BIMM 100 Lecture 10

BIMM 100 Lecture 10 - BIMM100: Lecture 10 ­ gene...

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Unformatted text preview: BIMM100: Lecture 10 ­ gene structure and organiza7on Reading: pages 217 ­226 (and 199 ­204) Exam pickup: aEer class tomorrow or at your sec7ons Exam issues •  Solu7ons to exam answers will be discussed during office hours and sec7ons ­ there WILL NOT be a formal answer key posted ­ please come and talk to the TAs (and me) about your ques7ons! •  If you believe that an error has been made in the grading of your exam, provide a short explana7on wriWen on a separate piece of paper –  Do not add any addi*onal wri*ng to your exam paper! Any altera7on of your actual exam will result in me not re ­grading it! •  Turn in your original exam and the explana7on to E. Weber (room 1100C at the west end of Pacific Hall, 8 ­11 am M–F) no later than next Thursday, August 25th. •  I will re ­read your exam and your explana7on and respond to your request for reconsidera7on as soon as I can Genes and chromosomes •  Molecular defini7ons •  Genomic content and organiza7on –  Gene families –  DNA repeats –  Repeats and polymorphisms: •  Gene and individual iden7fica7on Overview and defini7ons The “problem” with DNA: a single human cell can contain 2 meters of DNA! All of this must be contained in a cell that is, on average, only 10 microns in size! What it a gene? The DNA sequence required for the coding of a func7onal product (protein or RNA). However ­ it also includes control regions! Remember: only 1.5% of the human genome codes for func7onal products. Is the rest just junk? Bacterial versus eukaryo7c gene structure Remember: bacteria have genes arranged in operons (a collec7on of genes that make one transcript) that can be made into several protein products. Polycistronic Monocistronic Eukaryotes are more complicated: each mRNA encodes a single protein Eukaryo7c transcrip7onal units While bacterial genes are arranged into a single transcrip7onal unit, eukaryotes are not. “Simple:” the primary transcript is processed to make a single mRNA, which encodes a single protein. Eukaryo7c genes have introns and exons. Introns are usually much larger. Titan has an intron that is 17kb! Eukaryo7c transcrip7onal units can be influenced by muta7ons Muta7ons in exons, introns, and control regions can all affect the expression of the gene’s product! Muta7ons in a or b may affect the expression of the protein. Muta7ons in c may affect the protein (diminished ac7vity, etc). Muta7ons in d may affect splicing! Complex eukaryo7c transcrip7on units: more possibili7es! Alterna7ve splicing (Same 5’ and 3’ exons) Alterna7ve polyadenyla7on (Same 5’, different 3’ exons) Alterna7ve promoters (Different 5’ exon, same 3’ exons) Now it’s more complex when we think about muta7ons ­ depends where they are located! Solitary or unique genes •  25 ­50% of protein ­coding genes are represented only once in the haploid genome –  Example? Lysozyme (enzyme that cleaves bacterial cell walls). –  It has 4 exons, three introns, and spans 15kb. –  There are no other detectable mRNAs around it 1 2 3 15kb 50 ­60 kb 4 Not all genes are solitary ­ many reside in “families” •  Duplicated genes: have close but non ­iden7cal sequences that are located within 5 ­50kb of each other •  If the genes encode proteins that have similar but non ­iden7cal aa sequences, they are said to be a gene family. The proteins encoded from these genes make up a protein family. •  Examples? Kinases, immunoglobulins, cytoskeletal proteins, heat shock proteins, α and β globulins How can exons be duplicated in the genome? Homologous recombina7on! In the following example, between L1 sites (they are abundant in our genome). Can result in: func7onally dis7nct variants pseudogenes “true” gene duplica7on Homologous recombina7on also drives gene duplica7on Known as “unequal crossing over” Can ALSO result in: func7onally dis7nct variants pseudogenes “true” gene duplica7on A great example of these concepts? Globin gene cluster Gene family! NOTE: an 80kb fragment from a “higher” eukaryote is very different… In the yeast, almost everything is a coding open reading frame (ORF) Pseudogene: regions related to func7onal globin ­type genes, but are non ­func7onal Alu sites: 300bp noncoding repeated sequence abundant in the genome What are all these gene for? Gene family! Different β globin genes likely arose from a duplica7on of an ancestral gene. Likely from unequal crossing over during meiosis Some of these genes then accumulated beneficial muta7ons (fetal gene versions are only expressed when the need for oxygen binding is high, while adult versions are expressed when the oxygen affinity needs to be lower). Pseudogenes started off like these genes, but they had no selec7ve pressure to be func7onal, and currently just remain in the genome! Heavily u7lized genes are encoded by mul7ple copies rRNA rRNA rRNA rRNA rRNA Some genes occur in tandemly repeated arrays ­ different from gene families, because these are actual repeated versions of the same genes! Why? Needed to meet the great cellular demand for their transcripts! Example: during early development, certain cells must double every 24 hours and contain 5 ­10 million ribosomes. So, the cells have about 100 copies of ribosomal genes! What kind of DNA is in our genome? Humans aren’t the only organisms with nonfunc7onal DNA •  •  •  •  Humans: 3300Mb DNA in the genome Amphibians: way more! Amoeba dubia: 200x more DNA than humans! Why? Lots of noncoding DNA –  Only 1.5% of our genome is coding Since the synthesis of noncoding DNA takes 7me, nutrients, and energy, it is more likely that microorganisms have lost nonfunc7onal DNA over 7me due to selec7ve pressure. The energy invested in DNA synthesis for higher eukaryotes, on the other hand, is trivial compared to our other energy needs! Nonfunc7onal DNA: satellite repeats •  Also called “simple sequence” –  6% of human genome –  Perfect or nearly perfect repeats –  1 ­13 bp repeats are called “microsatellite” repeats •  Most are 1 ­4bp –  most satellite repeats are 14 ­500bp arranged in tandem arrays of 20 ­100kb. Nonfunc7onal DNA: satellite repeats Most are located in specific chromosomal loca7ons (like centromeres and telomeres) What is this assay? What are the probes? Mouse chromosomes with simple sequence repeats shown in green. Microsatellite repeats may arise from “slippage” in replica7on No change in numbers! Microsatellite repeats may arise from “slippage” in replica7on If backwards slippage occurs, more copies of the repeats may be made N+1 change in numbers! Microsatellite repeats may arise from “slippage” in replica7on If backwards slippage occurs, more copies of the repeats may be made If this mistake is not caught by DNA repair proteins before the next replica7on, then the extra copy is permanent! Microsatellite repeats have serious clinical consequences •  Some individuals are born with a larger number of repeats in specific genes than the rest of the popula7on. –  Implicated in 14 neuromuscular diseases! –  Can func7on like a recessive muta7on (interfere with just the func7on of a par7cular gene) –  Can func7on like a dominant muta7on (interfering with the func7on of all genes in a par7cular cell) •  Example: myotonic dystrophy pa7ents have 100 ­4000 copies of CUG compared to 50 ­100 in normal people that form hairpins that interfere with all cellular RNA processing DNA polymorphisms are used for many purposes •  Individual iden7fica7on –  Minisatellites •  Highly conserved among individuals •  Numbers of the repeats are variable, as are their length –  Why? Probably due to unequal crossing over –  You can use these for “fingerprin7ng” •  Occur in 1 ­5kb regions made of 20 ­50 repeat units each about 14 ­100bp in size –  Can detect these by PCR! •  Linkage mapping for disease genes and popula7ons –  RFLP ­restric7on fragment length polymorphisms DNA polymorphisms: who’s the daddy? Who commiWed the crime? Common uses of this technology ­ fast, easy way to iden7fy an individual! For paternity: Make several sets of primers to unique sequences flanking the satellite repeats. Do PCR, and run out the fragments. This is a “DNA fingerprint:” the fragments have different repeat lengths, so they run differently in the gel! For a crime? Same process. Include vic7m’s DNA, too ­ a great control! F1’s the daddy! Human disease mapping •  Human diseases can be inherited in the following ways: –  Autosomal (not sex ­linked chromosome) •  Dominant ­ when expressed in the heterozygote, usually the parent has the disease! If your parent’s have the allele, you have a 50% chance of geqng it. –  Usually shows up aEer reproduc7on has occurred. Otherwise, these traits would be selected against! Ex: Hun7ngton’s disease. •  Recessive ­ both parents must be carriers of the allele for a child to be at risk of contrac7ng it. So, if your parents are carriers, you have a 25% chance of geqng the disease. Ex: cys7c fibrosis. –  X ­linked (on X ­chromosome) •  Recessive ­ will be expressed in males only (they get the bad copy of the X chromosome, while females have 2 copies. Ex: duchenne muscular dystrophy. If your mother is a heterozygote, every male child has a 50% of geqng the allele! RFLP analysis: restric7on fragment length polymorphism 1.  Digest genomic DNA with mul7ple restric7on endonucleases 2.  Subject products to gel electrophoresis 3.  Examine the unique paWerns that arise due to polymorphisms in single genes or repe77ve regions of non ­coding DNA –  Used in paternity and maternity cases, also in forensics, and in iden7fica7on of humans RFLP mapping Using polymorphisms as gene7c markers ­ first type of molecular markers used in linkage studies (is a gene “linked” to a disease?). Two chromosomes shown at leE. Digest with different endonucleases and perform Southern blots with probe (green). The muta7on at site a2 dras7cally changes the Southern blot result! RFLP can be used to inves7gate whether an allele of gene is linked to a disease Can trace allelic differences throughout families ­ does one allele fragment sta7s7cally segregate with a disease? Every individual has 2 alleles: Some contain allele 2 on both chromosomes, while others are heterozygous for allele 2. Increasing resolu7on in gene7c mapping You’ve learned everything you need to know to perform these types of experiments! ...
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