number of recombinants that are different between the 2 loci of interest ○​ 17.7 centimorgans ​ The top is mom, the bottom is dad ​ The green is them coming together in meiosis 1 ​ Recall how 3 point crosses work and how you can get information on location of genes ​ Bivalent and Tetrad are the same for our purposes ​ Top right: metaphase, homologous chromosomes are starting to separate ○​ The normal meiotic division occurs, the would divide ○​ In the end, you get gametes (haploid) ​ Defects can occur in meiosis 1 and 2 ○​ Nondisjunction ○​ Top left, after the cell divides, you can have an increased number of ploidy, and you end with an abnormal number of chromosomes in the end ​ Depending on which chromosomes this affects, it can give a rise to a number of complications ○​ Down’s syndrome ○​ Robert syndrome ​ We have 23 pairs of chromosomes ​ They are ordered based on size ​ Karyotypes reveal defects in chromosomes ​ Characteristic banding patterns to see if locations are missing, inverted, etc ○​ Philadelphia translocation is one famous type ​ Nondisjuction even (possibly at meiosis 1) that results in 3 copies of chromosome 18 ​ Children who are born with these conditions ​ You never see a trisomy of 1,2 or 3 because they are lethal, they result in a spontaneous abortion ○​ Large chromosomes ​ Aneuploidy= any number other than diploid- 1 less, or 1 more ○​ Leading cause of miscarriage ○​ 25% of abortions ​ Many of the aneuploidy are from the defects in female gametes ○​ Therefore, as a woman ages, the chance of the offspring having aneuploidy increases  ​ Huge increase associated with aneuploidy as women get older 1/31 ​ ​ Amount of A is always the same as T, and the amount of G is always the same as C ​ These 2 DNA strands are reverse complements of each other ​ Semi-conservative- 2 existing strands are separated ○​ Each used as template for synthesis of new strand ​ Although Francis and Crick proposed this model, it was still unknown ​ They could distinguish N14 and N15- 2 different isotopes of Nitrogen ○​ Found in the nitrogenous bases ​ They wanted to come up with a method where they could identify and label the new strand that was synthesized ​ Replication of DNA, you need: ○​ DNA template, DNA polymerase, DNA primer, DNTs ​ DNA polymerase reads the template, but only in a particular direction ​ DNA is antiparallel 5’ to 3’ ​ How new nucleotides are added is DNA polymerase needs 3’ hydroxy group to add the next nucleotide ​ Double-stranded helix needs to be opened up ​ Our chromosomes are very long ​ If we asked DNA polymerase to start at one side, it would take 20 days ​ When DNA is replicated, it is “licensed” so that replication happens at both ends ​ As scientists, we use information from one species, to gain information about another species ​ AT rich island= sequence on the chromosome that has a lot of As and Ts ​ What’s needed to get the process started? ○​ RPA will bird to the single stranded DNA ○​ Without this, the DNA will want to reanneal, so we need this to keep it separate and open ​ We have our double stranded DNA ​ In order for DNA polymerase to add a new nucleotide it needs an addition at the 3’ hydroxyl group ​ PCNA keep strands together ​ In order for polymerase to get started, you need a primer ○​ Polymerase alpha primer ​ Ligase joins okazaki fragments ​ PCR allows you to synthesize many copies ​ Important for cloning ​ You want to be able to take that DNA sequence and put it into vectors, manipulate it, amplify it, and obtain desired results ​ Mullis and others devised ○​ Denaturization- making the strands come apart by increasing the temp ○​ Annealing- allow it to cool ○​ Extenstion- polymerase extends the primers ​ Know how this works!