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Mutations Permanent changes of DNA that can be passed on to offspring if they occur in cells that produce gametes Increase variation in populations Types of mutations 1. Chromosomal 2. Genetic 1) Chromosomal Mutations Chromosomal mutations include: • Change of number • Change of structure Changes in Chromosomal Number Monosomy: individual inherits only one homologue (one of the homologous pair) Trisomy: individual inherits 3 homologous chromosomes of one pair. • Occur in plants and animals and is generally lethal • Happens because of a mistake in meiosis called non-disjunction. Nondisjunction Non-lethal human monosomies and trisomies • Turner syndrome: female with single X chromosome • Down syndrome (trisomy 21): most common, three copies of 21 chromosome Polyploidy Offspring end up with more than two complete sets of chromosomes New zygote goes through multiple replication events during s-phase • Terms indicate how many sets of chromosomes are present (triploids[3n], tetraploids[4n]) • Major evolutionary mechanism in plants (few animals) responsible for 47% of flowering plants. Polyploidy Change in Chromosomal Structure Environmental factors (radiation, chemicals, and viruses) can cause chromosomes to break When? How? If broken ends do not rejoin in the same pattern = structural chromosomal mutation Chromosomal Mutations Genetic Mutations A permanent change in the sequence of bases in the DNA Consequences of Genetic Mutations They vary in their phenotypic consequences depending on how protein activity is affected. Germ-line mutations are passed on to offspring (raw material for evolution) Somatic-line mutations only affect that organism, but will not be passed on to other generations. Causes Some are spontaneous (no apparent reason) • Rare due to DNA polymerase proofreading new strand during DNA replication. (1 to every 1 billion nucleotide pairs) Some due to environmental mutagens (increase chances). • Examples include radiation (UV) and organic chemicals (nicotine). Types of Genetic Mutations 1) Point mutations: changes or substitutions in one or a few nucleotides in the sequence of DNA. - usually affect a single amino acid in the protein. Base–pair substitution. Mutations are changes in DNA, but they are represented here as they are reflected in mRNA and its protein product. Base–pair substitutions may lead to silent, missense, or nonsense mutations. Types of Genetic Mutations 2) Frameshift Mutations: When a nucleotide is either inserted or deleted from the sequence in the DNA. • By changing the position of codons, these mutations may change every amino acid the follows the mutation. Strictly speaking, the example at the bottom is not a point mutation because it involves insertion or deletion of more than one nucleotide. QOD The template strand of a gene contains the sequence 3′–TACTTGTCCGATATC–5′. Draw the double strand of DNA and the resulting mRNA, labeling all 5′ and 3′ ends. Determine the amino acid sequence. Then show the same after a mutation changes the template DNA sequence to 3′–TACTTGTCCAATATC–5′. What is the effect on the amino acid sequence? Answer The amino acid sequence of the wild–type protein is Met–Asn–Arg–Leu. The amino acid sequence of the mutant protein sequence would be the same, because the mRNA codons 5′–CUA–3′ and 5′– UUA–3′ both code for Leu. This would make it a silent point mutation DNA Technology 1) 2) 3) 4) Science behind Analyzing DNA molecules Gene Regulation Recombinant DNA Technology 1) Genetic Engineering The direct manipulation of genes for practical purposes. Applications include the manufacture of protein products (hormones, blood clotting factors, etc.) Biotechnology: the manipulation of organisms or their components to make useful products. Biology behind genetic engineering Made possible by the discovery (1960’s) of bacterial enzymes that cut DNA molecules at a limited number of specific locations. Restriction Enzymes: naturally occurring bacterial enzymes that are used to protect them from intruding DNA from other organisms (viruses, other species of bacteria). • They work by cutting up the foreign DNA or restricting their propagation Restriction Enzymes Work by catalyzing the hydrolytic reaction of sugar phosphate bonds between specific bases. This results in DNA fragments that have either Blunt or Sticky ends. Ex. The restriction enzyme BAM III cuts sugar-phosphate bonds between A and T or visa versa. TGATCAAGCTACG ACTAGTTCGATGC Sticky ends Ex. The rest. enzy. Eco RI always cuts sugar-phos bonds b/w G and A or visa versa. TGAATTCGC ACTTAAGCG 2) Analyzing DNA DNA fingerprinting (gel electrophoresis): the technique of taking DNA samples, cutting them with restriction enzymes to make DNA fragments of different sizes, and separating them based on their size Used to identify: • mutant varieties of genes, • genetic make-up of extinct organisms, • individual’s identity from trace evidence, • and paternity cases DNA Fingerprinting Gel Electrophoresis: Technique that uses a gel as a molecular sieve to separate nucleic acids or proteins on the basis of size, electrical charge, and other properties (friction). 1.DNA samples are treated with a restriction enzyme to cut it into fragments of different sizes called restriction fragment length polymorphisms (RFLP’s) Gel Electrophoresis 2. All RFLP’s from sample inserted into a well in a gel 3. An electrical charge is applied to the gel, which causes RFLP’s to migrate down gel according to length (how? chromatography), resulting in a distinct pattern of bands based on genetic code. 4. Gel’s have many wells so that different samples can be analyzed side by side. 3)Eukaryotic Gene Regulation We all begin life from a single celled zygote with a full compliment of 46 chromosomes. Yet, through development and mitosis, cells become specialized in structure and function. Genes that code for digestive enzymes are only active in digestive cells, but that gene is still located in all of your cells; it is just turned off. Regulation Regulation of metabolic pathways can happen by: (a) Feedback inhibition of actual pathway, rapid (b) Repress the expression of genes that code for the enzymes in the pathway, long term. Operons and Regulation A single promoter serves all genes that code all enzymes in a pathway. Thus transcription will give rise to one long mRNA for all enzymes. Has many start and stop codons within. Key advantage to this is having a single “onoff switch” to control many functionally related genes. Operons and Regulation The “on-off switch” on DNA is called an operator. All together: The promoter, operator, and the genes they control is an operon. The operon is controlled by a protein called a repressor. The repressor protein is coded from a regulatory gene located upstream of the operon. The trp operon (tryptophan) fig 16.1 Repressor proteins become active by end product of path way (tryptophan in this example) - This is known as a Repressible operon Inducible Operons Opposite from repressible; repressor protein keeps operon off. (AKA repressor is active by itself) When inducer is present, it will change shape of repressor so that it can’t bind to operator, thus turning operon “on” The lac operon (lactose) fig 16.2 Lactose metabolism: inducer is allolactose which is an isomer of lactose. Genome • Genome: all of a cell’s DNA including all genes that code for protein product • In eukaryotes, there is no universal regulatory mechanism that controls the expression of coding genes. (as apposed to prokaryotes) – Regulation is possible at any point in the pathway from gene to functional protein The Four Types of Regulation 1.) Transcriptional Control: which genes are transcribed or the rate to which they are transcribed. – Nucleus – Could involve organization of chromatin or the use of transcription enzymes The Four Types of Regulation (Cont.) 2.) Posttranscriptional Control: after [rimary mRNA transcript is formed. – Nucleus – Processing into mature mRNA, or speed with which mature mRNA leaves the nucleus The Four Types of Regulation (Cont.) 3.) Translational Control: – Cytoplasm – How long the mature mRNA lasts in the cytoplasm or ability to bind to ribosomes – Possible that certain mRNA molecules may need additional changes before translation occurs The Four Types of Regulation (Cont.) 4.) Posttranslational Control: – Cytoplasm – Occurs after protein synthesis – Polypeptide may have to go through additional changes before it is biologically functional – Could be subject to feedback control • Regardless of the mechanism of gene expression in eukaryotes, there is cellular control of the amount and activity of gene products. 4) Recombinant DNA (rDNA) rDNA is engineered to contain DNA from two different sources. To make rDNA engineers need to use a vector. Vectors are organisms or substances that insert their DNA into other host organisms Viruses or Plasmids Plasmids are small accessory rings of bacterial DNA used for sexual reproduction during conjugation rDNA To engineer rDNA, a foreign gene and a plasmid (vector DNA) are treated with the same restriction enzyme (one that produces sticky ends) The two segments are joined by using DNA ligase. Recombinant plasmids can be inserted back into bacteria, to reproduce asexually (how?) This will make many copies of the rDNA so we can harvest their protein product Recombinant plasmids can also be inserted into other cells to incorporate the rDNA to their genome Using viruses as vector DNA can also be employed to incorporate new genes into patients - Gene therapy