biotech test #3 dna things, protein synthesis, recombinant dna, tissue culture

Genes

- A gene is a specific nucleotide sequence that is transcribed into RNA. - Structural genes encode proteins but initial transcription product is mRNA. - All genes are made up of three regions: Promoter, Coding and Terminator. - Prokaryotic mRNA is devoid of introns while Eukaryotic mRNA contains introns and exons. - Multiple proteins can be made from a single prokaryotic gene because they can contain multiple Sigma Subunit / Promoter sequence regions.

Plasmid host cells require the following features:

- Absence of Restriction endonucleases - Cells do not carry out plasmid exchanges - Cells lack the endonuclease encoded by the End A1 gene. These cells have increased transformation frequencies.

tRNA and Translation

- Cells actively synthesizing proteins have about 60 different types tRNA. - tRNA molecules are from 75 to 93 nucleotides in length. - Each tRNA forms a folded, L shaped structure. -Transcription and translation are not spatially separated in prokaryotes. They occur concurrently. - In eukaryotes, a mature mRNA leaves the nucleus via special pores. - Introns are removed before mRNA leaves the nucleus. - Translation requires the interaction of mRNA, charged tRNAs, ribosomes, and a large number of proteins that facilitate the inititaion, elongation and termination of the polypeptide chain

Host Cell Plasmid Uptake

- Cells that can take up plasmids are "competent" - Competent cells normally take up plasmids during conditions of high cell density or starvation - Natural transformation sequence is: -Binding of double stranded DNA to components of the cell wall -Entry of the DNA into the Periplasm where it is protected from nucleases -One strand is transmitted into the cytoplasm and one strand is degraded -If the DNA is linear, it is integrated into the host chromosome -Competence can be induced to a cell via cold CaCl treatment or heat shocking - Transformation frequency and efficiency determine the success of transformations. - Transformation efficiency is about 0.1 % of cells

Cosmids

- Cloning vectors called cosmids can carry about 45 kb of cloned DNA and are maintained as plasmids in E. Coli. - They combine the properties of plasmids and bacteriophage λ vectors. - A Fosmid is a kind of cosmid that carries up to 40 kb of insert DNA and a cos site for in vitro bacteriophage λ packaging. - The origin of replication of a fosmid is derived from the E coli F factor (sex plasmid), hence the name fosmid. - Fosmids are very stable single copy vectors while cosmids are maintained at higher copy numbers, which leads to deletions or rearangements of parts of the insert DNA.

Evoluitonary Significance II

- DNA Ligase is needed to bind sticky ends together - DNA Ligase catalyzes the phosphodiester bonds and joins the blunt ends. - Sticky end ligations can occur at low temps while blunt end ligations need higher temps and require 10 to 100 X more T4 DNA ligase. - Ligated constructions must contain the info for cellular maintenance - Finally, you have to be able to screen for the applicable DNA constructs and perpetuate them.

DNA Probes

- DNA Probes are labeled with radioisotopes or fluorescent tagging systems - The random primer method of labeling uses a mixture of synthetic random oligonucleotides containing all possible combinations sequences of 6 nucleotides (hexamers) that act as primers for DNA synthesis. - dNTP is deoxyribnucleoside triphosphate - dNTPs that contain the isotope 32P in the alpha position phosphate. - These dNTPs are incorporated into DNA via PCR - Immunological assays are used to screen a library when a DNA probe is not available.

The action of the different hormones / regulators is not consistent:

- Different plants will respond to the same chemical differently. - Different plant parts from the same plant can respond differently

Exons

- Exon skipping mechanisms generate different gene products in different tissues from the same structural gene. - Exons tend to be 150 to 300 bases in length. - Spliceozymes are the enzymes involved in selection of the exons to be used in mRNA synthesis.

Plasmid formation sequence

- First, the plasmid cut with a restriction enzyme that lies within one of the antibiotic resistance genes - The linear molecules are combined with prepared target DNA - Mixture treated with T4 DNA ligase - Treatment with alkaline phosphatase removes unwanted ligation products - Alkaline Phosphatase removes the 5' phosphate groups from the linearized plasmid DNA - T4 then causes circularization of the plasmid Post transformation, nicks in DNA are sealed by host cell DNA ligase system.

Transcription Regulation in Eukaryotes

- Genes in particular organs are situated in specific locations in the Nucleus in order to make more of the proteins that give the organ its characteristics. - Transcription control is mediated by proteins that are collectively classified as transcription factors. - Operons are normally not found in Eukaryotes. - Each Eukaryotic structural gene has its own set of response elements. - DNA-protein interactions and protein-protein associations are important for regulating transcription. - A eukaryotic promoter consists of a TATA sequence (Hogness box) a CCAAT ("cat" box), and a sequence of repeated GC nucleotides.

Genomic Libraries

- Genomic libraries contain proteins for industrial, agricultural and medical applications. - In Prokaryotes, structural genes form a continuous coding region in the genomic DNA. - In Eukaryotes, exons are separated by introns - The process of subdividing genomic DNA into clonable elements and inserting them into host cells is called creating a library. - After a library is created, the clone(s) with the target sequence must be identified. - DNA probes target nucleotide sequences in a DNA sample - DNA hybridization is possible because DNA can be split into single strands by heat or alkali treatment - Heated strands need to be rapidly cooled to keep the strands separate - The process of heating and slowly cooling double stranded DNA is called annealing

Protein Secretion in Gram Positive Bacteria

- Gram Positive Bacteria have a single main secretory pathway. - In some situations, secretory proteins make direct contact with a membrane-bound assembly of proteins (a secretion complex or Sec complex) - For other Sec proteins a group of proteins called a signal recognition complex binds to a signal peptide. - This combination then attaches to a membrane bound signal recognition complex receptor before making contact with a Sec complex.

Protein Secretion in Gram-Negative Bacteria (Type II Pathway)

- Gram-negative bacteria have multiple pathways for the secretion of various proteins. - In the Type II Pathway the SecB protein binds to a secretory protein in the cytoplasm. - The SecB attaches to the Sec A protein that is part of the Sec complex of the inner membrane. - The Sec protein is then transported through the inner membrane. - The protein is properly folded in the periplasm. - Finally, the protein combines with the Gsp complex and is translocated into the external environment.

Vectors for Cloning Large Pieces of DNA

- High capacity cloning systems (15 to 20 kb) utilize bacteriophage λ Vectors - This comes from the E coli phage λ - After the bacteriophage λ infects E.coli by injection of its DNA, it can either cause a lytic cycle where after 20 minutes the cell lyses and 100 phages are released or a lysogenic cycle occurs where the DNA is incorporated into the Bacterial chromosome. - Under conditions of severe stress the phage DNA is excised and a lytic cycle ensues. - The bacteriophage DNA has about 20 kb that are essential for integration and excision of the DNA, which leaves about 20 kb that can be replaced by cloned DNA. This recombined DNA molecule can be perpetuated as a "recombinant" bacteriophage λ through compulsory lytic cycles.

Bacterial Operons

- In bacteria, gene regulation is done through operons. - An operon is a cluster of genes found in prokaryotes that are coordinately regulated. - Nucleotide sequences in and around the RNA polymerase binding site often play essential roles in determining whether an operon is transcribed. - Operons are regulated by effector proteins that are either normally found blocking transcription or normally are found unattached to the promoter sequence. - A repressor protein binds to an operator region and prevents promoter sequence binding.

Vegetative (asexual) Propagation Occurs in Nature

- Layering - a drooping lower branch contacts the soil (pressed down by snow or vegetation); roots form at point of soil contact forming a new genetically identical tree - When trees of some species are cut down, new shoots emerge from the stump - strawberries spread through sending out above-ground horizontal shoots called runners, also called stolons.

Micropropagation

- Many genetically identical plants are produced from ONE PLANT - A single node will produce a shoot within 4-6 weeks that has 4-6 nodes. - Each plantlet can be "subcultured" to produce another 4-6 plants each. - Hundreds of thousands of plants could be developed from one node - Since these are produced from axillary buds, the plantlets will be clones of the mother plant.

Bacterial Transcription

- Many bacteria are able to produce several sigma factors, each of which recognizes a different promoter sequence. These initiate different subsets of genes. - Repressor proteins bind to operons and prevent the associated gene from being transcribed. - Repressors release their hold once they encounter the molecule that the enzyme in question is coded for is available. It links to the repressor and it releases the DNA strand. - Once the molecule is no longer available, the repressor clamps again on the promoter sequence. - Use of a repressor is a negatively controlled system.

Transcription Regulation in Bacteria (Prokaryotes)

- Only the proteins that are needed for maintenance of basic cellular functions are expressed continuously. These are the "housekeeping" genes. All other genes have their expression regulated. - A Shine-Dalgarno sequence precedes each start codon. - For many bacterial structural genes, the promoter region has two DNA binding sites for RNA Polymerase. - These are recognized by the component of RNA pol known as the Sigma Factor. - The sites have the sequences TATAAT (a Pribnow box) and TTGACA.

DNA and Plasmid Mapping

- Physical maps that designate the relative positions of restriction endonuclease sites on a specific piece of DNA. - They are constructed by treated the DNA molecule singly with different endonucleases and then with combinations of the same Res. - The positions of the cleavage sites are determined from an analysis of fragment sizes. - The fragment sizes are determine by gel electrophoresis fragment separation.

Micropropagation vs. Regeneration (Micropropagation)

- Plants that develop from existing terminal or axillary buds are true clones of the "mother plant". - Meristematic buds tend to be protected from variations caused by environmental factors. - Little or no variation usually occurs when this method of tissue culture is used.

Plasmid Cloning Vectors

- Plasmids are double stranded circular DNA molecules that are independent extrachromosomal entities. - 1 to 9 copies per cell = low copy plasmids - 10 to 100 copies = high copy plasmids - Plasmid DNA makes up 0.1 to 5.0 % of the bacterial DNA - Some plasmids can only replicate in one species of host cell = narrow host range - Plasmids that can replicate in many cell types = broad host range - Shuttle Vectors are vectors with a second origin of replication that enables the plasmid to replicate in alternative species host cells.

Micropropagation vs. Regeneration (Regeneration)

- Regeneration using callus can produce variants of the original plant - Non-meristematic cells are not protected from variations caused by the environment. - When these cells experience a mutation, the plants that are produced from them will also show a difference from the "mother plant".

Protein Secretion Pathways in Prokaryotes

- Secretory proteins are needed for getting nutrients, communication, protection, and cell structures created within and outside of cells. - The main barrier to the release of these proteins is the cell membrane. - Gram-negative bacteria have inner membrane, periplasmic space, and an outer membrane, while gram-positive bacteria have only to move the proteins across a single cytoplasmic membrane. - Secretory proteins in eukaryotic organisms need many modifications, like glycosylation, sulfation, and phosphorylation.

Transcription

- Some repressed structural genes are activated by a cascade of events that is triggered by a specific extracellular signal, such as a temperature increase or the presence of a hormone. - Some proteins bind to response elements and prevent transcription. - In general, regulation of transcription in eukaryotes is complex. - Some structural genes are under the control of unique transcription factors. - Some proteins obstruct transcription by binding to the transcription process either before initiation or during the elongation process.

A Genetically Engineered Plant has a New Gene

- Squash gets coat proteins from Cucumber or zucchini virus to prevent viral disease (to vaccinate the squash) - Corn gets a gene from bacteria that allows it to survive in the presence of weedkiller (one that is less harmful to environment that other herbicides). - Corn gets a gene from different bacteria that allows it to produce its own pesticide (protects against insects). - After a new gene is given to a plant, scientists must figure out how to regenerate the transformed plant cell.

Transferring DNA into E. coli

- Standard practice is treating mid log phase cells with ice cold Calcium Chloride, then exposing them to a 2 minute bath at 42 degrees C. - This creates transient openings in the cell walls that allow DNA to enter the cytoplasm. - Transformation efficiency is at best 1 per 1,000 - Electroporation subjects the cell to a high voltage electric field in the presence of DNA. - Electroporation protocols vary by bacterial species - Electroporation is an effective way to transform E. coli with plasmids containing inserts longer than 100 kb.

Vector Function Strategies

- Suicide genes: Vector carries a gene that when expressed kills the cell - The cell killing gene is infused into the correct reading frame to the LacZ gene - Cells with a plasmid and no IPTG (an inducer of the lac operon) in the medium does not synthesize the suicide protein. - In this case, only surviving in the presence of IPTG and antibiotic carry a plasmid with a DNA insert - All vectors need to have a choice of cloning sites and an easy way of identifying cells with plasmid/cloned DNA Constructs

Regeneration

- The process whereby a part of a plant can be turned into a whole new plant - differentiated tissue: stems, leaves, roots, etc. - undifferentiated (embryonic) cells are totipotent: can become a whole new plant by differentiating into a whole new plant.

Protein secretion in Eukaryotes

- The signal sequence of the secretory protein is bound by a signal recognition particle during protein synthesis. - The signal recognition particle attaches to a receptor membrane of the ER, and the Secreted Protein passes thru a channel in the membrane as translation proceeds. - A signal peptidase removes the signal sequence, and the Sec Pro is released into the lumen of the ER, where it is folded and if needed glycosylated. - A vesicle containing the protein buds off from the ER and moves to the Golgi Ap, moves to the plasma membrane and then goes out into the environment.

Codons

- There are 64 Codons, 3 Stop Codons, - 1 Codon each for methionine and tryptophan. - 2 codons each for asparagine, lysine, cysteine, tyrosine, phenylalanine, glutamine, glutamic acid, aspartic acid, and histidine. - 3 codons each for isoleucine. - 4 codons each for alanine, glycine, valine, threonine and proline. - 6 codons each for serine, arginine and leucine. - Codon usage varies between Prokaryotes and Eukaryotes.

Restriction Endonucleases

- There are five types of restriction enzymes - Type II are the endonucleases used in transgenics - They bind to a DNA region with a specific palindromic sequence - Most commonly used is EcoR1. It is a homidimeric protein. (made of two identical proteins) - Recognition sequence has six base pairs - Cleaves between the oxygen of the 3' carbon of the sugar and the phosphate group attaches to the 5' carbon of the sugar of the adjacent nucleotide - Most Type II Res cut within their recognition site. - EcoR1 produces two 4 nucleotide sticky ends - Lengths of recognition sites can be 4,5,6,8 or more nucleotide pairs - We use mostly 4 and 6 nucleotide recognition site Type IIs for DNA recombination.

Evolutionary Significance of Restriction Enzymes In Molecular Cloning

- They cleave foreign DNA from bacteriophages - Bacteria have evolved systems that protect their own DNA from being cleaved - Protection involves methylation of cytosine residues, which protects them from attack - Many REs can bind to the same recognition sites and cleave at different positions. - An RE can cleave more than one site within a plasmid - REs are not the only proteins needed for successful molecular cloning.

Translation in Prokaryotes

- Translation begins by the binding of a small ribosomal subunit to and mRNA by a base pairing of a Shine-Dalgarno sequence (about 8 nucleotides long) that is located near the 5' end of the mRNA and the 3' end of the rRNA small sub-unit. - The translation begins with the 3'-AUC-5' anticodon of a specific initiator tRNA, fMet-tRNA. - A large ribosomal subunit then combines with this to form the initiation process.

Translation in Eukaryotes

- Translation is initiated by the binding of a particular charged tRNA, Met-tRNA, to the small ribosomal subunit. - The 5' capped end of an mRNA associates with the initiator tRNA . - The complex migrates along the mRNA unit until and AUG is encountered. - The migration stops, and the large subunit then joins the complex to form the initiation complex. - Elongation and termination are very similar in pro and eukaryotes.

Translation

- Translation occurs in a 5' to 3' direction along the mRNA at a rate of about 15 amino acids per second. - A single mRNA can be translated simultaneously by a number of ribosomes, with each ribosomes producing a polypeptide chain. - Elongation continues until a UAA, UAG, or UGA codon is encountered. - After translation, a protein may be modified in many ways. - In Prokaryotes and Eukaryotes, the methionine at the N terminus is cleaved from most proteins.

Protein Secretion in Gram-Negative Bacteria (Type III Pathway)

- Type III Pathways are use to secrete toxins and other proteins into the cytoplasm of eukaryotic host cells. - Type III Pathways are Sec protein independent. - These systems are made up of about 20 different proteins that form a continuous channel through the inner and outer membranes. - A hollow, needle like structure extends from the bacterial surface into the host cell.

Type IIS Restriction Enzymes

- Type IIS restriction endonucleases form a subgroup of the Type II category. - Type IIS Res are used for specific molecular studies, such as multiplex polony sequencing. - These Type IIS REs are unique in that they cut both strands a fixed number of nucleotides away from one end of the recognition site. - Any particular sequence of nucleotides may be present between the binding sequence and the cut (restriction) site - The cleavages for most type IISes are staggered - The notation for these is written as GGATG(9/13)

Gregor Mendel (1822-1864)

- Worked in monastery garden - Time period = 1856-1863 - Published: "Experiments on Plant Hybrids" in obscure journal - Worked 'discovered' in 1900

Strategies for Using Antibiotic Resistance

- You can use a double antiB res strategy to select out cells using a pBR322 system where the target DNA was inserted into the BamH1 site - Cells are incubated on antiB free media to allow the genes to become expressed - The BamH1 site of pBR322 is within the Tetr gene, so insertion of the DNA into this gene disrupts Tet resistance - Cells with recircularized DNA have both Amp and Tet resistance - Dilute the transformed cells - Plate on complete media with Ampicillin - Transfer to plate with complete media and Tetracycline - Keep the same locations on the plate for the colonies - Cells that form colonies on Tet plates carry recircularized pBR322 without insert DNA. - Cells that do not grow on Tet media carry the cloned DNA constructs - This is called replica plating

Callus

- a mass of unorganized cells - Callus cells are totipotent - Caused by Equimolar amounts of auxin and cytokinin stimulate cell division. - Requirement for support ensures that scale-up is limited (Ginseng saponins successfully produced in this way).

cDNA Libraries

- cDNA is a double-stranded DNA complement of an mRNA sequence; synthesized in vitro by reverse transcriptase and DNA polymerase. - A cDNA library is a collection of cDNA clones that were generated in vitro from the mRNA sequences of a single tissue or cell population. - A cDNA library is screened by DNA hybridization to identify clones that carry a specific plasmid-cDNA construct. - Standard cDNA synthesis produces complete (full length) and incomplete molecules.

Proteins

- consist of amino acids. Peptide bonds hold the amino acids in the chains together. - essential polymers that are involved in almost all biological functions. - range in length from about 40 to more than 1,000 amino acids. - four levels of protein structure

Advantages of Micropropagation

- economical in time and space - greater output -can produce millions of uniformly flowering and yielding plants - African Biotechnologies - fruit crops banana and indoor pot flowers- 6 million pieces per year - disease free - elite plants with exceptional characteristics - facilitates safer movements of germplasm across nations - In vitro germplasm assures the exchange of pest and disease free material - great for - vegetatively reproduced crops - crops which produce few seeds or highly heterozygous seeds.

Steps involved in Tissue Regeneration

- tissue must be sterile - completely free of any microoganisms; done using aseptic technique 1. Starting tissue is called an explant: differentiated cells (these cells have developed to be part of specialized tissue (root, leaf, stem, ovary, cotyledon, etc.). 3. Individual cells (or clumps of cells) of the callus are transferred aseptically to a different petri dish containing sterile medium that encourages the undifferentiated callus cells to become shoots and roots. - One mass of callus cells can be divided and transferred to many plates for development into shoots and roots. 4. Once shoots and roots have developed, they are transferred to soil and grown to maturity.

Advantages of tissue culture over intact plants

1. The biochemical engineer can grow plant cells in liquid culture on a large scale--Bioreactor 2. The production of dihaploid plants from haploid cultures shortens the time taken to achieve uniform homozygous lines and varieties 3. The crossing of distantly related species by protoplast isolation and somatic fusion increases the possibility for the transfer and expression of novel variation in domestic crops 4. Cell selection increases the potential number of individuals in a screening program 5. Micropropagation using meristem and shoot culture techniques allows the production of large numbers of uniform individuals of species from limited starting material 6. Genetic transformation of cells enables very specific information to be introduced into single cells which can then be regenerated

Totipotency

A cell characteristic in which the potential for forming all the cell types in the adult organism are retained.

Sterile

A culture that is free of viable microorganisms.

Pathogen

A disease-causing organism.

Petiole

A leaf stalk; the portion of the plant that attaches the leaf blade to the node of the stem.

Autoclave

A machine capable of sterilizing wet or dry items with steam under pressure. Pressure cookers are a type of autoclaves.

Chemically Defined Medium

A nutritive solution for culturing cells in which each component is specifiable and ideally of known chemical structure.

Medium (Media)

A nutritive solution, solid or liquid, for culturing cells

Node

A part of the plant stem from which a leaf, shoot or flower originates.

Horizontal laminar flow unit

An enclosed work area that has sterile air moving across it. The air moves with uniform velocity along parallel flow lines. Room air is pulled into the unit and forced through a HEPA (High Energy Particulate Air) filter, which removes particles 0.3 μm and larger.

Clonal Propagation

Asexual reproduction of plants that are considered to be genetically uniform and originated from a single individual or explant.

Contamination

Being infested with unwanted microorganisms such as bacteria or fungi.

Monoecious

Both sexes on same plant (e.g. corn, pecan)

Pathogenic

Capable of causing a disease.

Differentiated

Cells that maintain, in culture, all or much of the specialized structure and function typical of the cell type in vivo. Modifications of new cells to form tissues or organs with a specific function.

Adventitious

Developing from unusual points of origin, such as shoot or root tissues, from callus or embryos, from sources other than zygotes.

Aseptic

Free of microorganisms.

Hormones

Growth regulators, generally synthetic in occurrence, that strongly affects growth (i.e. cytokinins, auxins, and gibberellins)

Detergent

Increasing the efficiency of sterilization.

Somaclonal Variation

Phenotypic variation, either genetic or epigenetic in origin, displayed among somaclones.

Somaclones

Plants derived from any form of cell culture involving the use of somatic plant cells.

Clone

Plants produced asexually from a single source plant.

Aseptic technique

Procedures used to prevent the introduction of fungi, bacteria, viruses, mycoplasma or other microorganisms into cultures.

Dioecious

Sexes on different plants

Types of Propagation

Sexual: - Self-pollination -Wind pollination - Cross-pollination - Insect pollination Asexual: - Cuttings - Layering - Grafting - Biotechnology

Plant Tissue Culture

The growth or maintenance of plant cells, tissues, organs or whole plants in vitro propagation - Rapid (compared to outdoors) - Asexual Used for: 1.Micropropagation 2. Regeneration

Coconut milk

The liquid endosperm of coconut contain the cytokinin zeatin and will support the continued cell division of mature cells, leading to the formation of callus.

Sterile Techniques

The practice of working with cultures in an environment free from microorganisms.

Internode

The space between two nodes on a stem

Explant

Tissue taken from its original site and transferred to an artificial medium for growth or maintenance.

Plants that receive a new gene are called

Transgenic plants or GMOs

Haploid

a cell containing 1 (1n)set of chromosomes (pollen or ovule)

Diploid

a cell containing 2 sets (2n)of chromosomes (vegetative cell)

Polyploid

a cell containing more than 2 sets of chromosomes. Usually vegetatively reproduced.

Mutation

a heritable change in a gene

Agar

a polysaccharide powder derived from algae used to gel a medium. Agar is generally used at a concentration of 6-12 g/liter.

nucleoside

an organic nitrogen containing base molecule that is separate from the other nucleotide constituents

Explants

are plated on a sterile petri dish containing hormones and nutrients that promote the explant cells to develop into

Meiosis

cell division where nuclear complement (2n only) is reduced to 1n (gametes)

Mitosis

cell division where nuclear complement (1n or 2n) is maintained

Hybridization

controlled cross-fertilization

Independent Assortment

each chromosome operates independently during mitosis & meiosis

DNA Structure

five carbon sugar, an organic base molecule, and a phosphate group - 2'-deoxyribose is the sugar - A, T, C, G - Nucleotide subunits of DNA are joined by phosphodiester bonds - Alpha phosphate attaches to the 5', beta to the 3' - During DNA Replication, the beta and gamma phosphates are cleaved off - The chains in DNA are held together by hydrogen bonds. - The strands in a DNA molecule run opposite to each other. - DNA only goes thru replication while RNA also goes thru transcription and translation. - DNA is only found within organelles (nucleus, mitochondrion, chloroplast).

Chromosome

genetic material of cell organized in linear structures

Gene

genetic material on a chromosone determining a characteristic

Cytokinins

induces the production of shoots

Auxins

induces the production of roots

Polony sequencing

inexpensive but highly accurate multiplex sequencing technique that can be used to "read" millions of immobilized DNA sequences in parallel. This technique was first developed by Dr. George Church's group at Harvard Medical School.

Plant Hormones

naturally occurring chemicals that influence plant growth.

Culture

plant growing in vitro.

RNA

same structure as DNA, ribose is the sugar - A, U, C, G - Three main forms: m, t, and r RNA, rRNA has two subunits.

Growth Regulators

synthetic versions of hormones.

Genotype

the genetic makeup of an organism

Phenotype

the physical manifestation of the genotype modified by the environment