1/7/13 – Happy New Year! Warm-up – Big Idea practice question, Ecology slide 80 Makeup quizzes Pass in chem and cell review Quote from Michael Crichton.

1/7/13 – Happy New Year!• Warm-up – Big Idea practice question, Ecology slide 80• Makeup quizzes• Pass in chem and cell review• Quote from Michael Crichton in Jurassic Park – ““Just because science can

do something doesn’t mean that it should”- Example: pet cloning- Debate (I check Ch. 18 notes)

• Correct quizzes• Be sure to have transformation lab packet for tomorrow

Homework – 19.1-19.2, 20.1-20.2 Cornell notes and concept checks due Friday, online

assignments due next Monday (21.2 and 21.5 due next Wednesday, online Friday)

Cell Respiration and Photosynthesis Review Manual due next Tuesday, Molecular Biology due Tuesday 1/22

Final covers Ch. 2-21, 47.3, 51-56 – half are cumulative AP released questions, half are Molecular Biology, and there will be 1 long FRQ and 3 short

Animal Cloning Much Harder• In tadpoles, the ability of

the transplanted nucleus to support normal development is inversely related to the donor’s age.

• 1997 when Ian Wilmut and his colleagues cloned an adult sheep

• One, “Dolly,” of several hundred implanted embryos completed normal development. – Improper

methylation in many cloned embryos interferes with normal development.

1/8/13

• Warm-up – Big Idea practice question, Ecology slide 118• Test corrections• Transformation analysis and Transformation Efficiency• Check per 6 right side ch. 18 notesHomework- Finish transformation lab packet – final discussion

tomorrow – packets due and quiz Thurs.Mutation practice due tomorrow19.1-19.2, 20.1-20.2 Cornell notes and concept checks due

Friday, online assignments due next Monday (21.2 and 21.5 due next Wednesday, online Friday)

• Chem Big Ideas, slide 7 and 9 • Wrap-up transformation• Finish Ch. 17 and Mutations

Homework- Study for short transformation quiz tomorrow- will turn in

packet then19.1-19.2, 20.1-20.2 Cornell notes and concept checks due

• COMPARING EUK & PROK TRANSCRIPTION & TRANSLATION

• E – Compartmentalized with Transcription in nucleus, Translation in Cytoplasm and extensive RNA processing in between, also complicated mechanisms for targeting proteins to the appropriate organelle.

• P- no nuclei so transcription and translation can occur simultaneously - Ribosomes attach to the leading end of a mRNA molecule while transcription is still in progress, protein diffuses to where needed

Fig. 17.2a

Figure 17.3

mRNARibosome

Polypeptide

TRANSCRIPTION

TRANSLATION

TRANSCRIPTION

TRANSLATION

Polypeptide

Ribosome

Pre-mRNARNA PROCESSING

(a) Bacterial cell (b) Eukaryotic cell

Nuclearenvelope

INITIATION AND ELONGATION

E - The promoter also includes a binding site for RNA polymerase several dozen nucleotides upstream of the start point.

P - In prokaryotes, RNA polymerase can recognize and bind directly to the promoter region.

E - eukaryotes have three RNA polymerases (I, II, and III) in their nuclei.– RNA polymerase II is used for mRNA synthesis.

P - Bacteria have a single type of RNA polymerase that synthesizes all RNA molecules.

• STEP 3 - TERMINATION– E - RNA polymerase continues for hundreds

of nucleotides past the terminator sequence, AAUAAA.

– P - RNA polymerase stops transcription right at the end of the terminator.• Both the RNA and DNA are then released.

RIBOSOMES

• While very similar in structure and function, prokaryotic and eukaryotic ribosomes have enough differences that certain antibiotic drugs (like tetracycline) can paralyze prokaryotic ribosomes without inhibiting eukaryotic ribosomes.

• Transduction• Both generalized and specialized transduction use

phage as a vector to transfer genes between bacteria.

Fig. 18.13

• A transposon is a piece of DNA that can move from one location to another in a cell’s genome.

• Transposon movement occurs as a type of recombination between the transposon and another DNA site, a target site.– In bacteria, the target site may be within the

chromosome, from a plasmid to chromosome (or vice versa), or between plasmids.

• Transposons can bring multiple copies for antibiotic resistance into a single R plasmid by moving genes to that location from different plasmids.– This explains why some R plasmids convey

resistance to many antibiotics.

• The transposase enzyme recognizes the inverted repeats as the edges of the transposon.

• Transposase cuts the transposon from its initial site and inserts it into the target site.

• The simplest bacterial transposon, an insertion sequence, consists only of the transposase gene

Fig. 18.17

• Composite transposons (complex transposons) include extra genes sandwiched between two insertion sequences.– It is as though two insertion sequences

happened to land relatively close together and now travel together, along with all the DNA between them, as a single transposon.

• While insertion sequences may not benefit bacteria in any specific way, composite transposons may help bacteria adapt to new environments.– For example, repeated movements of resistance

genes by composite transposition may concentrate several genes for antibiotic resistance onto a single R plasmid.

– In an antibiotic-rich environment, natural selection favors bacterial clones that have built up composite R plasmids through a series of transpositions.

Mutations

• What is a mutation?

• What types of mutations are there?

• What can result? – 3 possibilities (same, worse, better)

• Mutations are changes in the genetic material of a cell (or virus).

• These include large-scale mutations in which long segments of DNA are affected (for example, translocations, duplications, and inversions).

• A chemical change in just one base pair of a gene causes a point mutation.

• If these occur in gametes or cells producing gametes, they may be transmitted to future generations.

5. Point mutations can affect protein structure and function

• For example, sickle-cell disease is caused by a mutation of a single base pair in the gene that codes for one of the polypeptides of hemoglobin. – A change in a single nucleotide from T to A in

the DNA template leads to an abnormal protein.

Fig. 17.23

• A point mutation that results in replacement of a pair of complimentary nucleotides with another nucleotide pair is called a base-pair substitution.

• Some base-pair substitutions have little or no impact on protein function.– In silent mutations, alterations of nucleotides

still indicate the same amino acids because of redundancy in the genetic code.

– Other changes lead to switches from one amino acid to another with similar properties.

– Still other mutations may occur in a region where the exact amino acid sequence is not essential for function.

• Other base-pair substitutions cause a readily detectable change in a protein.– These are usually detrimental but can

occasionally lead to an improved protein or one with novel capabilities.

– Changes in amino acids at crucial sites, especially active sites, are likely to impact function.

• Missense mutations are those that still code for an amino acid but change the indicated amino acid.

• Nonsense mutations change an amino acid codon into a stop codon, nearly always leading to a nonfunctional protein.

Fig. 17.24

• Insertions and deletions are additions or losses of nucleotide pairs in a gene.– These have a disastrous effect on the resulting

protein more often than substitutions do.

• Unless these mutations occur in multiples of three, they cause a frameshift mutation.– All the nucleotides downstream of the deletion

or insertion will be improperly grouped into codons.

– The result will be extensive missense, ending sooner or later in nonsense - premature termination.

Fig. 17.24

• Mutations can occur in a number of ways.– Errors can occur during DNA replication, DNA

repair, or DNA recombination.– These can lead to base-pair substitutions,

insertions, or deletions, as well as mutations affecting longer stretches of DNA.

– These are called spontaneous mutations.

• Mutagens are chemical or physical agents that interact with DNA to cause mutations.

• Physical agents include high-energy radiation like X-rays and ultraviolet light.

• Chemical mutagens may operate in several ways.– Some chemicals are base analogues that may be

substituted into DNA, but that pair incorrectly during DNA replication.

– Other mutagens interfere with DNA replication by inserting into DNA and distorting the double helix.

– Still others cause chemical changes in bases that change their pairing properties.

Mutation Practice

Agenda 1/10/13

1) Transformation Quiz 2) Go over Mutation assignment3) Ch. 18 Gene Regulation

Homework – 19.1-19.2, 20.1-20.2 Cornell notes and concept checks due

Me/TA’s – get micropipettes and food dye tubes ready for tomorrow, workstations for Monday

2 Ways they can regulate metabolism based on needs at the time:

1) Adjust the activity of enzymes already present (for example, by feedback inhibition).

2) Make more specific enzyme molecules by regulating gene expression (or stop making them if have all they need)

Gene Expression in Bacteria

Operon Model – Bacterial Gene Regulation through Negative Control (Repressors)

• An operon consists of 3 parts:

1) operator – controls access of RNA polymerase to genes (where repressor binds)

2) promoter – where RNA polymerase attaches

3) genes of the operon (often several enzymes in a biological pathway)

Let’s look at those parts -

Repressor protein has allosteric site –

1) What happens when tryptophan (corepressor) is present?

2) Tryptophan absent?

Here’s what it looks like

2 types of operon

1) Repressible (such as tryptophan)– Normally on, but can be turned off by repressor– Normally anabolic (building) and product acts as

corepressor to activate repressor and shut down operon

2) Inducible (lac operon)– Normally off (repressor active as synthesized, stays

bound), but can be activated by inducer inactivating the repressor

– Normally catabolic (breakdown) and substrate serves as inducer to turn pathway on only when needed

• INDUCIBLE – the lac operon– contains genes for enzymes that break down

(catabolize) lactose.– In the absence of lactose, this operon is off as

an active repressor binds to the operator and prevents transcription.

Fig. 18.21a

Fig. 18.21b

• When lactose is present in the cell, allolactose, an isomer of lactose, binds to the repressor.

• This inactivates the repressor, and the lac operon can be transcribed.

Positive Control of Gene Expression –

• Even if the lac operon is turned on by the presence of allolactose, the degree of transcription depends on the concentrations of other substrates.– If glucose levels are

low ATP low , then cyclic AMP (cAMP) high and binds

to catabolite activator protein (CAP),

higher level of transcription of

lac mRNA

NOTE – Glucose is the preferable energy source for the bacteria.

• If glucose levels are sufficient and cAMP levels are low (lots of ATP), then the CAP protein has an inactive shape and cannot bind upstream of the lac promotor.– The lac operon will

be transcribed but at a low level.

• For the lac operon, the presence / absence of lactose (allolactose) determines if the operon is on or off.

• Overall energy levels in the cell determine the level of transcription, a “volume” control, through CAP.

• CAP works on several operons that encode enzymes used in catabolic pathways.– If glucose is present and CAP is inactive, then

the synthesis of enzymes that catabolize other compounds is slowed.

– If glucose levels are low and CAP is active, then the genes which produce enzymes that catabolize whichever other fuel is present will be transcribed at high levels.

L.O. 3.21

• Two main differences from prokaryotes.– Typical multicellular eukaryotic genome is much

larger than that of a bacterium.

• Cell specialization limits the expression of many genes to specific cells.

Eukaryotic Gene Expression

Every step in gene expression can be regulated:

• Chromatin packing

• Transcription

• RNA processing

• Translation

• Alterations to the protein product

Fig. 19.7

Regulation of Transcription Initiation

• Chromatin-modifying enzymes provide initial control of gene expression by making a region of DNA either more or less able to bind the transcription machinery

• Genes of densely condensed heterochromatin are usually not expressed, presumably because transcription proteins cannot reach the DNA

• Chemical modifications of chromatin play a key role in chromatin structure and transcription regulation.

1) DNA methylation is the attachment by specific enzymes of methyl groups (-CH3) to DNA bases after DNA synthesis.– Inactive DNA is generally highly methylated compared

to DNA that is actively transcribed.– For example, the inactivated mammalian X

chromosome in females is heavily methylated (Barr body)

– Genes are usually more heavily methylated in cells where they are not expressed.

– Demethylating certain inactive genes turns them on.– However, there are exceptions to this pattern.

• This methylation pattern accounts for genomic imprinting in which methylation turns off either the maternal or paternal alleles of certain genes at the start of development.

2) Histone acetylation (addition of an acetyl group -COCH3) and deacetylation appear to play a direct role in the regulation of gene transcription.– Acetylated histones grip DNA less tightly,

providing easier access for transcription proteins in this region.

– Some DNA methylation proteins recruit histone deacetylation enzymes, providing a mechanism by which DNA methylation and histone deacetylation cooperate to repress transcription.

Epigenetic Inheritance• Although the chromatin modifications just

discussed do not alter DNA sequence, they may be passed to future generations of cells

• The inheritance of traits transmitted by mechanisms not directly involving the nucleotide sequence is called epigenetic inheritance

Animation: Initiation of Transcription

Right-click slide / select “Play”

• Bending of DNA enables transcription factors, activators, bound to enhancers to contact the protein initiation complex at the promoter.– This helps

position the initiation complex on the promoter.

Fig. 19.9

• Eukaryotic genes also have repressor proteins that bind to DNA control elements called silencers.

• At the transcription level, activators are probably more important than repressors, because the main regulatory mode of eukaryotic cells seems to be activation of otherwise silent genes.

• Repression may operate mostly at the level of chromatin modification.

• In contrast to prokaryotes, only rarely are eukaryotic genes organized together.– Genes coding for the enzymes of a metabolic

pathway may be scattered over different chromosomes.

– Even if genes are on the same chromosome, each gene has its own promoter and is individually transcribed.

• To coordinate gene transcription, a common group of transcription factors binds to them, promoting simultaneous gene transcription.– For example, steroid hormones enter a cell

and bind to a specific receptor protein in the cytoplasm or nucleus.

– After allosteric activation of these proteins, they function as transcription activators.

– Other signal molecules can control gene expression indirectly by triggering signal-transduction pathways that lead to transcription activators.

WHAT CHAPTER DID WE LEARN THIS IN?

RNA Processing• In alternative RNA splicing, different mRNA

molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns

• Gene expression may be blocked or stimulated by any post-transcriptional step.

• By using regulatory mechanisms that operate after transcription, a cell can rapidly fine-tune gene expression in response to environmental changes without altering its transcriptional patterns.

• microRNA’s (miRNA) and small interfering RNA’s (siRNAs) can bind to mRNA & degrade or block translation

Post-transcriptional mechanisms play supporting roles in the control of gene expression

(a) Primary miRNA transcript

HairpinmiRNA

Hydrogenbond

miRNA-proteincomplex

mRNA degraded Translation blocked

(b) Generation and function of miRNAs

Figure 18.15

•An increase in the number of miRNAs in a species may have allowed morphological complexity to increase over evolutionary time, also they play a big role in embryonic development

• Normal cell growth controls go bad

• Causes:– random spontaneous mutations – environmental influences such as chemical

carcinogens or physical mutagens.

• Cancer-causing genes, oncogenes, were initially discovered in retroviruses, but close counterparts, proto-oncogenes were found in other organisms.

1. Cancer results from genetic changes that affect the cell cycle

• Proto-oncogenes code for proteins that stimulate normal cell growth and division

• Oncogenes arise from a genetic change that leads to an increase in the proto-oncogene’s protein or the activity of each protein molecule.

• Mutations to genes whose normal products inhibit cell division, tumor-suppressor genes, also contribute to cancer.

Fig. 19.13

• Mutations in the products of two key genes, the ras proto-oncogene, and the p53 tumor suppressor gene occur in 30% and 50% of human cancers respectively.

• Both the Ras protein and the p53 protein are components of signal-transduction pathways that convey external signals to the DNA in the cell’s nucleus.

2. Oncogene proteins and faulty tumor-suppressor proteins interfere with normal signaling pathways

• The p53 gene, named for its 53,000-dalton protein product, is often called the “guardian angel of the genome”.

• Damage to the cell’s DNA acts as a signal that leads to expression of the p53 gene.

• The p53 protein is a transcription factor for several genes.– It can activate the p21 gene, which halts the cell

cycle by binding to CDK’s while DNA is repaired– It can turn on genes involved in DNA repair.– When DNA damage is irreparable, the p53 protein

can activate “suicide genes” whose protein products cause cell death by apoptosis.

Agenda 1/11/13• Intro Crime Scene Lab – do pages 23-26,4-5 together (30 min)• Practice Micropipetting (20 min)• Viruses?

Homework – HIGHLIGHT QUICKQUIDE - be ready to do restriction digest Mon!DNA Scissors activity with key on website if want extra restriction enzyme

practice (optional)19.1-19.2, 20.1-20.2 online assignments due Monday – 84 minutes (21.2 and

21.5 due next Wednesday, online Friday)Cell Respiration and Photosynthesis Review Manual due next Tuesday,

Molecular Biology due Tuesday 1/22

Me – TA’s pour gels and set up workstations for digest

Micropipetting practice• Work in pairs – one of you gets a P20 and one a P200• Loading tips & how to hold• 1st and 2nd stops• Set to 20 ul• Tap vial to get all liquid to bottom – pull up from there• Practice pulling up 20 ul with each micropipette- transfer to

empty tube and back – feel the difference between P20 and P200

• Practice getting no air bubbles – this will significantly affect the accuracy of your sample amount

• Can practice pulling up different amounts if you like• End by setting P20’s to 10ul – what we will use tomorrow

Biotech Vocab.

• genetic engineering - the direct manipulation of genes for practical purposes.

• recombinant DNA - genes from two different sources - often different species - are combined in vitro into the same molecule

• biotechnology - the manipulation of organisms or their components to make useful products

• gene cloning – preparing multiple identical copies of gene-sized pieces of DNA

• cloning vector - DNA molecule that can carry foreign DNA into a cell and replicate there

• Gene cloning and genetic engineering were made possible by the discovery of restriction enzymes that cut DNA molecules at specific locations.

• In nature, bacteria use restriction enzymes to cut foreign DNA, such as from phages or other bacteria.

• Most restrictions enzymes are very specific, recognizing short DNA nucleotide sequences and cutting at specific point in these sequences.– Bacteria protect their own DNA by methylation.

Restriction enzymes are used to make recombinant DNA

• Each restriction enzyme cleaves a specific sequences of bases or restriction site.– These are often a symmetrical series of four to eight

bases on both strands running in opposite directions.• If the restriction site on one strand is 3’-

CTTAAG-5’, the complementary strand is 5’-GAATTC-3’.

• Because the target sequence usually occurs (by chance) many times on a long DNA molecule, an enzyme will make many cuts.– Copies of a DNA molecule will always yield the same

set of restriction fragments when exposed to a specific enzyme.

• Restriction enzymes cut covalent phosphodiester bonds of both strands, often in a staggered way creating single-stranded ends, sticky ends.– These extensions will form hydrogen-bonded

base pairs with complementary single-stranded stretches on other DNA molecules cut with the same restriction enzyme.

• These DNA fusions can be made permanent by DNA ligase which seals the strand by catalyzing the formation of phosphodiester bonds.

• Restriction enzymes and DNA ligase can be used to make recombinant DNA, DNA that has been spliced together from two different sources.

Fig. 20.2

Viruses

• Evolved from plasmids or transposons most likely (mobile genetic material) – more closely related to host cell DNA than other virus DNA

• Probably entered damaged cells first, then once developed capsid/envelope, could enter undamaged cells also

Viruses - Relative size and structure

Viral Genome Possibilities

• The capsid is a protein shell enclosing the viral genome.

• Capsids are build of a large number of protein subunits called capsomeres, but with limited diversity.– The capsid of the tobacco

mosaic virus has over 1,000 copies of the same protein.

– Adenoviruses have 252 identical proteins arranged into a polyhedral capsid - as an icosahedron.

Fig. 18.2a & b

• Some viruses have viral envelopes, membranes cloaking their capsids.

• These envelopes are derived from the membrane of the host cell.

• They also have some viral proteins and glycoproteins.

Fig. 18.2c

• The most complex capsids are found in viruses that infect bacteria, called bacteriophages or phages.

• The T-even phages that infect Escherichia coli have a 20-sided capsid head that encloses their DNA and protein tail piece that attaches the phage to the host and injects the phage DNA inside.

Fig. 18.2d

• Can reproduce only within a host cell.• An isolated virus is unable to reproduce - or do

anything else, except infect an appropriate host.• Host range - “lock-and-key” fit between proteins on

the outside of virus and specific receptor molecules on the host cell’s surface.

• Some viruses (like the rabies virus) have a broad enough host range to infect several species, while others infect only a single species.

• Most viruses of eukaryotes attack specific tissues.

Viruses- Living or Not?

• Virus takes over the machinery of the host cell!

Fig. 18.3

Virulent Phages and the Lytic Cycle

• While phages have the potential to wipe out a bacterial colony in just hours, bacteria have defenses against phages.– Natural selection favors bacterial mutants with

receptors sites that are no longer recognized by a particular type of phage.

– Bacteria produce restriction nucleases that recognize and cut up foreign DNA, including certain phage DNA.• Modifications to the bacteria’s own DNA prevent its

destruction by restriction nucleases.

– But, natural selection favors resistant phage mutants.

Temperate Phages Use Lytic and Lysogenic Cycle

• Human immunodeficiency virus (HIV)

• Causes AIDS (acquired immunodeficiency syndrome)

• Includes an envelope with glyco-proteins for binding to specific types of white blood cells

Fig. 18.7a

Retroviruses

• After HIV enters the host cell, reverse transcriptase synthesizes double stranded DNA from the viral RNA.

• Transcription produces more copies of the viral RNA that are translated into viral proteins, which self-assemble into a virus particle and leave the host.

Fig. 18.7b

• Vaccines can help prevent viral infections– Edward Jenner and 1st vax for smallpox late

1700’s – used cowpox from milkmaid’s sore

• Antibiotics only work on bacteria, not viruses

• Antivirals interfere with viral nucleic acid synthesis– AZT interferes with reverse transcriptase of HIV.– Acyclovir inhibits herpes virus DNA synthesis.

3 Causes:1) Mutation of existing viruses

– RNA viruses tend to have high mutation rates because replication of their nucleic acid lacks proofreading.

– Some mutations create new viral strains with sufficient genetic differences from earlier strains • This is the case in flu epidemics.

2) Spread of existing viruses from one host species to anotherex. – Hantavirus from mice to people

3) Spread from a small, isolated population to a widespread epidemic.

ex. - AIDS

Emergence of New Viral Diseases

• Viroids, smaller and simpler than even viruses, consist of tiny molecules of naked circular RNA that infect plants.– When replicated by the host’s cellular enzymes,

they cause errors in the regulatory systems that control plant growth (stunt growth).

7. Viroids and prions are infectious agents even simpler than viruses

• Prions are infectious proteins that spread a disease.– They appear to cause several degenerative brain

diseases including scrapie in sheep, “mad cow disease”, and Creutzfeldt-Jacob disease in humans.

• According to the leading hypothesis, a prion is a misfolded form of a normal brain protein.

• It can then convert a normal protein into the prion version, creating a chain reaction that increases their numbers.

Fig. 18.10

• One basic cloning technique begins with the insertion of a foreign gene into a bacterial plasmid.

Fig. 20.1

DNA Technology- Gene Cloning

• The potential uses of cloned genes fall into two general categories.

• First, the goal may be to produce a protein product.– For example, bacteria carrying the gene for human

growth hormone can produce large quantities of the hormone for treating stunted growth.

• Alternatively, the goal may be to prepare many copies of the gene itself.– This may enable scientists to determine the gene’s

nucleotide sequence or provide an organism with a new metabolic capability by transferring a gene from another organism.

• Like unicellular organisms, the tens of thousands of genes in the cells of multicellular eukaryotes are continually turned on and off in response to signals from their internal and external environments.

• Gene expression must be controlled on a long-term basis during cellular differentiation, the divergence in form and function as cells specialize.– Highly specialized cells, like nerves or muscles, express

only a tiny fraction of their genes.

Animations of cell differentiation from stem cells:

– http://learn.genetics.utah.edu/content/tech/stemcells/scintro/

1. Each cell of a multicellular eukarote expresses only a small fraction of its genes

1/7/13 – Happy New Year! Warm-up – Big Idea practice question, Ecology slide 80 Makeup quizzes Pass in chem and cell review Quote from Michael Crichton.

ecology slide

short slide

cell respiration

online assignments

rna polymerases

molecular biology

cornell notes

cell review quote

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