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Lecture 14

Biology 2581B Lecture 14: Lec 14 – Gene and Expression
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Department
Biology
Course
Biology 2581B
Professor
David R Smith
Semester
Winter

Description
Lecture 14 – Genes and Gene Expression What is a gene? - BAD definitions - Not a good way to describe a gene - Basic unit of biological information - Segment of DNA encoding a protein - Discrete region of a chromosome encoding a protein or RNA SIMPLE EXAMPLE OF A GENE – cox1 - Human mitochondrial genome: 16.6 kb - Cox 1: 1,542 bp long of double stranded DNA o Found in human mitochondrial genome o On mitochondrial chromosome - When want to make the product, transcribe the gene, get single stranded mRNA (nucleotides – single stranded), translate using ribosome and get a protein - Protein product of cox1: cytochrome C oxidase subunit 1 - TO GET NUMBER OF AMINO ACIDS: o (Nucleotides/3) – 1 - Example: 900 nucleotide strand of mRNA  last triplet is a stop codon = 299 amino acids NOT 300 - Protein is found in the complex - Cytochrome C oxidase – in ETC - Cox1 is a subunit of cytochrome C oxidase DNA  RNA  PROTEIN – central dogma - mRNA sequence is identical to the coding strand except Ts were replaced by U - mRNA has start (AUG) and stop codon - Coding sequence is broken down into triplets (3 letter codons) o Look at the universal genetic code Universal genetic code - CAG = Gln - Codons to deduce an mRNA sequence o mRNA has uracil instead of thymine Polycistronic RNA transcript - Cox1 is part of a polycistronic RNA transcript o When it is expressed in the mitochondria, it is apart of a long polycistronic transcript o Transcript gets processed into smaller mRNAs - Polycistronic transcript: they are not all protein coding transcripts o Pieces of it code protein, rRNA, tRNA o Polycistornic transcript does not mean everything in it encodes protein - Cox1 gene o Intact and continuous ▪ Coding sequence is not broken up ▪ No breaks in the coding o Part of a polycistronic transcript o Encodes a protein Same gene different species –cox1 - Genes can be different even when dealing with the same one MUSHROOM - Mitochondrial genome: 135 kb o Larger than human mtDNA - Cox1: 29,902 bp long – double stranded DNA o 27,000 bp longer than human copy of the gene o 19 introns o 20 exons - Mushroom gene is longer than human copy because the gene is full of introns - How many intronic nucleotides? o 28, 315 – basically all intronic other than 5% of it o Chunk of introns - Introns have to get spliced out in the nucleus with a spliceosome (complex machinery) o Involves other RNAs and other proteins - In mtDNA and chloroplasts, the introns are DIFFERENT o Get mRNA from a gene with the introns in it and it is single stranded o Each of the introns fold into a complex secondary structure o The structure allows the intron to self-splice - SELF SPLICING INTRONS - splicing mechanism is due to secondary structure - Mature transcript is closer the length of the gene in humans o Gives same protein - cytochrome C oxidase subunit I - Mature product is 45 nucleotides longer than human mRNA = 15 amino acids longer Diplonema - Not photosynthetic unicellular eukaryote – isolated from sweater - Most abundant predator on earth o It is unicellular and it can eat things - mtDNA is broken up into many miniature chromosomes o Genome is broken up into many little circles - Cox1 gene is found on 9 different chromosomes o Fragmented o All of chromosomes contain one of the exons for Cox1 o 9 different circular chromosomes each contain a little piece of cox1 gene - When want to express cox1, have to transcribe each of the exons (9 separate exons with their own RNA) and then splice them together in order to make an INTACT transcript o Mature transcript with 9 pieces linked into one o THEN make the protein - Different than how humans do it – cox 1 is contained on one chromosome and there is no splicing - Trans-splicing: have distinct exons that are separated from one another and need to be joined post transcriptionally Trans-splicing - Exons located in distant regions or even on different strands or chromosome are transcribed separately and then joined together - Each of the exons has a fragment of an intron o When 2 exons are floating around each with a piece of an intron, the 2 introns can meet and fold into a secondary structure even though they are not joined o Intron gets spliced out and links the pieces together - Trans splicing is mediated by introns Perkinsus – ribosomal slippage - Unicellular eukaryote that parasitizes oysters - Normal cox1: start and stop codon – everything in between should be in frame o Mutations that shift the frame are lethal - The genome was sequenced: o Move along the sequence and find many frameshift mutations o Moving along the coding sequence and the frame is shifting - Frame shifts are found in the mature mRNA o 10 different frame shifts in the gene - Should not get a functional protein if coding sequence is not in frame… - Frameshifts always occur at identical motifs o 2 types of motifs: ▪ AGGY – Y can be T or C (or U or C in RNA) ▪ CCCCU - Ribosome goes along the mRNA, reads the triplets and when it hits a frameshift, the ribosome know that it has to jump o Hits a motif and jumps ▪ Jumps 1 nucleotide or jumps 2 nucleotides o Jumping restores the frame (doesn’t restore the sequence) but as the ribosome reads it and makes the amino acid sequence - Perikinesus: it is frame, get a shift (introduce a stop), occurring at a motif o Instead of it being lethal, the ribosome knows to jump one and restore the frame Magnusiomyce capitatus - transitional bypassing - Looking at the same gene: cox1 is giving the same protein with the same function in ETC o Only thing that is changing is how the gene is expressed - Parasitic fungus - Cox1 gene in mRNA level: have coding sequence in frame, non coding DNA, coding sequence in frame, non-coding DNA and more coding sequence o Looks like the non-coding DNA is intronic o Non-coding DNA interrupting the coding sequence = they must be intronic ▪ What the people who first studied it hought o Sequence don’t look like introns (don’t have the classic intronic sequence) o They do not get spliced out of the mRNA - “introns” were always present in the mRNA - As ribosome moves along the coding sequence, and approaches a non-coding DNA, it forms a stem loop o Non-coding DNA forms a loop o Stem loop flies off the mRNA and lands somewhere else - Gets a stem loop, goes off the mRNA and then lands back in the next coding section Translatio
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