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Lecture 1

BIOL 308 Lecture 1: Lecture 1 notes

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BIOL 308
John Heikkila

Definitions ▪ 5’ guanine cap: Phosphate-phosphate bond, where the cap is added in a reverse orientation so it won’t be degraded by ribonucleases ▪ Alleles: Multiple forms of one gene ▪ CDS: Amino acid coding sequence (see ORF) ▪ Chromosome: DNA and its proteins, can be uni/polyploid ▪ Cis regulatory regions: Referring to DNA/RNA ▪ Gene expression: Information flow process where gene  RNA/protein ▪ Gene: Unit of heredity needed to make RNA/proteins (have regulatory sequences – found upstream/downstream, exons – code for proteins, introns – removed in RNA) ▪ Genetic complementation: Test whether mutations in 2 strains are different genes (complementation won’t happen is the mutations are in the same gene) ▪ Genetic suppression: Second mutation restores the phenotype back to the original background mutation ▪ Genome: All genes in one gamete ▪ Genotype: All genes and alleles for an organism ▪ Heterochromatin: Chromosome heavily packed with proteins and histones ▪ Knockout: Inhibiting function of a gene in embryonic stem cells (ES) where the gene might be expressed but the protein won’t be created 1: Alter its coding region through DNA fragment insertion 2: Prevent it from being expressed at all morpholino antisense oligonucleotides to bind to mRNA and block it from being transcribed 3: Prevent translation of a specific mRNA to eliminate its function 4: Knockout promoter region – this is harder to do ▪ Mutant: A phenotypic genetic expression change ▪ Mutation: Permanent nucleotide sequence change ▪ ORF: Open reading frame (see CDS) ▪ Phenotype: Physical appearance of an organism due to genotype/environment ▪ Trans acting proteins: Transcription factors, proteins that bind to cis acting sequences ▪ Transcription factors: Need to bind to the promoter region to activate transcription ▪ Transgenic organism: Has foreign DNA introduced into its cells (random by injection into pro-nucleus or fertilized ovum which causes disruption of endogenous genes important for normal development) ▪ Transgenic studies: Analyze the promoter’s ability to direct tissue specific gene expression Eukaryotic gene expression regulation ▪ Promoter: Where transcription is initiated (before/upstream = cis regulatory regions) ▪ Transcriptional initiation site (+1): Acts as a number line to identify bases by numbers (left is negative, right is positive) ▪ Exon start: Where translation is initiated – the region that is coded for a particular protein ▪ Exon end: Where translation is terminated – translation beings on mRNA ▪ After exon: Polyadenylation site ▪ After polyA: Transcriptional termination site (after/downstream = cis regulatory sites) Gene Original Promoter – Exon 1 – Intron – Exon 2 Primary transcript Transcription Exon – Intron – Exon mRNA RNA processing 7 methyl guanine cap at 5’ – Exon 1 – Exon 2 – Poly A tail at 3’ Peptide Translation Nitrogen cap – Peptide - Carboxyl Protein Post translational modification 3D folding and modifications (glycosylation, etc.) Why is it important to understand gene expression? ▪ RNA/Proteins are the final products of gene expression and mutations could negatively affect an organism ▪ Proteins: Structural, enzymes, regulators (during DNA transcription), hormones, receptors, neurotransmitters (expressed as larger parental form and are cut into smaller neuropeptides or inhibiters), transporters (carry RNA to the site of transcription), antibodies ▪ Find the genetic causes for diseases, addictions, behaviours (heart disease, alcohol, shyness, etc.) ▪ DNA should be able to be repaired so that transcription/translation mistakes aren’t lethal ▪ Important to understand capping, splicing, ribozymes, post translational processing, processing of t/rRNA How do we study gene expression and protein/RNA function? Biochemical approach 1. Isolate protein 2. Find the partial amino acid sequence 3. Make oligonucleotide probes 4. Find cDNA or genomic clone in library 5. Sequence isolated gene ▪ Isolate protein based on function (assay under different conditions) ▪ AA sequence used to find nucleotide sequence Genetic approach 1. Isolate genetic clone (mutant .vs. not) 2. Genomic DNA  cDNA for the gene’s mRNA 3. Sequence cDNA to find ami
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