Intro to Life Processes - Chapter 22

DNA and Biotechnology

DNA

• deoxyribonucleic acid

• the chemical used to encode genetic information for most known life forms

Double helix

• the shape in which DNA is formed into

• Two helixes attached by complementary base pairs

Polynucleotide

• each helix is this

• composed of a series of nucleotides

Nucleotide

composed of three parts - phosphoric acid, pentose sugar (deoxyribose), a nitrogen-containing base (A, C, G, or T)

Complementary base pairing

• nucleotides exhibit this

• A and T form two hydrogen bonds

• G and C form three hydrogen bonds

• Allows for A and T and G and C, respectively, to pair

DNA Replication

copying the DNA before replication

• when cells divide, each cell gets an exact copy of the DNA

• the double-stranded nature of DNA allows for each strand to serve as a template for each copy of the DNA

Major Events in Replication

1. DNA helicase unwinds double-stranded DNA

2. New complementary DNA nucleotides are fit into place and joined by DNA polymerase

3. Antiparallel configuration means synthesis occurs in opposite directions (leading strand follows helicase enzyme, lagging strand forms Okazaki fragments)

4. DNA ligase seals and breaks in the sugar-phosphate backbone, and DNA returns to its coiled structure

5. The two double helixes are identical to the original DNA molecule

Mutation

when an error during replication occurs and is not corrected

• mutations are a source of novel diversity in populations

RNA

• ribonucleic acid

• made up nucleotides and has a ribose backbone

• has four nucleotide bases:

  • Adenine (A)

  • Uracil (U)

  • Cytosine (C)

  • Guanine (G)

DNA and RNA Similarities

• nucleic acids

• composed of nucleotides

• sugar-phosphate backbone

• four different types of bases

DNA and RNA Differences

DNA

• Found in the nucleus

• Stores genetic information

• Deoxyribose sugar base

• Bases are A, T, C, G

• Double-stranded

• Transcribed to produce RNA

RNA

• May be found throughout the cell

• Assists in the processing of genetic information

• Ribose sugar base

• Bases are A, U, C, G

• Single-stranded

• Involved in gene regulation/protein production

Types of RNA

messenger, ribosomal, transfer

Messenger RNA (mRNA)

• is produced in the nucleus, where DNA serves as a template for its formation

• carries genetic information from DNA to the ribosomes in the cytoplasm (where protein synthesis occurs)

Ribosomal RNA (rRNA)

• is produced using DNA as a template in the nucleolus, where DNA serves as a template for its formation

• the subunits then leave the nucleus and combine with proteins to form the large and small subunits of the ribosome

• the large and small subunits combine to form a ribosome complex (the ribosome)

• the ribosome is the location where protein synthesis in the cell takes place

Transfer RNA (tRNA)

• is produced in the nucleus, where DNA serves as a template for its formation

• transfers amino acids to the ribosomes in the order dictated by the mRNA

• at the ribosomes, the amino acids are bonded together in the correct order to form a protein

• clover shaped with a binding site for an amino acid

Structure and Function of proteins

• proteins are polymers composed of amino acids

• there are 20 different amino acids that make up most proteins

• proteins differ because the number and order of amino acids of which they are made

How does coding in DNA get turned into proteins in a cell?

transcription and translation

Transcription

the process by which an mRNA strand forms a complementary strand to the DNA template

Translation

the process by which an mRNA strand is read by a ribosome, and tRNA is used to build a protein based on the mRNA coding

Codon

three base sequence of mRNA that corresponds to a specific amino acid (there are 61 codons, and 20 amino acids)

Transcription - Forming mRNA

1. RNA polymerase: opens up the DNA helix so that complementary base pairing can occur

2. Processing mRNA: newly transcribed complementary strand needs to be packaged and edited before it is considered mature mRNA, and it is released from the nucleus

Exon

parts of the genome that are ultimately expressed

Intron

parts of the genome that are not part of any gene, and are ultimately not expressed

Anticodon

the part of tRNA that binds to the complementary mRNA codon

Translation Process

1. Initiation - mRNA binds to the smaller of the ribosomal subunits, and then the larger subunit attaches to the complex

2. Elongation - the large ribosomal subunit has three binding cites (A, P, and E)

3. Termination - when one of the three stop codons enters the A site, a protein called a release factor binds to the stop codon, and cleaves the polypeptide from the last tRNA

Pretranscriptional control

the DNA and the transcription enzymes have to get together in the nucleus

Transcriptional control

a number of mechanisms regulate which genes get transcribed, and how quickly they get transcribed

Posttranscriptional control

how mRNA is processed, and how fast it is processed before it leaves the nucleus

• can be altered to influence gene expression

Translational control

small RNAs may attach to the mRNA to enable it to bond to the ribosomes faster or slower (or not at all)

Posttranslational control

after the protein is formed, it may need to be adjusted before it is biologically functional

Transcription Factors

DNA-binding proteins that regulate how genes are expressed

• at extremes, transcription factors act like a light switch, turning certain genes on or off

• most of the time, the transcription factors act more like a dimmer switch, regulating to what degree a particular gene gets expressed

Biotechnology

the use of natural biological systems to achieve a purpose desired by humans

Genetic Engineering

the purposeful modification of the genomes of organisms (bacteria, plants, animals, etc.) to either improve the characteristics of the organism or make a biotechnology product

Polymerase Chain Reaction (PCR)

a laboratory process by which a targeted DNA segment (usually a few hundred base pairs long) can be amplified (copied)

• uses DNA polymerase (enzyme) to artificially run DNA replication in a test tube

PCR Steps

1. Denaturation - DNA is separated into two separate strands with heat (92 to 95 degrees Celsius)

2. Annealing - temperature lowered (50 to 60 degrees Celsius); oligonucleotide primers hybridize to each of the single DNA strands

3. Extension - temperature raised (~72 degrees Celsius); an engineered polymerase adds complementary base pairs to each of the single DNA strands, creating double-stranded DNA

4. Repeat steps 1-3 until there is sufficient DNA to accomplish your goal’ 30 to 35 cycles yields ~ 1 million copies of the DNA sample

DNA profiling (fingerprinting)

1. DNA is taken from a particular location (crime scene)

2. A non-coding section of DNA is identified, and the number of repeated segments in that section is determined

   • the sections used are called Short Tandem Repeats (STRs)

   • the number of copies that an individual has is directly inheritied from their parents

   • by looking at multiple STRs, we can establish a specific genomic pattern that is unique to an individual (unless they have an identical twin)

   • the FBI’s CODIS system uses 13 STRs and a marker for sex

3. DNA samples are taken from the suspects

4. PCR is run on the CS sample, and the suspects, and the resulting DNA is put into wells on an electrophoresis gel

Cloning

the production of identical copies of DNA, cells, or organisms through an asexual means

Gene Cloning

the process that produces many identical copies of the same gene

Recombinant DNA (rDNA)

DNA that contains genes from more than one source organism

Vector

• a means by which the genes of interest can be inserted into a host cell (such as a bacterium)

• the required technician in order to create recombinant DNA

Plasmid

• small accessory ring of DNA found in bacteria that often hold genes for antibiotic resistance

• ^ the ring is not part of the bacterial chromosome and replicates on its own, thus plasmids are commonly used as vectors to make rDNA

Restriction Enzyme

an enzyme used to cut a DNA molecule at a specific DNA sequence

• restrict the growth of viruses

DNA ligase

an enzyme used to seal breaks in the DNA backbone

• ligase is used to seal foreign DNA into a plasmid

Complementary DNA (cDNA)

a DNA molecule that has had the introns removed

Genome editing

targeting specific sequences in the DNA for removal or replacement

CRISPR

• clustered regularly interspaced short palindromic repeats

• first discovered in prokaryotes, where bacteria use it as a form of immune defense against invading viruses

• based on using an enzyme called Cas9 (breaks DNA at a specific point, but breaks both strands of the DNA at that location

Produce medical products - Genetically Modified Bacteria

• insulin

• clotting factor VIII

• human growth hormone

• tissue plasminogen activator (clot buster)

• hepatitis b vaccine

Produce agricultural products - Genetically Modified Bacteria

• frost - bacteria that prevent fruits from freezing

• root bacteria now produce toxins that are harmful to insects

Produce environmental products - Genetically Modified Bacteria

• bacteria that metabolize oil

• bacteria that can remove the sulfur from coal before it is burned

Agricultural Products - Genetically Modified Plants

• insect resistant everything

• herbicide resistant everything

• fruits with increased shelf life

• fruits that are damage resistant

• crops with higher yields

• heat resistant crops

• drought resistant crops

• crops with greater nutrients

Medical Products - Genetically Modified Animals

• salmon that grow faster

• sterile mosquitos

• cattle/goats with health products in their milk (human growth hormone, treatments for cystic fibrosis, cancers, blood diseases, etc.)

• non-human animal research models that mirror the test results of human trials

• xenotransplantation livestock

Xenotransplantation

the use of non-human animal organs from transplant into humans

Functional genomics

the study of how our genes are controlled and how they work together to form an organism

• identifies coding vs non-coding areas

• determines which genes do what

• determines which non-coding areas play a role in the production of small RNAs and the regulation of genes

Comparative genomics

the study of how genes of different related organisms differ from each other, and how these differences evolved

Proteomics

the study of the structure, function, interaction of cellular proteins

• a single gene can code for several hundred proteins

• all of the proteins that the human genome codes for is called the human proteome

• computer modeling is required to determine tertiary and quaternary structures for many proteins

• essential to understanding the causes and treatments for many diseases

Bioinformatics

the application of computer technology to the study of the genome

Gene therapy

• the insertion of genetic material into human cells for the treatment of a disorder

• has been successfully used to treat:

  • genetic errors that regulate metabolism

  • cardiovascular disease

  • some cancers

  • one form of inherited blindness

Ex Vivo Gene Therapy

• the removal, genetic alteration, and reinsertion of stem cells from a patient

• has been used to successfully treat:

  • hemophilia A

  • Alzheimer’s disease

  • Parkinson’s disease

  • Crohn’s disease

  • and some cancers

SCID

• sever combined immunodeficiency disorder

• causes patients to be unable to produce an enzyme (ADA) that is necessary for B and T white blood cells to mature

• has been successfully treated with ex vivo gene therapy using a retroviral vector

In Vivo Gene Therapy

new genes, carried in a vector, are injected directly into a patient to treat a genetic disorder

• uses a retrovirus or an adenovirus as a vector to carry the corrective DNA

• can be injected, sprayed in the nose, or placed in the lower repsiratory tract

• minimally invasive and generates quick results, but effects are not as long lasting as ex vivo therapy