Biology 202
Unit 2A
Genetic Control and Genetic Engineering

B. Krumhardt, Ph.D.

Genetic Control

Regulatory Genes in Bacteria

¥¥encode for proteins that regulate the activity of structural genes

¥¥makes cells more efficient - they make only the RNA/proteins they need

¥¥e.g. - the enzyme for metabolism of the sugar galactose is not made if galactose is not present

Operon

¥¥group of structural genes coding for a enzymes in a metabolic pathway

OPERON

¥¥DNA: transcription 
REGULATOR...... PROMOTOR-OPERATOR-STRUCTURAL
 GENES FOR
  ENZYMES
 RNA 
mRNA polymerase REPRESSOR
 binds binds - prevents REPRESSOR transcription

¥¥Operator

––on/off switch for transcription of structural genes

––located upstream of structural genes

Operon examples

¥¥Inducible operon model

––metabolite binds repressor; this combo can't bind operator, transcription ensues

––metabolite = inducer (it builds up when enzymes coded are needed)

––e.g. lac operon LAC

¥¥Repressible operon model

––repressor binds only when corepressor metabolite attached

––product of the enzymes coded for by the structural genes

––E.g. trp operon TRP

Organization of Eukaryotic DNA

¥¥Chromatin packing

––Only ÒunpackedÓ chromatin can be transcribed

––Only partially unpacked during interphase

––Coarse transcription control

¥¥Post-transcriptional processing

––Introns - non-coding (for protein) sequences in DNA

––allows for alternative processing of exons (transcribed and translated regions)

––processing in nucleus - splicing (post-transcriptional)

¥¥ introns removed, can be site of control

¥¥Repetitive Sequences

––sequences of repeated DNA

––Can be introns

––May be essential for cell division (sequences at centromeres and telomeres

––Perhaps allows reorganization of DNA without disruption of operons
e.g.. cross-over

Genetic Mutations

¥¥Rearrangement of genes - may\may not affect expression

¥¥Genetic Mutations - changes in DNA sequence in cells

––if new bases change codons  different amino acid sequence  maybe a nonfunctional protein

––Germinal mutation - in germ cell line - passed to offspring

––Somatic mutation - in cell line other than germ cell - not passed to offspring

––point mutation - only one DNA base wrong

Cancer

¥¥uncontrolled growth that spreads throughout body

––in plants - callus  mass of undifferentiated cells

––in animals, spreading = metastasis  form unspecialized cell masses, tumors; lose contact inhibition  more tumors and loss of normal functions

¥¥Oncogenes - cancer causing genes

––normal genes, but the regulation is changed due to a mutation - before loss of regulation - "proto-oncogenes"

––normal gene required for tissue\organ development - usually turned off in specialized cell

¥¥Mutagens - carcinogens - cause genetic mutations

––e.g. chemicals, viruses, x-rays

––e.g. - retroviruses

¥¥RNA not DNA is genetic material

¥¥Brings its own enzyme, reverse transcriptase, to make DNA from RNA to the infected cell; has no editor, mistakes occur

¥¥then DNA inserts into host DNA  may cause cancer because of disruption of normal genetic control

¥¥Usually additional mutations are necessary for cancer to occur

Control of Normal Cell Growth

¥¥growth factors - proteins made by cells

–– bind cell membrane proteins of other cells

–– they can either stimulate or inhibit cell cycling (mitosis)

––usually occurs between different types of cells

¥¥contact inhibition - cells touch  inhibition of mitosis

¥¥hormones can work as growth factors

Basics of
Genetic Engineering

Genetics technology advancing at the Òspeed of lightÓ

Restriction Enzymes

¥¥enzymes made by bacteria that break DNA into discrete pieces

¥¥very specific

¥¥E.g. Double strand DNA cut by a restriction enzyme, EcoR1:
C G|A A T T C T A C C
G C T T A A|G A T G G

––Notation - G/AATTC

––After digest, Òsticky endsÓ are left

¥¥Enzyme looks for specific strand and always cuts it the same way

¥¥Several spots in the genome of an organism can be cut by the same restriction enzyme  restriction fragments

¥¥Restriction digest

¥¥Electrophoresis - separates DNA by size, smaller pieces move further through the gel toward the positive electrode as current passes through a buffer

––Ethidium bromide makes bands (discrete pieces of DNA) fluoresce under UV light

––Electrophoresis

Restriction Fragment Analysis

e.g. Restriction digest of Viral DNA

¥¥Characteristic length pieces of DNA made when normal viral DNA is with a certain restriction enzyme

¥¥Mutant virus may have a restriction enzyme cutting site lost

¥¥"DNA Finger printingÓ
or Restriction Fragment Length Polymorphisms (RFLP)

¥¥E.g.
Viral Mutant
DNA viral DNA
- -
-
- -
-
-
- -

¥¥RFLP markers may indicate the site of alleles associated with genetic diseases

Human DNA fingerprinting

¥¥Different restriction enzymes used to cut human DNA

¥¥each individual has a characteristic chromatogram  a "fingerprint"

Polymerase Chain Reaction

¥¥amplifies small quantity of DNA, just need to know primer sequence

¥¥Process

––1. Melt DNA with heat

––2. Primers base-pair as tube cools

––3. High temperature stable DNA polymerase  duplicates the DNA

––4. melt, prime, cool, duplicate....

¥¥One copy produces billions in a few hours

¥¥PCR

ÒCloningÓ

¥¥Using these techniques in a different way:

––use restriction enzyme to cut out Ògene of choiceÓ from donor DNA; insert this into a ÒvectorÓ

––Vectors used to transfer gene to a host cell that can express the gene

––Cloning a gene

Vectors

¥¥plasmids

––most commonly used vectors

––extra-chromosomal DNA in bacteria containing antibiotic resistance genes

¥¥in nature they can transfer antibiotic resistance from one bacterium to another

––Commercially-available plasmidÕs restriction sites & antibiotic resistance genes are mapped

––use same restriction enzyme to cut plasmid and cut out gene of choice

¥¥may cut out a ÒmarkerÓ gene

¥¥Same restriction enzymes used, same sticky ends on both plasmid and Ògene of choiceÓ

––Put together with DNA ligase  gene of choice now in plasmid - called "recombinant DNAÒ

––Put recombinant plasmid in bacteria

––Bacteria multiply quickly  possibilities

¥¥bacteria make the protein coded for by your gene of choice - isolate it

¥¥"cloning" = when plasmid has multiplied  multiple exact copies of gene - "clones"

¥¥Can accumulate lots of the cloned gene of choice to use as research tools (cut it out of plasmid with same restriction enzyme)

––e.g. Heat plasmid -- DNA double strand will separate, use it as a probe of unknown DNA--it will anneal only with specific DNA sequence by base-pairing--used to detect presence/absence of gene

Other Vectors

––Disabled virus

¥¥some viruses insert into host DNA

¥¥Used to introduce new genes into plants

¥¥Gene therapy with disabled and engineered viruses - take out disease-causing genes & replace them with working human genes  give to person who doesn't have the working gene - potential use in all genetically inherited diseases

––Bacteriophages

¥¥Viruses of bacteria

¥¥Cut with DNA with restriction enzyme and cut other DNA with same enzyme

¥¥Ligate and infect the bacterium with the recombinant phage

¥¥Useful tool for production of a genomic library

Human Genome Project

¥¥Linkage Mapping - ID location of 3000 genetic markers using RFLPÕs

¥¥Chromosome walking - order fragments made with different restriction enzymes

¥¥Sequence fragments  common database

¥¥Faster, alternative strategy - shotgun approach

––Cut DNA with several restriction enzymes, sequence all fragments, use computer to determine overlaps and overall sequence

¥¥Analyze genomes of other species to develop techniques, strategies, and help with interpretation

Determine gene expression

¥¥Using Microarrays - ties genes to physiology

¥¥Scientists working

BIOL 202 Unit 2B

Photosynthesis and Leaves

B. Krumhardt, Ph.D.

Photosynthesis

¥ Light - captured (absorbed) by chlorophyll

– 2 kinds of chlorophyll:

a & b

¥ violet, blue, blue-green, orange, and red light are absorbed

¥ green, yellow, some orange are reflected so plants look green

Chloroplasts

¥ Double membrane - inner membrane in flattened sacs - ÒthylakoidsÓ

in stacks - ÒgranaÓ

in fluid ÒstromaÓ

– chlorophyll in thylakoid membranes

–thylakoid compartment within

¥ many photosynthesis enzymes in stroma

¥ Overview of photosynthesis:

CO2 + H2O ¬ Carbohydrate + O2

solar energy

¥ Chloroplasts are found mainly in mesophyll cells forming the tissues in the interior of the leaf

¥ O2 exits and CO2 enters the leaf through microscopic pores, stomata, in the leaf

¥ Veins deliver water from the roots and

carry off sugar from mesophyll cells to other areas of the plant

¥ A typical mesophyll cell has 30-40 chloroplasts

Light reactions

¥ occur on the thylakoid membrane

¥ two photosystems (Ps)

– PsI - chlorophyll A (peak absorbance 700nm)

– PsII - chlorophyll B (peak absorbance 680nm)

– both contain other pigments, "light harvesting antennae", which harvest solar energy - light - and transfer it to the chlorophylls

¥ chlorophyll a and b have electrons which are energized by the solar energy ¬ transferred to acceptor molecules

¥ two electron pathways (of e- transfer) - both occur at once

¥ Noncyclic photophosphorylation

–PsII (peak absorbance 680 nm) absorbs light ¬ e- energized

– e- leaves chlorophyll b

¥ the hole left by the e- leaving is freed by H2O ¬ 2 H+ + 2e- + 1/2 O2

¥ O2 diffuses off

¥ H+ into thylakoid space

– e- transported in membrane via pigments--cytochromes ¬ p700PsI

– H+ move to thylakoid compartment when e- pass through cytochromes

– the increased H+ in thylakoid space provides proton-motive force of chemiosmotic phosphorylation

ATP synthase

(ADP + P ¬ ATP)

– increased H+ in stroma (from H+ through ATP synthase and e- at p700) drives NADP+ reductase:

¥ NADP+ + 2e- + 2H+ ¬ NADPH

¥ NADPH used for synthesis of sugars, fatty acids, etc. later

– non-cyclic photophosphorylation produces O2, NADPH & ATP

¥ Cyclic photophosphorylation

– e- leave PsI without reaction with NADP, so

– ¬ ¬ return to PsI (p700) ¬ more ATP made as light stimulates e- transfer to e- acceptor and H+ moves to thylakoid compartment when e- pass through cytochromes ¬ more chemiosmotic phosphorylation

Dark Reaction

¥ occurs in the stroma

¥ Dark reaction is really very light dependent

– occurs in light most of the time because it uses the products of the light reaction

– does occur in the dark too

– (the light reactions only occur in light)

¥a.k.a. Carbon Fixation

¥ Calvin Cycle - C3 Pathway:

– first enzyme: RUBISCO--ribulose bisphosphate carboxylase, most abundant enzyme on Earth

– 3 ATP + 3 NADPH + 3 Ribulose bisphosphates + 3 CO2) ¬ 3-6C sugars ¬ spontaneous ¬ 6-3C G3P's (glyceraldehyde 3-phosphate)

–5 of the G3P's are used with 3 ATP to re-synthesize the Ribulose bisphosphate to be used in the next round of cycle, leaving one extra G3P formed

– extra G3P enzymatically converted to carbohydrates, fats, amino acids, nucleic acids, etc., using NADPH from light reaction

¥ this is the organic molecule gained due to the Calvin cycle

¥ C4 pathway

– special mesophyll cells, bundle sheath cells, capture CO2 with special enzyme (PEPCO)

– the C is then transferred to Calvin cycle (RUBISCO)

– 1st molecule formed with CO2 fixation has 4C

– found in tropical plants especially grasses

¥e.g. corn

¥CAM pathway

– trap the CO2 at night - keep their stomata closed in day to conserve H2O

– found in succulents, cactus

Photorespiration

¥ O2 & CO2 compete for active site on RUBISCO; so if increased O2 & decreased CO2, RUBISCO is inhibited

¥ This causes photorespiration to occur:

– Ribulose bisphosphate + O2 ¬ PGA + phosphoglycolic acid

– The phosphoglycolic acid breaks to make two CO2 ¬ increasing the CO2 in cell ¬ stimulation of RUBISCO

¥ Photorespiraton is evolutionary baggage

¥ When RUBISCO first evolved, the atmosphere had far less O2 and more CO2 than today

– Then, the inability of the active site of RUBISCO to exclude O2 would have made little difference

¥ Today it makes a significant difference

– Photorespiration can drain away as much as 50% of the carbon fixed by the Calvin cycle on a hot, dry day

¥ C4 plant species have evolved alternate modes of carbon fixation to minimize photorespiration

¥ C4 plants - PEPCO unaffected by increased O2, so they are more efficient, do carbon fixation even when increased O2

¥ Sugar made in the chloroplasts supplies the entire plant with chemical energy and carbon skeletons to synthesize all the major organic molecules of cells

– About 50% of the organic material is consumed as fuel for cellular respiration in plant mitochondria

– Carbohydrate in the form of the disaccharide sucrose travels via the veins to nonphotosynthetic cells.

– There, it provides fuel for respiration and the raw materials for anabolic pathways including synthesis of proteins and lipids and building the extracellular polysaccharide cellulose

¥ Plants also store excess sugar by synthesizing starch

– Some is stored as starch in chloroplasts or in storage cells in roots, tubers, seeds, and fruits

¥ Heterotrophs, including humans, may completely or partially consume plants for fuel and raw materials

¥ On a global scale, photosynthesis is the most important process to the welfare of life on Earth

– Each year photosynthesis synthesizes 160 billion metric tons of carbohydrate per year

Biology

202 Unit 2C

Plant Form and Function

B. Krumhardt, Ph.D.

¥ Plant morphology – study of external structures

¥ Plant anatomy – study of internal structures

Angiosperms

¥ Most diverse and widespread

¥ 275,000 plant species extant

¥ Reproduction and seed dispersal adaptations – flowers and fruits

Two Plant Groups

¥ Monocots

– One cotyledon

– Parallel venation

– Vascular bundles complexly arranged

– Fibrous root system

– Floral parts in multiples of 3

¥ Dicots

– Two cotyledons

– Netlike venation

– Vascular bundles arranged in a ring

– Taproot system

– Floral parts in multiples of 4 or 5

Plant Organs

¥ Roots depend on shoots for sugars and other organic nutrients

¥ Shoots depend on roots for minerals, water, and support

– Leaves on shoots provide photosynthesis

– Flowers are shoots modified for reproduction

Roots system

¥ Subterranean – roots anchor plants

¥ Roots absorb water and dissolved minerals from soil

¥ Sunlight cannot penetrate the soil – plants store food in roots

¥ Tap roots

– One large vertical root (taproot)

– Many small secondary roots

– Firm anchorage

– Some are modified to store reserve food

– Penetrate soil more deeply

– Often store food for plant (e.g. carrot)

– Characteristic of dicots

¥ Fibrous roots

– Fine threadlike roots

– Extensive exposure to soil

– Mostly shallow roots, anchoring the top of the soil

¥ Prevent erosion

– Characteristic of most monocots and some dicots

¥ Root hairs

– Extensions of epidermal cells on root surface

¥ NOT secondary roots

– Increased surface area provides increased water and mineral absorption

¥ Enhanced by mycorrhizae

¥ Adventitious roots

– Root in an atypical place

¥ Adventitious means a plant part in an atypical place

– E.g. Prop roots of corn

Shoots

Shoot system

¥ Stems, leaves, and flowers

¥ Air is source of CO2

– Air is less than 1% CO2

¥ Dry terrestrial environment provides challenges

Stems

¥ Nodes – point of attachment for leaves

¥ Internodes – stem between nodes

Buds

¥ Terminal – at the tip (apex, apical) of the shoot

– Site of most growth in young shoots

– Apical dominance – terminal bud produces hormones that inhibit the growth of axillary buds

¥ Axillary – in the angle of the leaf attachment

– Dormant in young plants

– Potentially can form a branch when apical dominance diminished

Modified Stems

¥ Stolons – grow atop soil surface – reproduces asexually, forming small plants at each node

¥ Rhizomes – horizontal stems growing underground

¥ Tubers – swollen rhizomes specialized for food storage

¥ Bulbs – vertical underground stems with swollen underground leaves specialized for food storage

Leaves

¥ Blade – main photosynthetic structure

¥ Petiole – attaches the blade to the stem at the node

– Monocots generally lack petioles – instead the blade wraps the stem in a sheath

Leaf Veins

¥ Monocots – major veins are parallel

¥ Dicots – major veins are multi-branched

Leaf Division

¥ Simple leaves – consist of one undivided blade

¥ Compound – consist of divided leaflets

¥ Doubly compound – leaflets are divided

¥ Division minimizes loss due to damage

Modified Leaves

¥ Tendrils – provide support

¥ Spines – provide protection from grazing

¥ Succulent leaves – modified to store water in dry environments

¥ Brightly colored modified leaves to attract pollinators to minimized flowers in some plants

Plant Tissues

¥ Dermal tissue – epidermis

– Single layer of tightly packed cells, form the skin of the plant

– Root hairs are extensions of these cells

– Cuticle – waxy coating secreted by stem and leaf epidermal cells

¥ Vascular tissues

– Xylem conveys water and dissolved minerals to the shoots

– Phloem conveys food from shoots to roots and other nonphotosynthetic parts

¥ Also from storage roots to actively growing shoots

¥ Ground tissue

– All plant tissue that is not dermal or vascular

– Functions in photosynthesis, storage and support

Vascular tissue specialization

¥ Xylem – specialized for water transport

– Gymnosperms

¥ Tracheids – function in both support and water transport

– Angiosperms

¥ Tracheids of gymnosperms specialized into 2 cell types

– Vessel elements – short tubular cells aligned to transport water – found only in angiosperms

– Fiber cells – lignified cells provide support – found in some conifers and in angiosperms

Xylem

¥ Tracheids and vessel elements

– Conduct water in xylem

– Dead at functional maturity – only thickened cell walls remain with pits at the ends of the cells

¥ Pits consist of only primary cell walls

¥ Tracheids

– Long thin cells with tapered ends with pits to allow water flow

– Secondary cell walls thickened with lignin – tracheids provide support too

¥ Vessel elements

– Wider, shorter, and thinner-walled than tracheids

– Aligned end to end with perforated ends to form xylem vessels

¥ Water flows freely through these ÒpipesÓ

¥ Both tracheids and vessel elements cells stop elongating when dead at maturity

¥ Wood in wood plants consists mostly of tracheids and vessel elements

Phloem

¥ Sieve-tube member cells

– Transport sucrose and other nutrients

– Alive at functional maturity

¥ Lack nucleus, ribosomes, and vacuole

– Sieve plates at the ends of these cells have pores for nutrient transport

¥ Companion cells

– Connected sieve-tube members by plasmodesmata

¥ Pores in cell walls

¥ Connect the cytoplasm of one cell to another

¥ Allow functional elements of companion cells to serve the sieve-tube member

–E.g. Endoplasmic reticulum is continuous through these pores

Ground Tissue

¥ Dicots

– Pith – internal to vascular tissue

– Cortex – external to vascular tissue

– Functions – photosynthesis, storage, and support

¥ Monocots

– Vascular bundles are scattered throughout the ground tissue

Plant Cells

¥ Protoplast surrounded by a cell wall

– Contains cytoplasm and organelles

¥ Tonoplast encloses the vacuole containing cell sap