* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download bio_task_9-1 - andrewbartaseniorscienceat2
Survey
Document related concepts
Biochemistry wikipedia , lookup
Genetic engineering wikipedia , lookup
Plant virus wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Gene expression wikipedia , lookup
Silencer (genetics) wikipedia , lookup
Epitranscriptome wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Biosynthesis wikipedia , lookup
Endogenous retrovirus wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Transcript
Using Bioinformatics to Crack the Flu Code 1 All About Inflenza Influenza virus particles dry out (half-life a few hours at room temperature). In humans, the virus infects cells lining the respiratory tract (nose, throat and lungs). Cold and dry weather allows the virus to survive longer outside the body than in warm weather. Once infected it takes 1-3 days to get sick. Courtesy of Centers for Disease Control and Prevention It spreads easily by coughing and sneezing. 2 3 Flu Types: Influenza A Spreads fast Influenza B Influenza C Mild infections 3 Effect on Society Deaths Medication and health care loss of working hours nationally. Around 75 million working days/year are lost due to influenza. Table showing the mortality/10 million deaths caused by Influenza and Pneumonia (a complication associated with infection with influenza). Source: http://www.cdc.gov/ncidod/EID/vol10no1/02-0705.htm 4 ‘Getting the Flu’ Just about everyone has been sick with ‘the Flu’ at some stage in their lives. So, why do people get sick again and some die when they ‘catch the Flu’ again? Shouldn’t they have immunity to the Flu virus? 5 Antigens identify Flu strains Influenza viruses are named according to the antigens (proteins) sticking out of their virus coat. (H) There are two types of antigen = N and H. In different virus strains, the shapes of N and H are different. (N) There are 9 known N and 16 known H types. 6 The role of flu virus antigens The N antigen is required to cut the virus away from the host cell so it can spread to infect more cells. The N shown above has its cutting site blocked by a drug designed stop the flu from spreading. The H antigen is like a key that allows the virus to enter into cells with a matching lock. This allows the virus to replicate inside the cell. Bird Flu H allows the virus to infect bird intestinal cells. Human Flu H allows the virus to infect human lung cells. 7 Virus N cuts the links between the viruses H attaches to cell surface and the cell surface so virus particles proteins so virus can enter cell are free to go and infect more cells. Proteins on cell surface Virus genes are released into the cell. The lung cell is ‘tricked’ into using these genes to make new Human Lung Cell virus particles. 8 How we respond to antigens… The H and N antigens are like the ‘face’ of a flu virus. If the virus strain has infected you before, the virus ‘face’ is recognised and your immune system goes to war fast! The virus is killed off and sometimes you don’t even get sick. If the virus ‘face’ changes slightly (<1% = genetic drift), it can still be recognised quite quickly and your immune system will fight fast. You may be sick for a few days. If the virus ‘face’ changes radically (genetic shift = up to 50%), it is not recognised. it takes longer for your immune system to prepare for war. The virus takes hold and can make you very sick. Major changes to the shape of the virus ‘face’ can cause a Pandemic 9 The 1918 Spanish Flu a bigger killer than WWI The Spanish Flu pandemic killed more than 40 million people! The virus antigens were extremely different to those encountered previously. People carried no immunity to this virus strain so they were highly susceptible to illness and even death. It started in America and spread with soldiers going to war. 10 What causes a Pandemic? Genetic Shift! Let’s use the H5N1 Bird flu as an example…. H5N1 bird flu occasionally infects humans but at this stage humans do not pass this infection on to other humans. The spread of infection in birds means more humans will come into contact with and be infected by H5N1 bird flu. The concern? Eventually a pig will be infected with a human flu and a bird flu at the same time. They will serve as a ‘mixing pot’ for the two flu types to swap genes. The Result? A new flu subtype can emerge which easily spreads from person to person. An influenza pandemic would then occur with severe symptoms, like the lethal leakage of fluid into the lungs caused by the 1918 Spanish flu. This process of repackaging of viral genes is called reassortment. It is illustrated in the next slides. 11 Reassortment Influenza A infecting a human. Can spread from human to human due to H and N proteins on surface. Pig can become infected easily with bird flu and/or human flu. Serves as a mixing pot! Influenza A infecting a chicken. Can occasionally infect humans but cannot spread from human to human due to H and N proteins on surface. 12 Virus coats break down and RNA genes move to the cell nucleus to be copied and transcribed. Repackaging of genes creates a virus that can now transfer from human to human! PANDEMIC? Viral genes are copied and prepared for packaging into new virus particles. 13 Naming the Flu A/chicken/Korea/01(H9N2) Influenza A virus was isolated from a chicken in Korea in 2001. The antigen types were H9 and N2 Try some yourself: A/swine/Ehime/80(H1N1) A/Tokyo/67(H2N2) A/duck/Hainan/2004(H6N2) B/Nanchang/97 NB. Occasionally you will find more in the name. For our purposes today, ignore those letters &/or numbers Answer question 3 in your worksheet 14 The Influenza Genome All of the genetic material found in the virus is known as its genome. The genome is divided into 8 ribonucleoprotein (RNP) segments. The genetic material is (-) sense RNA (this is complementary to mRNA) Source: http://www.omedon.co.uk/influenza/influenza/ 15 Influenza has 8 gene segments Sequencing has revealed the genome for influenza along with the proteins it codes for. Segment Size (nucleotides) 1 2341 PB2 Subunit of polymerase: Host cap binding and endonuclease 2 2341 PB1 Catalytic subunit of polymerase 3 2233 PA Subunit of polymerase, active in vRNA synthesis 4 1778 HA Haemagglutinin 5 1565 NP Nucleoprotein: Part of transcriptase complex 6 1413 NA Neuraminidase: release of virus 7 1027 M1 Matrix protein: Major component of virion M2 Integral membrane protein: Ion channel NS1 Anti-interferon protein. Effects on cellular RNA transport NS2 RNP nuclear export 8 890 Polypeptide Function Source: http://www.omedon.co.uk/influenza/influenza/ 16 The importance of Flu Chasers…. Scientists monitor the Flu viruses circulating in the population by looking at changes to the virus H and N antigens. To identify the strain of flu virus…… Send a sample off to the laboratory for Gene Sequencing. This is an accurate way to find out the sequence of nucleotides in the viral RNA. After sequencing the H and/or N genes they can compare them with the gene sequences from other strains of the virus. This lets them look for the mutations that can cause epidemics and pandemics. 17 Mutant gene = Mutant Protein? Yes or No? But does a change or mutation in the gene sequence always mean there will be a change in the protein or antigen? To work this out we must determine the gene sequence and then the amino acid sequence for the protein. 18 What is Gene Sequencing? Gene sequencing is identifying and determining the order of the base pairs in a segment of RNA or DNA A G T G C C T T A A A T A T G AG T A A T GG AGAA GA A C TT T . . . Answer question 1 in your worksheet. 19 The 1918 Spanish Flu N Gene Sequenced and Translated The underlined sequence codes for the signal peptide. Boxed amino acids indicate potential glycosylation sites. Circled amino acids indicate the active site residues (3). Reid, Ann H. et al. (2000) Proc. Natl. Acad. Sci. USA 97, 6785-6790 Copyright ©2000 by the National Academy of Sciences 20 Gene Expression This is not how transcription happens in the influenza virus! The flu virus genome is RNA NOT DNA! But, it still makes mRNA and so we can use the sense DNA genetic code. 21 Transcription – copy DNA into RNA Try to work out the following: 1. 2. The second strand of DNA (complementary strand). Remember the base pairing rule in DNA, A pairs with T and G pairs with C. The code in RNA after copying the 1st DNA strand (the sense strand). Be careful. In RNA, T is replaced by U. An A in the DNA sense strand will see U added to the growing RNA strand. Write your answer down and then click to check your answer. DNA: AAT CTG GGG AAC TCG TTT CGC CCC CGA TTA GAC CCC TTG AGC AAA GCG GGG GCT mRNA: UUA GAC CCC UUG AGC AAA GCG GGG GCU 22 mRNA containing the genetic code copied from the (-) sense RNA virus genome moves into cytoplasm of the host cell. Ready for……. Translation 23 5’ 3’ Codon = 3 bases in mRNA Anti-codon = 3 bases in tRNA Amino Acid TRANSLATION: 1. mRNA locks onto a ribosome. 2. The ribosome reads the mRNA message 3 bases at a time = CODON 3. Transfer RNA (tRNA) molecules carry amino acids. Each tRNA has an anticodon that will only base pair with the correct codon on mRNA. 4. Base pairing occurs between mRNA and tRNA and the new amino acid is added to a growing chain. Source: http://genetics.nbii.gov/Basic1.html 24 A series of three nucleotides coding for an amino acid in DNA is a triplet Nucleotides in the genetic code correspond to sense strand DNA or mRNA. T T C A TTT Phe [F] TTC Phe [F] TTA Leu [L] TTG Leu [L] TCT Ser [S] TCC Ser [S] TCA Ser [S] TCG Ser [S] TAT Tyr [Y] TAC Tyr [Y] TAA Ter [end] TAG Ter [end] CCT Pro [P] CCC Pro [P] CCA Pro [P] CCG Pro [P] CAT His [H] CAC His [H] CAA Gln [Q] CAG Gln [Q] ACT Thr [T] ACC Thr [T] ACA Thr [T] ACG Thr [T] AAT Asn [N] AAC Asn [N] AAA Lys [K] AAG Lys [K] F i r CTT Leu [L] s CTC Leu [L] t C CTA Leu [L] CTG Leu [L] P Back to page 28 o ATT Ile [I] s i A ATC Ile [I] t ATA Ile [I] i ATG Met [M] o n GTT Val [V] GTC Val [V] G GTA Val [V] GTG Val [V] GCT Ala [A] GAT Asp [D] GCC Ala [A] GAC Asp [D] GCA Ala [A] GAA Glu [E] GCG Ala [A] GAG Glu [E] G TGT Cys [C] T TGC Cys [C] C TGA Ter [end] A TGG Trp [W] G T h CGT Arg [R] T i CGC Arg [R] C r d CGA Arg [R] A CGG Arg [R] G P o AGT Ser [S] T s AGC Ser [S] C i AGA Arg [R] A t AGG Arg [R] G i o GGT Gly [G] T n GGC Gly [G] C GGA Gly [G] A GGG Gly [G] G Source: http://psyche.uthct.edu/shaun/SBlack/geneticd.html 25 The Genetic Code Since RNA is constructed from four types of nucleotides, there are 64 possible codons (4x4x4). Three of these codons specify the termination of the polypeptide chain = STOP codons. That leaves 61 codons to specify only 20 different amino acids. Most amino acids have more than one codon. Exceptions to this rule are the START transcription amino acid Methionine (Met) and the amino acid Tryptophan (Trp) The genetic code is said to be degenerate. NB/ The sequences coding for the protein N in this activity are shown as sense DNA rather than mRNA. 26 Cracking the Flu Code STUDENT ACTIVITY Now you will use Comparative Genomics to Look for changes or mutations in the gene for influenza N antigen. Use the student instructions And Answer the questions in your student worksheet as you complete these activities. 27 N Sequences Click on the ‘i’ button above to open the text file containing the N sequences you will use in this activity. Save this file to your desktop for later use in Biology Workbench. Click the URL below to open The Biology WorkBench to run sequence alignments: 1. 2. 3. http://workbench.sdsc.edu/ Follow the student instructions to: Import the N sequences from the text file on your desktop into Biology WorkBench, and Run a multiple sequence alignment. Genetic Code The button on the left will take you back to the slide containing the genetic code used for translating your protein sequences. 28 INVESTIGATING MUTATIONS TEACHER LED DISCUSSION 29 Mutations Mutations are events which change the sequence of DNA. They can be very small changes (1 base pair) or very large changes (hundreds of base pairs). Mutations can involve base substitutions, deletions or insertions. They can also involve sequence inversions. Mutations can: • affect how genes are expressed • how RNAs fold • how mRNAs are spliced (introns removed), and • how chromosomes segregate. We will discuss the impact of mutations on proteins: Substitution, deletion, insertion and inversion events Diagram showing different mutation events in DNA. Source: http://www.uoguelph.ca/mbgwww/courses/94200/Toxicology3.html 30 Point Mutations Single nucleotide mutations or Point Mutations and their Consequences on the genetic code: -Synonymous mutation: code for same amino acid (i.e. the genetic code is degenerate) GCU – Alanine GCC - Alanine -Missense mutation: codes for different amino acid - Conservative: chemically similar amino acid (Lys Arg) AAG – lysine AGG – Arginine - Nonconservative: chemically different amino acid (Phe Ser) UUC – Phenylalanine UCC - Serine -Nonsense mutation: stop codon UGG – Tryptophan UAG - STOP 31 Frameshift Mutations Frameshift mutations occur when nucleotides are inserted into or deleted from a coding sequence. The worst mutations occur when insertions and deletions are not in multiples of three. A frameshift mutation in the sequence of the flu’s NA gene would alter the active site of this enzyme drastically. Sialic acid residues would no longer bind with the active site. The virus would stick to the host cell (see diagram below) so it could no longer spread. The reading frame changes and the amino acids being added downstream of the mutation alter drastically. This changes the shape of a protein and generally stops it from doing its job. Source: http://www.roche.fr/rochefr/planete/internet/in ternet.jhtml?ssRubrique=1700014 32 Investigating Mutations STUDENT ACTIVITY Go back into Biology Workbench to: Run a multiple sequence alignment using H3N2, H1N1 and H5N1 influenza A viral sub-strains. Use the sequence alignment to construct a phylogenetic tree. Follow the student instructions And Answer questions in the student worksheet 33