CHAPTER 16: Regulation of Gene Expression in Bacteria

Eukaryotic transcription initiation requires specific cis-acting sites

Cis-acting sequence: • Located on same chromosome as the gene that it regulates • Required for accurate regulated transcription of genes. All of the following are examples of "cis-acting sequences":• Promoters • Enhancers • Silencers

Interactive variance

Deviation from additive components that occurs when two or more loci behave epistatically

Plasmids used in D N A cloning

Genetically modified bacterial plasmids were the first vectors developed; engineered to contain: • A number of convenient restriction sites • A marker gene to select for presence in host cell (again, this is often an antibiotic resistance gene... hence, if antibiotic is applied, all bacteria WITHOUT the plasmid will die, leaving those with the "gene of interest", but also leaving the antibiotic resistance gene as well)

The HTT gene and its protein product

H T T gene• Located on chromosome 4• Encodes protein huntingtin that is essential to survival of certain neurons in adult brains• Facilitates vesicle transport at neuronal synaptic junctions• H T T is present at synapses• Transports molecules from nucleus to cytoplasm

Homeobox

Hox genes contain a 180-b p domain known as homeobox• Encodes D N A-binding sequence of 60 amino acids (homeodomain)• Expression of Hox genes is collinear with the anterior-to-posterior organization in embry

Transcription is under control of a single regulatory region

In prokaryotes, genes coding for enzymes with regulatory functions are organized in clusters • Regulatory regions usually located upstream of gene cluster they control • Regulatory region on same strand—"cis-acting" • Trans-acting element: Molecules (like proteins) that bind the cis-acting sitess:

Operon Model: Negative Control

Jacob and Monod (1960) • Proposed operon model: Involved negative control • Group of genes is regulated and expressed together as a unit • Proposed lacI gene regulates transcription of structural genes by producing allosteric repressor molecule

Narrow-Sense Heritability

Narrow-sense heritability (h2)• Proportion of phenotypic variance due to additive genotypic variance alone Genotypic variance can be divided into subcomponents—three kinds of gene action:• Additive variance• Dominance variance• Interactive variance

RNA interference ________

involves short RNA molecules that trigger degredation of mRNA

Tumor-Suppressor Genes

Tumor-suppressor genes Regulate cell-cycle checkpoints and initiate process of apoptosis Mutated tumor-suppressor genes Are unable to respond to cell-cycle checkpoints or undergo apoptosis This leads to more mutations and development of cancer

maternal effect gene products

may form gradients in the embryo and regulate zygotic genes

What is the term for undifferentiated cells that give rise to all more mature flower organs?

meristem

What type of RNAs have "Gene regulatory functions" in particular?

small noncoding RNAs

In the Lac Operon, the LacZ, LacY and LacA are all _____ that code for enzymes.

structural genes

Which of the following allows genes to become available for transcription?

Acetylation of histones

Gap Proteins

Act as transcription factors• Activate pair-rule genes, whose products divide embryo into smaller regions two segments wide• Anterior and posterior regions

Inducible enzymes

Bacteria adapt to environment by producing inducible enzymes only when specific substrates are present

What is true of siRNAs?

they are double stranded RNAs and arise from viral infection

zip code

a 3' untranslated sequence to which ZBP1 binds and blocks translation until mRNA is in correct location

disruptive selection

both extremes are selected for

Notch Signaling Pathway

Notch signaling pathway:• Works via direct cell-cell contact—controls developmental fate of interacting cells• Notch gene encodes signal receptor (transmembrane protein) embedded in plasma membrane• Notch signal only works between adjacent cell

mRNA localization and translation initiation are highly regulated

mRNAs are not always translated immediately but stored for translation at a later time or in response to certain cues (such as in an egg that just got fertilized) . Example: • Cis-regulatory element in the 3′ UTR called a cytoplasmic polyadenylation element (CPE) • Recognized and bound by the cytoplasmic polyadenylation element binding protein (CPEB) • CPEB recruits poly-A specific Ribonuclease (PARN) and Maskin (the mRNA is now dormant

hypermethylation of a DNA repair gene

predisposition to mutations

homeotic genes

these genes specify the actual identity of each segment

Cell Cycle Control and Checkpoints

G1/S, G2/M, and M checkpoints: Three distinct checkpoints where the cell monitors external signals and internal equilibrium Cells decide whether to proceed to the next stage of the cell cycle

CRISPR loci include spacer sequences derived from portions of bacteriophage genomes.

bacteriophage

phosphatase

enzyme that removes phosphate group

Expression of the trp operon would allow _________ to get made

enzymes capable of synthesizing tryptophan "from scratch"

PCR

in vitro DNA replication

The presence of lactose would ________ expression of the structural genes of the ____ Operon.

induce ; Lac

What is the term for "continually produced" enzymes?

inhibited inducible constitutive CORRECT activated

In the Lac Operon, the gene products beta-galactosidase, permease, and transacetylase are translated from __________

one polycistronic mRNA strand

Remember that this equation can be useful in calculating genotype frequencies and allele frequences:

p2 + 2pq + q2 = 1

The type of GMO called "Golden rice" is designed to _______

produce extra vitamin A

Autosomal S T R D N A Profiling

S T R profile of individual is expressed as the number of times the S T R sequence is repeated

Cancer

Spectral karyotype of a normal cell. (b) Karyotype of a cancer cell showing translocations, deletions, and aneuploidy—characteristic features of cancer cells

mRNA Localization and Localized Translational Control

There is an mRNA 3′ UTR sequence termed a zip code: A cis-regulatory element that serves as a binding site for a RBP (ribonucleoprotein) called zip code binding protein 1 (ZBP1) Blocks translation until mRNA is localized to site of cell where it is needed...... (such as actin mRNA... actin is a protein that can rapidly polymerize to form pseuodopods in cells such as amoebas and immune cells, for their movement

Variable expression

Variable expression of yellow phenotype (and associated obesity) in genetically identical mice caused by diet-related epigenetic changes in the Avy allele.

Alternative splice site: Types of Alternative Splicing

Within an exon that may be upstream or downstream of the normally used splice site

q

frequency of allele a

p2

frequency of the genotype AA

driver mutation

gives growth advantage to cancer cell

Maternal Gene ProductS

• Placed in egg during oogenesis• Activated immediately after fertilization• Establish anterior-posterior axis of embryo• Encode transcription factors:• Activate transcription of gap gene• Expression of gap gene divides embryo into head, thorax and abdominal regions of adulT

Viral Vectors for Gene Therapy

• Successful gene therapy requires a delivery system to transfer genes into patient's cells• Genetically engineered viruses have been used as vectors• Viral vectors carry therapeutic DNA as payload so that the virus will infect target cells and successfully deliver the DNA

The regulation of gene expression in eukaryotes. After the initiation of transcription, there are many opportunities for posttranscriptional regulation

. After the initiation of transcription, there are many opportunities for posttranscriptional regulation.

Effects on transcription levels of different point mutations in the promoter region of the b-globin gene.

. Each line represents the level of transcription produced in a separate experiment by a single-nucleotide mutation (relative to wild type) at a particular location. Dots represent nucleotides for which no mutation was tested. Note that mutations within specific elements of the promoter have the greatest effects on the level of transcription

How the multiple-factor hypothesis accounts for the 1 : 4 : 6 : 4 : 1 phenotypic ratio of grain color...

...when all alleles designated by an uppercase letter are additive and contribute an equal amount of pigment to the phenotype.

After transformation procedures are carried out on plant cells in the process of creating a GMO crop about how many such cells get successfully transformed?

1 in 1000

Pathways of Degradation:

1) Exoribonuclease enzymes shorten length of poly-A tail... this is called Deadenylation-dependent decay ▪ Binding of poly-A binding protein to tail stabilizes mRNA 2) Decapping enzymes removes 7-methylguanine cap; that mRNA is now unstable 3) Endonuclease cleaves mRNA internally

eQTL variants control gene expression through three major pathways:

1) direct effects on expression that control the amount of mRNA produced, (2) chromatin-mediation effects, including transcription factor binding, histone modification, and DNA methylation, and (3) direct effects on splicing of pre-mRNA, producing variant mRNA molecules. The contribution of each pathway is indicated by the thickness of arrows. Over 60% of eQTL variants regulate gene expression via chromatin modifications.

There are three major classes of MAE

1.Parent-of-Origin Monoallelic Expression: Imprinting 2. Random Monoallelic Expression: i.e. Inactivation of the X Chromosome 3. Random Monoallelic Expression of Autosomal Genes

C R I S P R-Cas is an Adaptive Immune System in Prokaryotes 2

2. c r R N A biogenesis- C R I S P R loci are transcribed starting at the promoter within the leader, into long R N A transcripts- Processed into short C R I S P R-derived R N As (c rR N As), each containing a single spacer flanked on both sides by repeat sequences

Segment formation and body plans in Drosophila and mammals

20 segmentation genes identified and classified based on mutant phenotype• Gap gene mutations delete a group of adjacent segments and cause gaps in normal body plan• Pair-rule gene mutations affect every other segment and eliminate a specific part of segment• Segment polarity gene mutations cause defects in segment portion

Use this equation and the data to answer the question below: p2 + 2pq + q2 = 1 Let's say you know the frequency of genotype AA is 25%, and the frequency of aa is also 25%. What is the frequency of the heterozygotes in the population?

50% Correct! Since AA basically IS "p2", and "aa" IS "q2", we can then plug the percentages into the equation to find "2pq" (which is the same thing as Aa... just a different way of representing Aa): p2 + 2pq + q2 = 1 0.25 + (2pq) + 0.25 = 1 2pq = 1 - 0.25 - 0.25 2pq = 0.5

Use this equation and the data to answer the question below: p2 + 2pq + q2 = 1 Let's (again) say you know the frequency of genotype AA is 25%, and the frequency of aa is also 25%. You found out the frequency of Aa in the last question. But you may be wondering what the usefulness is of representing "Aa" as 2pq. That is because knowing that Aa is 2pq can tell you the allele frequency, too. Given the above info, what is the allele frequency of p?

50% Correct! Since we know the frequency of genotype AA is 25% (or 0.25)... and we know AA is represented by p squared, we can find p by taking the square root of 0.25. The square root of 0.25 is 0.5, or 50%. Hence, the frequency of the p allele is 50%. Since p + q = 1, we can also find q: 1 - p = q 1 - 0.5 = 0.5

Portion of plasmid pV-GMGT04 used to create Roundup-Ready soybeans

A 1365-bp fragment encoding the EPSPS enzyme from Agrobacterium CP4 was cloned downstream from the cauliflower mosaic virus E35S promoter and the petunia chloroplast transit peptide signal sequence (ctp4). CTP4 signal sequences direct the EPSPS protein into chloroplasts, where aromatic amino acids are synthesized. The CP4 epsps coding region was cloned upstream of the nopaline synthase (nos) transcription termination and polyadenylation sequences. The CP4 epsps sequences encode a 455-amino-acid 46-kDa ESPSP protein.

A P C Gene

A P C (adenomatous polyposis) gene product Tumor suppressor that controls cell-cell contact and growth inhibition F A P (Familial adenomatous polyposis) is due to one mutant copy of A P C gene Heterozygous A P C mutation Causes formation of many rectal polyps or adenomas in early life Second A P C mutation leads to later stage of cance

A P C Tumor-Suppressor Gene

A P C gene: Adenomatous polyposis coli Is a tumor-suppressor gene Encodes protein involved in normal differentiation of intestinal cells In colorectal cancers, adenoma or polyp formation requires inactivating mutations in A D P gene

Five Populations of Drosophila

A d h genes from individuals representing five natural populations of Drosophila were cloned.. .• Cloned genes contained total of 43 nucleotide variations in A d h sequence of 2721 base pairs • Out of 14 variations in coding regions, only one leads to amino acid replacement

Accessing H G P

A gene map for chromosome 12 from the NCBI database Map Viewer. (b) Partial map of disease genes on human chromosome 21. Maps such as this depict genes thought to be involved in human genetic disease conditions. (But keep in mind, some of these are merely "associated" with the stated disease and may not cause all cases of it.)

Many Disease-Associated Genome Variations Affect Regulatory Regions

A genome-wide association study (GWAS) • Identifies genetic variations that are significantly enriched in the genomes of patients with a particular disease and are not found in those without the disease Many GWASs evaluate single base-pair changes or single nucleotide polymorphisms (SNPs)

Long Noncoding RNAs and Posttranscriptional Regulation

A lncRNA regulates muscle differentiation by sequestering miRNAs. (a) In Duchenne muscular dystrophy cells, linc-MD1 expression is aberrantly low and miR-133 and miR-135 downregulate mRNAs for the muscle-promoting transcription factors MEF2C and MAML1. (b) When linc-MD1 is introduced (using a virus vector), the miRNAs are bound and MEF2C and MAML1 mRNAs are upregulated, leading to increased muscle differentiation

The Human Epigenome Project:

A multinational, public/private consortium • Identify, map, and establish the functional significance of all D N A methylation patterns in the human genome • Analysis may show that genetic responses to environmental cues mediated by epigenetic changes are pathways to disease

The emerging idea of the "Pangenome"

A pangenome attempts to visualize all genomic segments and gene variations found in a species. Notice that there are variations in individual genomes not represented in the reference genome, but these variations are included in the pangenome

Structural Genes of trp Operon 2

A repressible operon. (a) The components involved in regulation of the tryptophan operon. (b) In the absence of tryptophan, an inactive repressor is made that cannot bind to the operator (O), thus allowing transcription to proceed. (c) When tryptophan is present, it binds to the repressor, causing an allosteric transition to occur. This complex binds to the operator region, leading to repression of the operon

Third-generation sequencing (TGS)

A simplified version of one approach to TGS is shown here. in this example, a DNA polymerase molecule anchored within a nanopore binds to a single strand of DNA. As the polymerase incorporates fluorescently labeled nucleotides into a new DNA strand (shown in pink), each base emits a characteristic color that can be detected Rapidly decreasing costs have enabled rapid expansion of eukaryotic and prokaryotic genome sequencing. The number of eukaryotic and prokaryotic genomes sequenced (blue lines) has increased dramatically during the past decade due to improvements in high-throughput sequencing technologies, and sharply decreasing costs (green line) of sequencing large numbers of ba

The search for the Huntington gene

About 150 R F L P s distributed across the genome could be used with pedigrees to detect linkage anywhere in the genome between an R F L P marker and a disease gene of interest• Means it is possible to map a disease gene with no information about the gene, its gene product, or its function—- An approach referred to as reverse geneticsSee Chapter 22 for discussion of R F L P s

Accessing H G P

Accessing the Human Genome Project • Can access databases on Internet that display maps for all human chromosomes H G P's most valuable contribution was the identification of disease genes and development of new treatment strategies • Extensive maps developed for genes implicated in human disease conditions

Histone Modification

Acetylation by histone acetyltransferase (HAT) opens up the chromatin structure • Makes genes available for transcription Removal of the acetyl groups by histone deacetylase (HDAC) closes the configuration • Silences genes by making them unavailable

Acute Transforming Retroviruses

Acute transforming retroviruses: Carry cell-derived oncogenes Infect and transform cells into cancer cells So YES, remember... some viruses can "steal" genetic information from other cells and even other species (transduction)

Additive Alleles

Additive alleles are the basis of continuous variation:• Contribute to single quantitative character• Phenotypic contribution of each additive allele is approximately equal• Additive alleles together produce substantial phenotypic variation

Regulation of mRNA Stability and Degradation

Adenosine-uridine rich element (ARE): This is a cis-acting sequence element that regulates mRNA stabilitty

Advantages/Disadvantages to Retrovirus Vectors

Advantages and disadvantages to retrovirus vectors for gene therapy delivery:• Advantages: Provide long-term expression of delivered genes• Therapeutic genes are integrated into genome of patient• Disadvantage: Produce severe toxicity in some cases due to insertional mutations

Mitochondrial D N A Profiling

After P C R amplification, D N A sequence is determined by automated D N A sequencing• Matched to crime/suspect samples• m t D N A samples (high in copy number) are useful in cases where crime samples are small, old, or degraded Disadvantage of m t D N A• Like Y-chromosome profiles, it does not differentiate m t D N A from maternal relatives or siblings Mitochondrial D N A analysis is useful in wildlife forensic cases, especially in illegal wildlife trade

Epigenetic modifications of the genome help determine cell identity during development

After fertilization, global demethylation converts cells to a totipotent or pluripotent state. Two populations of pluripotent cells are created by a new cycle of DNA methylation in cells of the inner cell mass: embryonic stem cells (ESCs) and embryonic germ cells (EGCs). The ESCs will form the 200 different cell types in the body, and the EGCs will form primordial germ cells (PGCs), which migrate to the gonads and become precursors to sperm and eggs.

Histone Modification

Again: 3 ways by which chromatin remodelers, such as the SWI/SNF complex, alter the association of nucleosomes with DNA. (a) The DNA-histone contacts may be loosened, allowing the nucleosomes to slide along the DNA, exposing DNA regulatory regions. (b) The path of the DNA around a nucleosome core particle may be altered. (c) Components of the core nucleosome particle may be rearranged, such as by swapping in and out variant histone proteins.

Agricultural Biotechnology

Agricultural biotechnology• The goal is often to introduce insect resistance, herbicide resistance, or nutritional characteristics into crops• These goals can be accomplished via natural / selective breeding, although it may be a bit slower than producing a GMO to fulfill a similar goal: G M Os: Genetically modified organisms• Includes genetically modified foods:• Transgenic crop plants• Alfalfa, corn, rice, potatoes, tomatoes, tobacco, wheat, and cranberries are some examples (although there are ALSO non-GMO and organic versions of all of these to

Lac operon structural genes = lac Z, lac Y, and lac A

All three are transcribed as single unit of mRNA • Results in "polycistronic" mRNA • Cistron: Part of nucleotide sequence coding for single gene • This single mRNA strand is translated into three gene products

Types of Alternative Splicing Mutually exclusive exons

Allows for swapping of protein domains encoded by different exons Splicing is co-regulated for a cluster of two or more adjacent exons such that inclusion of one exon leads to the exclusion of the others in the same clusteR

Epigenetics and Cancer

Altered in cancer cells: •Epigenetic changes, including selective hypermethylation and gene silencing •But wait, didn't we just say HYPOmethylation was a trend in cancer? HYPERmethylation is the opposite (hypo vs. hyper are opposites, meaning too little vs. too much). YES but NOTICE in blue it says "selective hypermethylation." That means the exact genes exposed to either process can really matter! (This is a good example of why it's not productive to take an "all or none" approach to understanding complex processes!) ●Example: Silencing of the DNA repair gene MLH1 by hypermethylation is a key step in the development of some forms of colon cancer... MLH1 illustrates how epimutations can be involved in tumor formation... Alone or in combination with genetic changes

Riboswitches

Alternative forms of mRNA secondary structure • Bind with small ligands; cause conformational change and induce second RNA domain • Create antiterminator or terminator structure

Regulation of alternative splicing determines which RNA spliceforms of a gene are translated

Alternative splicing: • Generates different forms of mRNA from identical pre-m RNA (increases number of proteins compared to what you may think) • Expression of "one" gene actually gives rise to numerous proteins with similar yet different functions • Increases number of different proteins made from one gene called isoforms

Caution must be used since ethidium bromide is a mutagen

An agarose gel containing separated DNA fragments stained with a DNA-binding dye (ethidium bromide) and visualized under ultraviolet light. Smaller fragments migrate faster and farther than do larger fragments, resulting in the distribution shown. Molecular techniques involving agarose gel electrophoresis are routinely used in a wide range of application

epigenetic trait

An epigenetic trait is a stable, mitotically and meiotically heritable phenotype that results from changes in gene expression without alterations in the DNA sequence Epigenetics is the study of the ways in which these changes alter cell- and tissue-specific patterns of gene expression The epigenome refers to the epigenetic state of a cel

Protein-Coding Regions

Annotation (identifying functional elements) of sequence would reveal several identifiable features indicating that the sequence contains a protein-coding gene • Example:• Promoter sequence• Initiation sequence• Three exons [for example], two areas between exons represent introns; later spliced out during m R N A processing

Protein-Coding Regions

Annotation of a cDNA sequence containing part of the human β-globin gene. (a) The location of genes, if any, in this sequence is not readily apparent from a cursory glance. b) The analyzed sequence, showing the location of a promoter sequence (green). Open reading frames for three exons of the gene are shown in blue. (c) Diagrammatic representation of three exons for the human β-globin gene encoded by the sequence shown in (a) and (b

The first disease treated with gene therapy was SCID , using a(n) retrovirus vector.

Answer 1:SCID Answer 2:retrovirus

Antennapedia and bithorax

Antennapedia (A N T-C) complex• Contains five genes that specify structures in head and first two thoracic segments Bithorax (B X-C) complex• Contains three genes that specify structures in• Second thoracic segment• Entire third thoracic segment• Abdominal segment

Homeotic Genes and Mutants

Antennapedia (Antp) mutation in Drosophila. (a) Head from wild-type Drosophila, showing the antenna and other head parts. (b) Head from an Antp mutant, showing the replacement of normal antenna structures with legs. This is caused by activation of the Antp gene in the head region

Control of Apoptosis

Apoptosis = Programmed cell death Occurs when D N A or chromosomal damage is too severe to repair (and which could risk the cell becoming cancerous... so apoptosis can definitely be good!) Cells halt progress through cell cycle Prevents cancer Eliminates cells not contributing to final adult organism (so apoptosis also normally occurs as a developmental process, such as the webbing between fingers)

Two important domains within riboswitch

Aptamer: Binds to ligand •Expression platform: Capable of forming terminator structure

Reciprocal chromosomal translocations:

Are characteristic of many cancers Include white blood cell cancers such as leukemias and lymphomas Example: Burkitt lymphoma: Reciprocal translocation between chromosome 8 and chromosomes 2, 14, and 22

Metastasis-suppressor genes:

Are mutated or disrupted in metastatic tumors Less than a dozen identified Affect growth and development of metastatic tumors, not primary tumor Example: C D82 protein In metastatic tumors, expression of C D82 is lost

Omics

Areas of biological research having an "omics" connection are continually developing Some examples of "Omics"• Proteomics• Metabolomics• Glycomics (aspects of the organism's sugars such as how they are metabolized)• Toxicogenomics and many more

Artificial Selection

Artificial selection (fast and easy compared to technique on next slide) • Choosing specific individuals with preferred phenotypes from initially heterogeneous population for future breeding • Purpose: To develop population containing high frequency of individuals with desired traits

Artificial Trait Selection

Artificial trait selection focuses on traits that are polygenic and multifactorial (slower and more complex) • Traits of economic importance fit this description:• Crops and livestock• Grain yield in plants• Weight gain or milk yield in cattle• Speed or stamina in horses

Important to note:

As the textbook will say, the "heavy metals" this gene can protect against... include "zinc" BUT zinc only acts like a harmful heavy metal at "TOO-HIGH" concentrations! At lower, HEALTHY concentrations in natural whole, healthy foods, zinc it is actually an essential nutrient! (As will be apparent from its upcoming described role!)

DNA Methylation

Associated with decreased gene expression • Methylation occurs most often on cytosine of CG doublets in DNA • Methylation can repress transcription by binding to transcription factors of DNA

Autosomal S T R D N A Profiling

Autosomal STR DNA profiling:• Small samples of DNA amplified using PCR; enables generation of DNA profiles Majority of forensic DNA profiling is done using commercial kits, which amplify regions of DNA known as microsatellites or short tandem repeats (STRs)STRs used in forensic profiling: STRs: short tandem repeats• Similar to VNTRs but repeated motif is shorter• 2-9 base pairs repeated 7-40 times Hundreds of STR loci are present in human genome FBI and other law enforcement agencies selected 13 STR loci to be used as core for forensic analysis

"Normal metabolism creates oxidative end products that can damage DNA... 10,000 damaging DNA lesions a per day due to the actions of oxygen free radicals."

BUT notice how this quote makes the possibility of cancer seem "uncontrollable"- I DO NOT AGREE WITH THIS mindset, BECAUSE: As the author FAILS TO MENTION immediately after this quote... that is why we need to be eating antioxidants (ideally from whole foods, and which are more abundant in organic foods): These antioxidants are naturally designed to COUNTERACT all of these "oxidative end products"!

Disadvantages to Using Bacteria

Bacteria are unable to process and correctly modify many eukaryotic proteins• Cannot add carbohydrates or phosphorylate like eukaryotic organisms Solution (for SOME products)—many biopharmaceuticals are now produced in eukaryotic hosts • Bioreactors or biofactories: Living factories, such as a herd of goats or cows • Baculovirus: Gene delivery system in which a virus used to infect insect cell

The NIH Roadmap Epigenomics Projec

Based on the idea that many aspects of health and susceptibility to disease are related to epigenetic regulation or misregulation of gene activity Focused on how epigenetic mechanisms controlling stem cell differentiation and organ formation generate biological responses to external and internal stimuli that result in disease

Benign versus. Malignant

Benign tumors Result from unregulated cell growth that forms a multicellular mass Removed by surgery, causing no serious harm Malignant tumors Result from metastasized cells invading other tissue and causing life-threatening problems

Golden Rice

Beta-carotene pathway in Golden Rice 2. Rice plant enzymes and genes involved in beta-carotene synthesis are shown on the right. The enzymes that are not expressed in rice endosperm are indicated with an "X." The genes inserted into Golden Rice 2 are shown on the left.

Control of Eye Formation

Binary switch genes in Drosophila:• Wild-type allele of binary switch gene programs eye formation instead of antenna• Mutant allele eyeless: Eyes are reduced in size and have irregular facets

Binary switch genes and regulatory networks program genomic expression

Binary switch genes• Allow only two alternative developmental fates for cell• Initiate complete development of organ or tissue type Gene-regulatory networks• Binary switch genes in conjunction with signaling pathways

Components of the Notch signaling pathway in Drosophila

Binding of Delta to Notch triggers a proteolytic-mediated activation of transcription. The fragment cleaved from the cytoplasmic side of the Notch protein, called the Notch intracellular domain (NICD), combines with the Su(H) protein and moves to the nucleus where it activates a program of gene transcription.

Repression Loop

Binding of repressor to operators O1 and O3 creates repression loop • Prevents access of RNA polymerase to promoter

DNA sequence analysis relies on bioinformatics applications and genomic databases

Bioinformatics: • Uses computer-based approaches to organize, share, and analyze data related to• Gene structure• Gene sequence and expression• Protein structure and function

Reproductive Isolation

Biological barriers that prevent or reduce interbreeding between populations• Mechanisms may be• Ecological• Behavioral• Seasonal• Mechanical• Physiological (i.e. non-compatible egg/sperm receptors, non-viable embryo etc)

Biopharming

Biopharming • Production of proteins in genetically modified plants and animals (the goal and product of interest is just the molecule, not the whole GMO organism) Biopharmaceutical products • Many are recombinant proteins from GMOs• May be therapeutic proteins that treat disease• Insulin is one example: Today's insulin comes from bacteria that express it (which they do not naturally do).

U V Light and Ionizing Radiation

Both U V light and ionizing radiation (X rays and gamma rays) induce D N A damage: U V sunlight can cause skin cancer Radon gas Responsible for up to 50% of ionizing radiation exposure of U.S. population and contributes to lung cancer... ... may be responsible for much "indoor air" apparently contributing to cancer... and may explain those studies showing higher levels of cancer in people with "indoor-only" jobs compared to those with a mixture of indoor and outdoor activities.

Disruptive Selection

Both phenotypic extremes are selected for• Intermediates are selected against• Results in population with increasingly bimodal distribution for trait• Example: Applied selection for low and high bristle number in Drosophila population• Other examples could include contrasting colors for camouflage with bimodal environmental colors

Summary of Operon Model 2

Both the I− and OC constitutive mutations interfere with the molecular interactions • In these cases, the structural genes are always turned on: I− mutant: The repressor protein is altered or absent and cannot bind to the operator region • The structural genes are always turned on O^C mutant: The nucleotide sequence of the operator D N A is altered and will not bind with a normal repressor molecule • The structural genes are always turned on

Broad-Sense Heritability

Broad-sense heritability (H2) • Measures contribution of genotypic variance to total phenotypic varianceH2 VG / VP Genotypic variance component includes all types of genetic variation in population Broad-sense heritability estimates assume genotype-by-environment variance component is negligible

ONE specific insect-resistant GM variety: Bt toxin-producing crops

Bt (Bacillus thuringiensis) is a group of soil-dwelling bacteria that naturally only parasitize insects; they produce Cry proteins, which are toxic to insects• Bt crops are engineered to produce the Bt toxin on their own, inside the actual crop plant, instead of the toxin being controlled by the bacteria that naturally make it: cry genes introduced into plant cells...• The GM crop plant now can manufacture its own Bt Cry proteins, which kills the target pest when it eats the plant's tissue (immediately). But when soil bacteria do it naturally, they actually WAIT for an insect to eat them first. (Natural, "whole bacteria" Bt is allowed in organic farming, but is not harmful to humans or animals in that form, because of the way the gene expression gets controlled by the bacterium).• On the other hand, there is evidence that GMO Bt crops may harm certain people (from all the basic science I understand, I believe that people who do not produce enough stomach acid are more likely to get harmed).

Limitations in Twin Studies

By the time they are born, M Z twins do not necessarily have identical genomes • Gene-expression patterns in M Z twins change with age; this leads to phenotypic differences

C D Ks

C D Ks: Cyclin-dependent kinases Regulation of cell-cycle progress is mediated by molecules called cyclins and cyclin-dependent kinases (C D Ks) They regulate synthesis and destruction of cyclin proteins

C R I S P R-Cas9

C R I S P R = "clustered regularly interspaced palindromic repeats" -Cas system Gene editing method that involves the use of specifically engineered D N A-modifying enzymes (nucleases) that create changes in a specific sequence to remove, correct, or potentially replace a defective gene or parts of a gene Gene editing is based on using different nucleases to create breaks in the genome in a sequence-specific manner HOWEVER... as the below (recent, year 2018) article in "Nature Biotechnology" shows, this procedure is not without its risks; many kinks apparently still need to get worked out: The CRISPR-Cas9 system allows for gene editing in eukaryotes by targeting specific DNA sequences (more details in the relevant "special topic" section of our textbook):

C R I S P R-Cas has been Adapted as a Powerful Tool for Genome Editing

C R I S P R-Cas Infidelity: Cas9 not only cuts at the intended target but also at off-target sites in the genome• Off-target edits may be due to an s g R N A having more than one perfect match in the genome Improving the specificity of C R I S P-Cas edits to the human genome will be important for the safety of medical applications of this technology (again see link to a concerning paper on earlier slide)

CRISPR-Cas technology has diverse applications

C R I S P R-Cas as a Tool for Basic Genetic Research: The ability to efficiently and quickly delete a gene from the genome has only recently become possible with C R I S P R-Cas technology Reverse genetics:• Learn about the function of a gene is to delete it and observe the consequences

CRISPR-Cas has been adapted as a powerful tool for genome editing

C R I S P R-Cas9 In Vitro: Hybrid R N A molecule called a single guide R N A (s g R N A)• A 20-nucleotide-long targeting sequence of a c r R N A joined to minimal sequences of the trac R N A necessary for Cas9 function Any sequence, with a P A M, could be targeted for cleavage in vitro with just two components:1. An s g R N A containing customized c r R N A and tracr R N A-derived sequences2. Cas9

Notch signal system—C. elegans

C. elegans used for genetic studies• Genetics are well known• Genome has been sequenced• Adults contain small number of cells that follow highly deterministic developmental program

The catabolite-activating protein (CAP) exerts positive control over the lac operon

CAP: Catabolite-activating protein = • Exerts positive control over lac operon • Diminishes expression of operon when glucose is present (catabolite repression) • When glucose is absent, CAP binds to CAP-binding site, facilitating RNA polymerase binding at promoter and facilitating transcription

Methylation:

CH3 is the "methyl group"

C R I S P R-Cas9 Genome Editing of Euk Cells

CRISPR-Cas9 Genome Editing of Eukaryotic Cells = Endogenous double-strand break repair pathways in the eukaryotic cell: Nonhomologous end-joining (NHEJ)• Ligation of broken D N A fragments• This process is error prone• Often results in small insertions or deletions (indels) at the repair site Homology-directed repair (HDR)• Uses an undamaged homologous chromosome or sister chromatid as a template to correctly repair a broken chromosome• Can be "tricked" into using an artificial donor template (instead of the homologous chromosome) to make complex substitutions, deletions, or additions• Less error prone

Frequencies of X-Linked Traits

Calculating frequencies of X-linked traits using Hardy-Weinberg equation: • Calculate allele and genotype frequencies for X-linked traits in mammals • Frequency of X-linked allele in gene pool equals frequency of males expressing X-linked trait......Since each male has only one X chromosome • Female with an allele on both X chromosomes will be q2

Bacterial small noncoding RNAs have regulation functions:

Can be negative regulators of gene expression by binding to mRNAs and preventing translation by masking the ribosome-binding site (RBS) • Can be positive regulators of gene expression by binding to mRNAs and preventing certain secondary structures [that would otherwise mask an RBS] and enable translation

Chromatin Remodeling and Histone Modification and Cancer

Cancer cells also show disrupted histone modification profiles ●Mutations in genes encoding members of the histone-modifying proteins histone acetyltransferase (HAT) and histone deacetylase (HDAC) are linked to the development of cancer •Rubinstein-Taybi syndromec

Precision Oncology

Cancer cells express many proteins that are specific to the tumor and have the capacity to be recognized by the patient's immune system as nonself antigens Neoantigens:• The novel, nonself antigens that are contained within mutated proteins in tumor cells Tumor-infiltrating lymphocytes (T I Ls) T cells are known to associate with tumors and are able to recognize tumor neoantigens• But, often not able to destroy tumor cells

Proto-oncogenes and tumor-suppressor genes are altered in cancer cells

Cancer cells may metastasize: To metastasize from primary tumor, cancer cells must digest components of extracellular matrix and basal lamina Extracellular matrix and basal lamina normally separate the body's tissues and inhibit migration of cells

Cancer cells contain genetic defects affecting genomic stability, DNA repair, and chromatin modifications

Cancer cells show higher than normal rates of: Mutation Chromosomal abnormalities Genomic instability Mutator phenotype: High level of genomic instability in cancer cells

Cancer Stem Cells

Cancer stem cells have been identified in: Leukemia Brain cancer Breast cancer Colon cancer Ovary cancer Pancreatic cancer Prostate cancer

Carcinogens

Cancer-causing agents (i.e. chemicals) There is a delay (often a large time delay) between exposure to carcinogen and appearance of cancer... that is also an indication of the multistep process Example: Leukemia from radiation exposure (Hiroshima) had an incubation period of five to eight years This is ALSO why it is SO DIFFICULT to "prove" that certain substances were carcinogens (i.e. RoundUp / glyphosate has been used since the 1970's... and it was not declared a carcinogen until very recently- in 2018 the fact started finally finding its way into [only some] biology textbooks! (so, that took 48 YEARS!). For comparison, asbestos ALSO took 40 years to become known as harmful.

Cancer: Multistep Process That Requires Multiple Mutations

Cancer: Multistep process requiring multiple mutations: Age-related incidence of cancer indicates that cancer develops from accumulation of several mutagenic events in a single cell Incidence of most cancers rises exponentially with age Independent and random mutations are necessary for cell to become malignant

Precision Oncology

Cancers may use many different strategies to suppress T-cell responses including:• The synthesis of molecules that bind to T cells and repress their activity• Have less M H C on surface for immune cells to detect• The presence of tumor-associated regulatory T cells called T-regs (including suppressor T cells)• Role is to repress the activities of activated T cells• The presence of other tumor-infiltrating cells such as macrophages and monocytes

Environmental agents contribute to human cancers

Carcinogens: Any substance or event that damages D N A and causes mutations to occur in proto-oncogenes or tumor-suppressor genes Carcinogens include chemicals, radiation, some viruses, and chronic infections Can be natural or human-made Our environments (and especially synthetic substances) contain abundant carcinogens

Types of Alternative Splicing

Cassette exons: May be excluded from the mature mRNA by joining the 3′ end of the upstream exon to the 5′ end of the downstream exon Skipping of cassette exons is the most prevalent type of alternative splicing in animals • Accounting for nearly 40 percent of the alternative splicing events

C. Elegans (a roundworm) serves as a model for cell-cell interactions in development

Cell-cell interactions:• Influence transcriptional programs and developmental fate of neighboring cells during embryonic developmentSignaling pathways in development:• Regulate development• Act both independently and in coordinated networks• Send and receive developmental signals and elicit specific transcriptional response

Merozygotes

Cells diploid for certain added genes • Analysis of lac expression in absence or presence of lactose partial diploid merozygotes used to support operon model for the lac operon

Natural Carcinogens

Certain natural substances and natural processes are potentially carcinogenic Aflatoxin Mold on bread and corn; one of the most carcinogenic chemicals known Nitrosamines Meat preservative; known to cause cancer (most often added in overabundance to processed meats, and this practice is NOT allowed in certified organic meat) Certain naturally occurring and synthetic pesticides and antibiotics can be carcinogenic

Expression Q T Ls (e Q T Ls)

Changes in locule number during tomato domestication. The ancestral species S. pimpinellifolium contains two locules. At some point, a high-locule allele of lc was introduced and probably appeared before the introduction of the present-day fas allele, which further expanded locule number. These two QTLs are the major loci controlling locule number. As alleles of other loci controlling locule number were introduced into domesticated varieties, phenotypic diversity in the modern-day species S. lycopersicum expanded even further.

Chromosome Territory

Chromosome territory • In the interphase nucleus, each chromosome occupies a discrete domain and stays separate from other chromosomes (staining methods have visualized this!) • Interchromosomal domains -Channels between chromosomes that contain little or no DNA Chromosome structure is continuously rearranged; transcriptionally active genes get cycled to edges of chromosome territories

C R I S P R-Cas Technology Has Diverse Applications

Clinical Use of C R I S P R-Cas to Treat or Cure Disease Gene therapy: correct a disease-causing mutation in the genome to treat or cure the disease Safety concerns:• Can a disease-causing mutation be corrected without other unwanted changes to the genome?

Clonal Origin of Cancer Cells

Clonal origin All cancer cells in primary and secondary tumors are clonal... Clonal: Originated from a common ancestral cell that accumulated numerous specific mutations

Coactivators and Enhanceosome

Coactivators- • Interact with proteins and enable activators to make contact with promoter-bound factors • Coactivators form complex "enhanceosome" Enhanceosome-- • Interacts with transcription complex • Repressor proteins at silencer elements decrease rate of PIC assembly and RNA Pol II release

One important protein with a zinc finger domain:

Coded for by "Myt1L gene": Found in developing neurons: "Mutations in this gene have been associated with an autosomal dominant form of cognitive disability and with autism spectrum disorder. Alternative splicing results in multiple variants."

The Human Epigenome Project:

Collects and catalogs data on a set of human epigenomes to serve as reference standards • This atlas allows researchers to perform integrative and comparative analysis of epigenomic data across genomic regions or entire genome

Comparative genomics analyzes and compares genomes from different organisms

Comparative genomics • Compares genomes of different organisms to answer questions about genetics and other aspects of biology Prokaryotic genomes sequenced are from organisms causing human diseases:• Cholera, tuberculosis, leprosy, and lyme diseases• Genome size and gene number in various prokaryotes are highly variabl

c D N A Library

Complementary D N A (c D N A) library • Contains complementary D N A copies made from m R N As present in cell population • Represents genes active transcriptionally at the time cells were collected for m R N A isolation (so it tells you about the DNA that was actually DOING something- remember, all DNA does not undergo expression at the same time, otherwise all our cells would look alike!)

Complex Origins of Our Genome

Complex origins of our genome• Genetic diversity in m t D N A used to infer where our species, Homo sapiens, originated• Highest levels of diversity in African populations• Non-African populations had origins 50,000 years ago• H. sapiens originated in Africa from earlier species belonging to the genus Homo

Computer-automated DNA sequencing using the chain-termination (modified Sanger) method.

Computer-automated DNA sequencing using the chain-termination (modified Sanger) method. The inset box at upper right illustrates dideoxynucleotide (ddNTP) structure. (1) A primer is annealed to a sequence adjacent to the DNA being sequenced. A reaction mixture is added. (2) All four ddNTPs are added to the same reaction tube. During primer extension, the polymerase occasionally (randomly) inserts a ddNTP instead of a dNTP, terminating the synthesis of the chain because the ddNTP does not have the OH group needed to attach the next nucleotide. Over the course of the reaction, all possible termination sites will have a ddNTP inserted, thus all possible lengths are produced. Products of the reaction are added on a capillary gel (3), bands read by a detector and imaging system (4), from the newly synthesized strand.

Computer-Automated D N A Sequencing

Computer-automated high-throughput D N A sequencing • Since early 1990s, D N A sequencing has been done through computer-automated Sanger reaction-based technology • Generates large amounts of sequence D N A • Enabled rapid progress of Human Genome Project

Divergence

Conclusions from this study:• Denisovans and Neanderthals are not direct ancestors of our species• Denisovans and Neanderthals and members of our species interbred• Denisovans and Neanderthals contributed to our genome (even without being "direct ancestors")• Supports the [newer] idea that human / hominid evolution was, at the least, most definitely NOT "linear" like so many inaccurate diagrams / icons seem to portray!• Just because these other, extinct species were very similar to us does not mean they were "us" (i.e. our direct ancestors)

Homeobox

Conservation of organization and patterns of expression in Hox genes. (Top) The structures formed in adult Drosophila are shown, with the colors corresponding to members of the Hox cluster that control their formation. (Bottom) The arrangement of the Hox genes in an early human embryo. As in Drosophila, genes at the (3′ end) of the cluster form anterior structures, and genes at the (5′ end) of the cluster form posterior structures

lacI Repressor Gene

Constitutive mutations can happen to the lac I [repressor] gene: • Located close to but not part of lac operon structural genes • Normally produces repressor molecule, which regulates transcription of structural genes • Allosteric repressor-- • Interacts reversibly with another molecule • Causes conformational change in three-dimensional shape and change in chemical activity

Knockout (K O) Mice

Construct targeting vector: Creates segment of D N A for introduction into cell • Undergoes homologous recombination with gene of interest and renders it nonfunctional • Target vector has mutated copy of gene of interest • Foreign D N A disrupts reading frame and produces nonfunctional protein

Leader region

Contains two tryptophan codons

Controversy Regarding Golden Rice

Controversy regarding Golden Rice• Clinical trials show that the beta-carotene in Golden Rice 2 was efficiently converted into vitamin A• Golden Rice 2 is undergoing biosafety testing• Critics of G M foods suggest that Golden Rice will make farmers too dependent one one type of food• There are concerns about long-term health or environmental effects

Core-promoter elements found in focused promoters:

Core-promoter elements are usually located between -40 and +40 nucleotides, relative to the transcription start site, indicated as +1 . BRE is the TFIIB recognition element, which can be found on one side or other of the TATA box. TATA is the TATA box, Inr is the Initiator element, MTE is the motif ten element, and DPE is the downstream promoter element

Histone Modification

Covalent bonding of functional groups onto N-terminal tails of histone proteins • Most common: acetyl, methyl, phosphate • Acetylation decreases positive charge, which reduces affinity of histone to DNA ("loosens" the DNA) • Histone acetylation of nucleosome is catalyzed by histone acetyltransferase enzymes (HATs): associated with increased transcription

Covariance and Correlation Coefficient (r)

Covariance statistic• Measures how much variation is common to two quantitative traits Correlation coefficient (r) • Can standardize covariance• Indicates if two traits both increase or decrease together (r is positive), or if one increases as the other decreases (r is negative)

Methods used to create GM plants

Creating G M plants: Two approaches1. Biolistic method2. Agrobacterium tumefaciens-mediated transformation Both methods target plant cells growing in vitro• Plant tissue is cultured that is grown in presence of nutrients or hormones• Cultured cells form clumps that will form roots• The rooted plant develops into a normal plant

Environmental Effects

Critics of G M foods state that GMOs released into the environment have potential consequences• Advocates argue that potential consequences can be identified and managed Both textbooks and documentaries such as "Genetic Roulette" often at least address both sides of these arguments. • Emerging herbicide resistance:• Despite "reduced" amount of herbicide use, herbicide insecticide resistance is emerging (as admitted by our own Genetics textbook)• Spread of GM crops into non-GM crops:• Outcrossing (gene flow): The transfer of transgenes from GM crops or wild plants• Confers undesired phenotypes

Crystal Structure Analysis

Crystal structure analysis • Determination of the crystal structure of lac repressor, repressor bound to inducer and operator DNA • Genetic and biochemical data complement missing structural interpretation In vivo, all three operators must be bound for maximum repression

Cystic Fibrosis

Cystic fibrosis (autosomal recessive)- Incidence of 1/2600 0.0004 in people of northern European ancestry• Frequency of recessive allele Since p + q = 1, p = 1 - q = 1 - 0.02 = 0.98 2pq = 2(0.98)(0.02) = 0.04, or 4%

Fertilized Egg

Cytoplasm of fertilized egg• Organized into series of maternally constructed molecular gradients that play key roles in determining developmental fates of nuclei After fertilization (in Drosophila, remember):• Zygote nucleus undergoes nuclear division without cytokinesis• Resulting huge, multinucleated cell: Syncytium

DNA libraries are collections of cloned sequences

D N A libraries • Represent a collection of cloned D N A • Two main types: Genomic and complementary (c D N A) Genomic library • Contains at least one copy of all sequences in genome of interest • Constructed by cutting genomic D N A with restriction enzyme and ligating fragments into vectors

Q T L Mapping

D N A marker linked to Q T L: • Correlation between phenotypic expression of trait with different genotypes is indicated by D N A markers in F2 Q T L mapping population

D N A Methylation

D N A methylation is responsible for: Gene silencing associated with parental imprinting Heterochromatin gene expression X chromosome inactivation

DNA-based vaccines

DNA encoding proteins from pathogen inserted into vector• Triggers immune response if individual is exposed to pathogen in future• Trials are underway for HIV and ZIKA• However there are many legitimate concerns, as per below paper:"The major concerns with regard to safety are integration of the plasmid DNA into the host genome, adverse immunopathological effects, the formation of anti-DNA antibodies resulting in autoimmune disease...."For the even newer mRNA vaccines, while it IS true that mRNA can't "change your DNA", HOWEVER, there are actual, very legitimate concerns that have NOT YET BEEN addressed (such as, "How does the cellular machinery know when to turn off the translation of the proteins?"), as this immunology paper outlines:

Which of the following decreases gene expression?

DNA methylation

Southern Blot

DNA to be analyzed are digested with restriction enzymes and the fragments are separated by gel electrophoresis. The pattern of fragments in the gel is visualized and photographed under ultraviolet illumination. The DNA fragments in the gel are denatured by soaking the gel in an alkaline solution. Then the gel is placed on a sponge wick that is in contact with a buffer solution and covered with a DNA-binding membrane. layers of paper towels or blotting paper are placed on top of the membrane and held in place with a weight. Capillary action draws the buffer through the gel, transferring the pattern of DNA fragments from the gel to the membrane. DNA fragments are fixed onto the membrane and then hybridized to a labeled DNA probe. The membrane is washed to remove excess probe and overlaid with a piece of X-ray film for autoradiography or detected with a digital camera with chemiluminescence probes. Only fragments hybridizing to the probe are visualize

Compiling

Decision to designate sequence as "final" is dictated by the amount of error genome scientists are willing to accept as a cutoff (so yes, this means even once something is "sequenced"... it may not be an "exact, set-in-stone" sequence!) • Once compiled/error checked, the genome is analyzed to identify:• Gene sequences• Regulatory elements• Features that reveal important informatio

The Uniqueness of D N A Profiles

Decreasing probability number• As an increasing number of loci is analyzed, there are smaller probabilities of a random match• Core set of 13 S T R loci to generate D N A profiles is used by law enforcement• Frequency of profile would be 1 in 10 billion! Twins: An exception to uniqueness• Identical twins have identical D N A and therefore virtually identical profiles Sibling share one allele at any D N A locus• When D N A profiles come from two closely related people, profile probabilities must be adjusted

C R I S P R-Cas is an Adaptive Immune System in Prokaryotes

Defenses are part of Adaptive immunity if the defense mechanism uses past exposure to a pathogen to stimulate improved defense against future exposure to the same pathogen C R I S P R-Cas is an adaptive immune system in bacteria Discovery of C R I S P R : A C R I S P R is a genomic locus in bacteria that contains clustered regularly interspaced short palindromic repeats C R I S P R loci have been identified in 40 percent of bacteria species and in 90 percent of archaea, another type of prokaryote

CRISPR-Cas is an adaptive immune system in prokaryotes

Defenses are part of innate immunity if they are not tailored to a specific pathogen Bacterial innate immunity:• Express endonucleases (restriction enzymes), which cleave specific D N A sequences• Restriction enzymes destroy foreign bacteriophage D N A, while the bacterium protects its own D N A by methylating it• Block phage adsorption• Block phage D N A insertion• Induce suicide in infected cells

Computer-automated D N A sequencers:

Designed for high-throughput sequencing• Made genomics possible• Essential for Human Genome Project• Sequences contained multiple capillary gels• Generated over 2 million b p per day

Future challenges and ethical issues

Despite progress, questions remain• What is the proper route for delivery?• What amount of therapeutic gene products must be produced?• Can gene therapy treat diseases with multiple genes involved?• Can the expression and timing of therapeutic genes be controlled?• Will gene editing approaches become more widely used?

Southern Blot

Determines whether a clone contains all or part of gene • Ascertains size and sequence organization of gene or D N A sequence of interest • Two steps: • Separation of D N A fragments by gel electrophoresis • Hybridization by using labeled probes

Interpreting DNA profiles

Determining the significance of a D N A profile match• Once a suspect's profile does match, interpretation becomes complicated• Necessary to estimate the probability that two profiles are a random match• Profile probability or random match probability method gives a numerical probability that a person chosen at random has same D N A profile as suspect profile

Tumorigenesis

Development of malignant tumor Result of two or more genetic alternations Progressively release cells from normal controls on cell proliferation and malignancy

Differentiated states develop from coordinated programs of gene expression

Development: Attainment of differentiated state by all cells in an organism Developmental geneticists study:• What genes are expressed, and when• In what part of developing embryo genes are they expressed• How expression is regulated• Results of defective genes

Dominance variance

Deviation from additive components that results when phenotypic expression in heterozygote not precise

Precision medicine and disease diagnostics

Diagnostic tests = Detect the presence or absence of gene variants linked to suspected genetic disorder in a symptomatic patientPredictive tests = Detect a gene mutation in patients with a family history of having a known disorder (e.g., Huntington disease)Carrier tests = Help identify patients carrying a mutation linked to a disorder that may be passed to offspring (e.g., Tay-Sachs or cystic fibrosis)Prenatal tests = Detect potential genetic disease in a fetus (e.g., Down syndrome)Preimplantation tests = Performed early on embryos in order to select embryos that do not carry a suspected disease

DNA sequencing is the ultimate way to characterize DNA at the molecular level

Dideoxynucleotide chain-termination sequencing (Sanger): • Most common method of D N A sequencing • Based on using dideoxynucleotides = Deoxynucleotides with a hydrogen at 3′ instead of an O H • Dideoxynucleotide causes D N A synthesis to terminate

Phenotypic Variance (Vp)

Differences in yield between two wheat varieties at different soil fertility levels. (a) No genotype-by- environment, or G * E, interaction: The varieties show genetic differences in yield but respond equally to increasing soil fertility. (b) G * E interaction present: Variety A outyields B at low soil fertility, but B yields more than A at high fertility levels

Variable gene activity hypothesis

Differentiation is accomplished by activating and inactivating genes at different times in different cell types• The hypothesis assumes:• Cell contains entire genome• Different transcription of selected genes controls development and differentiation of each ce

C R I S P R-Cas is an Adaptive Immune System in Prokaryotes

Discovery of CRISPR = CRISPR spacer sequences were identical to fragments of bacteriophage genomes Viral sequences within CRISPR loci serve as a "molecular memory" of previous viral attacks?Evidence that CRISPRs are important for adaptive immunity:• Exposed S. thermophilus to a specific phage• Bacterial cells that survived became resistant to the same phage strain, but not to other phage strains• The resistant bacteria possessed new spacers within their CRISPR loci with an exact sequence match to portions of the phage genome they had been exposed to• Deletion of new spacers in the resistant strains abolished their phage resistance• Experimental insertion of the same new viral sequence-derived spacers into the C R I S P R loci of sensitive bacteria rendered them resistant

Histone Modifications

Disrupted in cancer cells Genes that encode histone acetylases, deacetylases, methyltransferases, and demethylases are often mutated or aberrantly expressed in cancer cells

GWAS Identified SNPs

Do not directly confer disease susceptibility • Are located near the [likely-existing] causative variant in the genome and, thus, the two are inherited together (hence, they are "linked") From the mapping data: • Over 90 percent of disease-associated variations in the human genome are located within regulatory regions that likely impact transcription (hence, they are not mutations in the "proteins of interest" themselves!)

Driver and Passenger Mutations

Driver Mutations Give a growth advantage to a tumor cell The total number that occur in any particular cancer is small—between 2 and 8 Passenger Mutations Have no direct contribution to the cancer phenotype Acquired over time possibly from the high levels of D N A damage that occurs in cancer cells

Genetic analysis of embryonic development in Drosophila reveals how the animal body axis is specified

Drosophila development:• Ten-day cycle• Five distinct phases before adult stage:• Embryo• Three larval stages• Pupal stage• Adult stage

Introduction

During its life span, an organism has one genome, but this genome can be modified in diverse cell types at different times to produce many epigenomes (which can affect "what that DNA is actually doing")

Environmental Induction of Epigenetic Change

During the 1944-1945 famine in the Netherlands: •On average, adult calorie intake was only 400-800 calories per day (recommended amount is 1800-2000 calories)! •Immediately after the famine, mortality rates doubled (mostly attributed to malnutrition) ●Women pregnant during the 1944-1945 famine in the Netherlands had children with increased risk of diseases such as diabetes and coronary heart disease ●The F2 generation also had abnormal health patterns ●Important to note that even though this was a result of calorie restriction, harmful chemicals that often go along with "high calorie" foods can yield similarly unhealthy results

Bacteria regulate gene expression in response to environmental conditions

E. coli gene regulation has been studied extensively :• Highly efficient genetic mechanisms have evolved that turn transcription of specific genes on and off depending on metabolic need for gene products • Expression also responds to changes in environment • Regulated gene activity includes: Replication, recombination, DNA repair, and cell division

ENCODE data are transforming our concepts of eukaryotic gene regulation

ENCODE: Encyclopedia of DNA Elements Project • Goal: Identify all functional DNA sequences and determine how elements regulate expression

Genetic and Epigenetic Regulation of Development

Each differentiated cell type in the adult has a distinct pattern of gene expression At fertilization, an egg and sperm fuse to form a single totipotent cell, the zygote• Can become any cell type including placenta Embryonic stem cells (E S C s) form from blastula• These cells are pluripotent and can form any of the 200 or so different cell types found in adult

A model of randomly determined epigenetic gene regulation.

Early in development, random monoallelic regulation of homologous alleles on a cell-by-cell basis results in a population of cells within a tissue expressing combinations of allele expression: neither allele, only maternal, only paternal, or both alleles (biallelic expression). These states are passed on, resulting in a mosaic pattern of expression.

Target Vectors Introduced into Cells

Embryonic (E S) stem cells • Using E S cells, scientists introduce targeting vectors into cells via electroporation • E S cell takes in targeting vector and endogenous enzyme recombinase catalyzes homologous recombination • Recombinant E S cells are injected into mouse embryo • Results: Chimeras

Gal4 Protein

Encoded by GAL4 gene • Negatively regulated by Gal80 protein • Has DNA-binding domain that recognizes and binds to sequences in UASG • Binding of phosphorylated galactose to Gal80p ("I'm here- don't suppress my role in feeding the yeast!") and/or Gal4p exposes activation domain of Gal4p

lac A

Encodes enzyme transacetylase, which may be involved in removal of toxic by-products of lactose digestion from the cell

C R I S P R-Cas9 Genome Editing of Eukaryotic Cells:

Engineered plasmid expression vectors carrying genes encoding Cas9 and an s g R N A with a specific D N A targeting sequence Introduced them into mammalian cell culture C R I S P R-Cas9 cut precise sequence Endogenous D N A repair mechanisms fixed break but created indels (insertions/deletions) or added new donor sequence

Transcripts and Noncoding RNA

Enhancer RNA: • Genomic sequences encoding enhancer RNAs often contain bona fide (real, actual) promoter elements that initiate transcription from the enhancer and extend outward for several kilobases • The most active enhancers in a cell type produce the most enhancer RNA • Unknown functional relevance

Enhancers

Enhancers regulate transcription of eukaryotic genes: • They are also cis-acting transcription regulatory elements Enhancers: Located on either side of gene, some distance from gene, or even within gene • Important in reaching maximum level of transcription

Environmental Induction of Epigenetic Change

Environmental agents including variations in nutrition, chemicals (this is one category of "agents" that I personally "love to hate"!), and physical factors such as temperature can alter gene expression by affecting the epigenetic state of the genome

Constitutive enzymes

Enzymes are continuously produced regardless of chemical makeup of environment

Epigenetic alterations to the genome

Epigenetic changes occur through three major mechanisms: • Methylation • Reversable; addition or removal of methyl groups • Histone modification and chromatin remodeling • Alter the accessibility of genes for transcription • Noncoding RNA

Chromatin Modifications and Cancer Epigenetics

Epigenetics: Study of factors that affect gene expression but do not alter nucleotide sequence of D N A Epigenetic effects may be present in somatic or germ-line cells Examples of epigenetic modifications: D N A methylation Histone acetylation and phosphorylation

Broad-Sense Heritability

Estimate provided by comparison of phenotypic variances for same trait in identical and fraternal twin sets

Narrow-Sense Heritability

Estimates of narrow-sense heritability are expressed as percentage values for traits among different organisms

Organization of the eukaryotic cell facilitates gene regulation at several levels

Eukaryotic gene regulation is more complex than that in prokaryotes: • Great amount of DNA is associated with histones and other proteins • mRNAs must be spliced, capped, and polyadenylated prior to transport from nucleus • Genes on numerous chromosomes are enclosed in a double membrane nucleus

Evolutionary conservation of developmental mechanisms can be studied using model organisms

Evolutionary mechanisms for differential development of organisms include:• Mutation• Gene duplication and divergence• Assignment of new functions to old genes• Recruitment of genes to new developmental pathways Evo-devo = evolutionary developmental biolo

Speciation and Evolutionary Origin

Evolutionary origin of Arrow cichlid • Phylogenetic, morphological, and ecological analyses has been done • Sequence analysis of m t D N A indicates two species are from monophyletic group • Concluded that Arrow cichlid evolved from[and ancestor of the] Midas cichlid

Human metallothionein IIA gene (hMTIIA)

Example of how a gene can be transcriptionally regulated due to interplay of: • Promoters • Enhancer elements • Transcription factors that bind to them

Detecting Genetic Variation

Example: Domestic dog... Genetic and archeological evidence indicates domestication of dogs took place at least 15,000 years ago (there was selection of desired traits present in genetic variation in wild wolves)The size difference between a Chihuahua and a Great Dane illustrates the high degree of genetic variation present in the dog genome

Proteolytic Enzymes

Example: Metalloproteinases Present at higher than normal levels in highly malignant tumors Malignant cells are not susceptible to normal controls conferred by regulatory molecules such as tissue inhibitors of metalloproteinases (T I M Ps)

Speciation

Example• Lake Apoyo in Nicaragua• Formed within past 23,000 years• Home to two species of cichlid fish• Midas cichlid: Common in region• Arrow cichlid: Found ONLY in this lake

Different types of alternative splicing eVENTS

Exons are indicated by boxes with introns depicted by solid thick lines between them. Alternative splicing is indicated by thin red lines either above or below the pre-mRNA. Transcription start sites (bent arrows) and polyadenylation signals (poly A) are indicated in the alternative promoters and polyadenylation examples

Two-Allele System

Expected genotypic frequencies for population in Hardy-Weinberg equilibrium• Calculated based on allele frequencies in gametes and random mating

One sub-category of transgenic animals: Knock-in animals

Express or overexpress particular gene of interest (transgene) • Vector with transgene undergoes homologous recombination into host genome • Vector with transgene can also be put into E S cells and injected into embryos • Allows for study of effects on appearance and function in mice

Segment Polarity Genes

Expression of segment polarity genes:• Controlled by transcription factors encoded by pair-rule genes• Segment polarity genes become active in single band of cells that extends around embryo's circumference to divide embryo into 14 segment The 14 stripes of expression of the segment polarity gene engrailed in a Drosophila embryo

P G P and H M P

Extensions of H G P • P G P: Personal Genome Project = Proposed personal genome approach • H M P: Human Microbiome Project = Determine if people share core human microbiome (the good / normal microbes such as bacteria in the body)• Understand correlations between microbiome & health• Use bioinformatics for analysis of microbiome Genome mosaicism• Cells in an individual don't all contain identical genomes• Can result from errors in D N A replication, creating aneuploidy, and other variations that accumulate as cells divide during development

M A D S-box proteins

Family of transcription factors in Arabidopsis homeotic genes• Sequence of 58 amino acids not similar to amino acid sequence in Hox genes• Homeotic genes of Arabidopsis do not share sequence homology with Drosophila Hox genes

Transcription Factory

Feature within nucleus that may contribute to gene expression • Nuclear sites that contain most of the active RNA polymerase and transcription regulatory molecules • Dynamic structures that form rapidly and disassemble upon stimulation and repression of transcription

Focused vs dispersed promoters

Focused promoters (a) specify one specific transcription initiation site. Dispersed promoters (b) specify weak transcription initiation at multiple start sites over an approximately 100-bp region. Dispersed promoters are common in vertebrates and are associated with housekeeping genes. Transcription start sites and the directions of transcription are indicated with arrows

Transcription Levels

For Maximum transcription of structural genes: • Repressor must be bound by lactose• Does not repress operon expression • CAP must be bound to CAP-binding site (no glucose is present when this is possible)

E

For example, BRCA1 is hypermethylated & inactivated in breast cancer and ovarian cancer •BRCA1 is a tumor suppressor gene •Therefore, if it is hypermethylated, it cannot get expressed very well... •And therefore, it wouldn't be able to "suppress tumors" very well. ●The combination of mutation and hypermethylation often occurs in familial forms of cancer •CDKN2A mutation in bladder cancer

Activators and repressors interact with general transcription factors and affect chromatin structure

Formation of RNA Pol II initiation complex: General transcription factors (GTF): • Are proteins • Required at promoter to initiate basal or enhanced levels of transcription • Assembly of proteins in specific order forms pre-initiation complex (PIC) • PIC provides platform for RNAP II to recognize transcription start sites

Antiterminator hairpin structure

Forms in absence of tryptophan; ribosome stalls at codons (but that allows the structural genes downstream to get translated)

terminator hairpin:

Forms in presence of tryptophan...ribosome has tryptophan to do translation with, at the LEADER sequence, BUT that causes a DIFFERENT hairpin to form elsewhere which prevents the structural genes from getting translated

Terminator hairpin

Forms in presence of tryptophan; ribosome proceeds through the leader sequence (but resulting different hairpins prevent structural gene translation)

Insulators

Found between an enhancer and a promoter for a nontarget gene • allow some enhancer-promoter interactions and block others

Calculating Frequencies of Alleles

Frequencies for multiple alleles:• Calculated by adding additional variables to Hardy-Weinberg equation • Example: Situation involving three alleles • p +q + r = 1 • Frequencies of genotypes given by(p + q + r)2 = p2 + q2 + r2 + 2pq + 2pr + 2qr = 1

Functional genomics establishes gene function and identifies regulatory elements in a genome

Functional genomics• Study of gene functions, based on resulting R N A s• Based on possible proteins and regulatory elementsFunctional genomics establishes gene function and identifies regulatory elements in a genome B L A S T searches• Used to screen databases and compare a sequence to a known sequence Similarity searches• A genome sequence statistically similar to gene with known function likely encodes for protein with similar function ; Example:• Compare portions of human leptin gene (L E P) with its homolog in mice (o b/L e p)

p53 and the Cell Cycle

Functional p53 can arrest cell cycle at several phases, but: Cells lacking p53 are unable to arrest at cell-cycle checkpoints or enter apoptosis Cellular stress events increase p53 levels, such as: D N A damage Double-stranded breaks in D N A Presence of D N A-repair intermediates due to U V light All of those are examples of what could predispose to cancer... so if p53 is not functional to halt that... it spells extra trouble.

G M Crops

G M crops are grown in approximately 30 countries:• 90 percent are grown in the United States, Brazil, Argentina, Canada, and India• 93 percent of soybeans• 88 percent of corn• 70 percent of processed foods in the United States contain G M crop ingredient

GM Foods [attempt to] Address Malnutrition

G M foods [attempt to] address malnutrition• More than 200 different G M foods have been created• Golden Rice: A variety of rice that contains the vitamin A precursor• Purpose: To alleviate vitamin A deficiencies in developing world• Protestors argue that Golden Rice is a threat to human health and biodiversity• Some may validly argue that there may be many other types of solutions to vitamin A deficiency (for example, there do exist many types of crop plants that naturally produce rich sources of vitamin A)

Gene Regulation in a Model Organism: Transcription of the GAL Genes of Yeast

GAL gene system in yeast- • Composed of four structural genes and three regulatory genes • Products of structural genes transport galactose into cell for metabolism Positive control:- Activator protein must be present to turn on gene transcription

GAL Genes are Inducible

GAL genes are inducible: • Transcription is regulated by presence or absence of substrate (galactose) • Absence of galactose: GAL structural genes not transcribed • Galactose added to medium: Transcription begins immediately

Transcription is Positively Regulated

GAL1 and GAL10 genes: • Transcription of both genes is positively regulated • Controlled by central control region, UASG (upstream activating sequence of GAL genes) • Contain four binding sites for Gal4 protein (Gal4p) • UAS is functionally similar to enhancers in eukaryotes

Health and Safety

GM food advocates state that there is "no evidence" of adverse health effects. However, existing observations and facts that could be considered evidence may be subjective- some scientists may indeed consider it "evidence" while other entities may claim it is not sufficient evidence, such as in this paper:)Also, here is a question to think about: If sufficient funding is unavailable to conduct extensive studies (i.e. cancer in a human decades later) and thus studies are impossible to conduct (or if they are done, but publication gets suppressed)... and for that reason there is "lack of evidence for harm"... is everyone going to consider that "a good enough reason to assume it is safe"? Probably not.In the analysis of the history of discovery of the toxicity / carcinogenicity of substances such as lead, asbestos (and more recently RoundUp specifically as well as certain sunscreens that ironically cause cancer)... the history of discovery has taught us that potential harm unfortunately cannot be detected right away, and all too often the truth can take 40+ years to get fully unveiled (!) In light of history such as that, I would never blame anyone for being skeptical of anything which they may suspect in the slightest as being possibly harmful to them- people are free to avoid anything they deem even slightly potentially harmful to themselves

GM foods controversies

GM foods have increased farm productivity [according to certain studies, yet not others, as per cases expressed in the documentary Genetic Roulette... see the video starting at 51:21] =• Critics claim GM foods are unsafe (many specific, legitimate details are presented in that same video above, but also between pages 5-10 of the "Theory of..." paper in the Supplemental Module, as well as the original sources cited, at the end of that paper)• However it is also true that every specific GM crop or organism has different expression sequences (although, as mentioned before, ONE feature common to all GMOs is the "inter-species genes" characteristic)• Thus, making general statements about all GM foods is not usually possible or accurate (unless, of course, you are talking about that one property of having "inter-species" genes, which IS common to all GMOs!)

Core Promoter TFIID

GTFs (general transcription factors) that assist RNAP II at a core promoter include: TFIIA, T FIIB, TFIID, TFIIE, TFIIF, TFIIH, and a large multi-subunit complex called Mediator Core promoter TFIID- • General transcription factor that assists RNA Pol II at core promoter TFIID • First step in forming PIC—binding of TFIID to TATA box via TATA binding protein (TBP)

Summary

Gap genes are activated to subdivide embryo into broad bands2. Gap genes activate pair-rule genes, which further divide embryo into segments3. Segment polarity genes further divide each segment into anterior-posterior axes, which are then given identity by Hox genes

GenBank & BLAST

GenBank:• Maintained by National Center for Biotechnology Information (NCBI)• Each sequence deposited in GenBank receives an accession number• Accession number is used to access / retrieve sequence for analysis• The largest publicly available database of D N A sequences BLAST (Basic Local Alignment Search)• Software application used to compare a segment of genomic DNA (which may have been newly discovered, and its potential function and other implications unknown) to sequences throughout major databases• Identifies portions that align with or are the same as existing sequences (similarity score)• "E Value": Expect value• Based on number of matching sequences in database expected by chance

Creating knockout and transgenic organisms for studying gene function

Gene targeting • Concept: To manipulate specific allele, locus, or base sequence and learn its function on gene of interest • Manipulates specific gene in genome Gene knockout • Concept: To disrupt or eliminate specific gene/genes and see "what happens" • Knockout mice in particular have revolutionized research

Genetic testing, including genomic analysis, is transforming medical diagnosis

Gene testing = One of the first successful applications of recombinant D N A technology• Currently >900 gene tests in use• Detect D N A mutations associated with single-gene diseases• Sickle-cell anemia, cystic fibrosis, Huntington disease, hemophilia, muscular dystrophies Autism spectrum disorder (A S D):Whole genome sequencing (W G S) revealed A S D has more than 100 different genes involved (not necessarily causing the disorder though, as SOME revealed inherited mutations but OTHERS were identified as sporadic de novo mutations (i.e. possibly from harmful chemicals

What genetic conditions are candidates for treatment by gene therapy?

Gene therapy:• The delivery of therapeutic genes into patient's cells• Goal is to correct genetic disease conditions caused by faulty genes• Gene therapy is the ultimate goal of translational medicine—translating scientific discoveries into effective therapy Criteria for gene therapy:• The gene or genes involved in causing disease have been identified• The gene can be cloned or synthesized in lab• Human Genome Project• Identification of human disease genes and their specific D N A sequences increased the number of candidates for gene therapy trials

Analysis of Developmental Mechanisms

General processes in development:• How adult body plan is laid down in embryo• Program of gene expression turns undifferentiated cells into differentiated cells• Role of cell-cell communication in developmen

Genetic drift causes random changes in allele frequency in small populations

Genetic drift = • Significant random fluctuations in allele frequencies in small populations (due to death of individuals for example...but NOT due to migration, despite what the term "drift" may seem to indicate)• Possible by chance alone• Degree of fluctuation increases as population size decreases • Can also [more specifically] occur as result of: • Founder effect • Genetic bottleneck

Genetic bottleneck

Genetic drift can also occur as a result of genetic bottleneck... • Develops when large population undergoes drastic but temporary reduction in numbers • Populations may recover, but with greatly reduced genetic diversity (that matches the low number of survivors)

Genetically engineered organisms synthesize a variety of biological and pharmaceutical products

Genetic engineering• Alteration of organism's genome using recombinant D N A technologies to add or remove gene from genome• Produces genetically modified organisms (G M Os) Biotechnology• Utilizing living organisms to create a product or process that helps improve life for humans or other organisms (this is a BROADER term that actually INCLUDES "genetic engineering" but ALSO includes OLDER techniques such as natural breeding).

Gene Maps

Genetic mapping • When many Q T Ls for a given trait are located, a genetic map gives positions of genes involved on different chromosomes Q T L mapping • Extensively used in agriculture• Corn, rice, wheat, and tomatoes (Table on next slide)• Livestock: Cattle, pigs, sheep, and chickens• Phenotypic effects in tomatoes

Neutral theory of molecular evolution:

Genetic variation due to mutation and drift• Proposes mutations leading to amino acid substitutions are detrimental; only a small fraction is favorable• Alternative explanation for high genetic variation is natural selection• Enzyme or protein variations maintained by adaptation to certain environmental conditions• Sickle-cell anemia (having the sickle cell trait renders one less vulnerable to malaria)

C R I S P R-Cas Technology Has Diverse Applications

Genetically attaching base-editing enzymes to d Cas9, like cytidine deaminases, to d Cas9 enables a researcher to convert the base cytosine into uracil, which is then replaced with thymine by the cell's endogenous base excision repair mechanismDoes not induce cuts in the D N A, it may avoid the indels common to imprecise double-strand break repair

What are GM foods (genetically modified foods)?

Genetically modified foods = Are / derived from "genetically modified organisms" (GMOs). Genetically modified organisms = Many sources define them as: "Plants and animals whose genomes have been altered / modified via genetic engineering or recombinant DNA technologies"......HOWEVER, one ADDITIONAL part of the definition of a GMO is the fact that the 'recombinant DNA' in this sense means DNA that has been put together in the same genome, from at least two different species. Hence, all GMOs share ONE main property: They all contain "inter-species gene sequence patterns." Beyond that, each type of GMO can vary quite a bit. As pages 5-9 of the "Theory of..." paper (in the Supplemental Module) mentions, the "inter-species genes" feature may be relevant when it comes to potential health differences if they get eaten. Unfortunately, that important part of the definition sometimes gets omitted / left out by certain sources... usually when they are attempting to justify the argument for "no significant difference" between the genetics of a GMO vs. that of a "natural hybrid" (i.e. from meiosis and random fertilization). But there is indeed ONE difference: Recent and intentionally-induced, inter-species gene sequences.

Genomic analysis before modern sequencing methods involved classical genetics approaches and cloning to map one or a few genes at a time

Geneticists typically followed a classical genetics approach to identify / characterize all of the genes in an organism's genome :1) They identified spontaneous mutations or collected mutants induced by chemical or physical agents 2) They generated linkage maps using these mutant strains Genomics allows sequencing of entire genomes: Genome = Complete set of D N A in a single cell of an organism Genomics: The study of genomes• Structural genomics• Functional genomics• Comparative genomics• Metagenomics

Genome-wide association studies identify genome variants that contribute to disease

Genome-wide association studies• Quest to identify genes that influence disease• Result: 700 publications linking 3000 genetic variations to about 150 traits How G W A S is carried out:• Compares genomes of thousands of unrelated individuals with particular disease to and those without disease• Identifies variations that confer risk of developing disease

Cancer

Genomic alternations associated with cancer include single-nucleotide substitutions, chromosomal rearrangements, amplifications, and deletions Cancer caused by mutations in somatic cells Only 5% of cancers are associated with germ-line mutations (hence only 5% of cancers are inherited!)

Genomic Instability

Genomic instability in cancer cells manifests in gross defects Translocations Aneuploidy Chromosome loss D N A amplification Chromosomal deletions

Changes Lead to Speciation

Geographic changes can lead to speciation• Example: Ancestors of snapping shrimp• Prior to formation of Isthmus of Panama, members were of a single species When the isthmus closed, each of the seven ancestral species was divided into two separate populations

Stress-Induced Behavior is Heritable

Glucocorticoid receptor (GR) expression levels were associated with differences in histone acetylation and DN A methylation levels GR gene promoter:• Low-M N rats with low expression of GR • Had significantly higher levels of promoter methylation • High-MN rats with high expression of GR • Had low levels of promoter methylation

Promoter Diversity

Great diversity exits in eukaryotic promoters, in structure and function: • Focused promoters- • Specific transcription initiation at ONE start site • Major type of initiation for lower eukaryotes • Dispersed promoters- • Direct initiation from several weak transcriptional start sites

Alternative Splicing

H G P revealed that number of genes is lower than number of predicted proteins • Many genes code for multiple proteins through alternative splicing• Produces incredible diversity of proteins beyond number of human genes • Alternative splicing patterns can generate multiple m R N A molecules = multiple proteins

The Relationship Between H D and Other Neurodegenerative Disorders

HD also shares some features with some non-polyQ neurodegenerative diseases Including Alzheimer disease (AD) and Parkinson disease (PD)• Are incurable disorders characterized by the same symptoms seen in Huntington disease:• Adult onset• The formation of misfolded protein aggregates• Neurodegeneration• Disease-specific behavioral changes Different proteins form aggregates in each of these disorders:• Tau in Alzheimer disease• Huntingtin in Huntington disease• Alpha-synuclein in Parkinson disease Aggregates first appear in different brain regions

Fitness (w)

Hardy-Weinberg analysis allows fitness (w) to be examined for each genotype: • Fitness: Individual's genetic contribution to the future generations (so, it's really "reproductive fitness") • Homozygous recessive individual who dies before producing offspring would have a fitness (w) = 0 • Frequency of such a recessive allele would be expected to decrease in each generation

Heterozygote Frequencies

Hardy-Weinberg law allows estimation of frequency of heterozygotes in population Frequency of recessive trait determined by first counting individuals in population sample who are affected• Example: Cystic fibrosis

The Hardy-Weinberg Law can be applied to human populations

Hardy-Weinberg law applied to humans: • Analysis of susceptibility to H I V-1 infection • Based on C C R5 gene... Encodes protein C C R5, a receptor for strains of H I V-1The Hardy-Weinberg Law can be applied to human populations The organization of the CCR5 gene in region 3p21.3 of human chromosome 3. The gene contains 4 exons and 2 introns (there is no intron between exons 2 and 3). The arrow shows the location of the 32-bp deletion in exon 4 that confers resistance to HIV-1 infection.

Hardy-Weinberg Predictions

Hardy-Weinberg law makes two predictions: • Frequency of alleles in gene pool doesn't change over time• After one generation of random mating, genotype frequencies for two alleles calculated as:p2 + 2pq + q2 = 1 • p = frequency of allele A • q = frequency of allele a ALSO: p2 = frequency of the genotype AA 2pq = frequency of the genotype Aa q2 = frequency of the genotype aa

Lac (lactose) operon

Has three structural genes (genes coding for primary structure enzyme): lac Z, lac Y, and lac A Operon has upstream regulatory region consisting of operator and promoter Entire gene cluster functions to provide rapid response to presence or absence of lactose

Types of Alternative Splicing Alternative promoters

Have more than one site where transcription may be initiated Produces pre-mRNAs with different 5′ exons, which may be alternatively spliced to downstream exons

Herbicide-Resistant G M Crops

Herbicide-tolerant G M crops are the most widely planted• 70 percent of G M crops are engineered to be tolerant to the herbicide glyphosate• They [RoundUp Ready crops] contain a weed / antibiotic resistance gene that confers tolerance to the broad-spectrum herbicide glyphosate (because, in addition to killing plants, glyphosate kills all bacteria, including probiotics, in the same way...a bit of knowledge not readily advertised... but the company is currently being pushed to put that fact on their RoundUp labels, since it adversely harms good bacteria in our intestines)• Glyphosate = The active ingredient in Roundup

An electropherogram showing the results of a DNA profile analysis using the 24-locus STR profile kit shown in the prior figure

Heterozygous loci show up as double peaks, and homozygous loci as single, higher peaks. The sizes of each allele can be calculated from the peak, locations relative to the size axis shown at the top of each panel.

Eye formation on the antenna (circled) of Drosophila induced by action of the mouse version of the eyeless gene (Small eye)

High magnification of the induced eye (circled) showing that the eye structure on the antenna is normal.

Style of maternal care is transmitted across rat generations through epigenetic events that take place early in postnatal life

High maternal nurturing induces high levels of serotonin in the brain, leading to DNA hypomethylation, histone acetylation, and increased expression of GR. In adulthood, high levels of GR expression increase adaptation to stress and, in females, passes on the high-MN phenotype. Rat pups experiencing low levels of maternal nurturing had higher levels of promoter methylation and reduced levels of GR expression. In adulthood, this led to poor stress adaptation and, in females, perpetuation of low levels of nurturing her pups.

Histone Modification

Histone modification is an important epigenetic mechanism of gene regulation Chromatin is composed of DNA wound around an octamer of histone proteins to form nucleosomes Amino acids in the N-terminal region of the histones can be covalently modified by acetylation, methylation, and phosphorylation

Histone Modification

Histones in nucleosomes have their N-terminal tails covalently modified in epigenetic modifications that alter patterns of gene expression. Ac = acetyl group Me = methyl group P = phosphate group

Homeotic selector genes specify body parts of the adult

Homeotic genes (selector genes)• Activated as targets of zygotic genes• Selector genes: Determine which adult structures will be formed by each body segment—antennae, mouth parts, legs, wings, thorax, and abdomen Homeotic mutants• Structure formed by one segment is transformed into that formed by another segmen

Homeotic Selector (Hox) Genes

Homeotic selector (Hox) genes:• Drosophila genome contains two clusters on chromosome 3• Antennapedia complex and bithorax complex• They encode transcription factors (see Table)

Alternative splicing example

How many different polypeptides can be produced through alternative splicing of the same pre-m R N A given the below numbers of each exon as described below? Each mRNA will contain one of the 12 possible exons for exon 4, one of the 48 possible exons for exon 6, one of the 33 possible exons for exon 9, and one of the 2 possible exons for exon 17 Can produce 38,016 different proteins!

SNPs

Human genomic sequence is 99.9% same with most genetic differences resulting from: • Single-nucleotide polymorphisms (S N P s)• Single base changes in genome• Variations may be associated with disease conditions • Copy number variations (C N V s)• Segments of D N A duplicated or deleted

Twin studies allow an estimation of heritability in humans

Human twins:• Useful subjects for examining phenotypic variance for multifactorial traits due to genotype as opposed to environment • Underlying principle:• Monozygotic (identical twins) are derived from single zygote • Dizygotic (fraternal twins) are derived from two separate fertilization events

The relationship between HD and other neurodegenerative disorders

Huntington disease is one of 14 trinucleotide repeat diseasesThe relationship between HD and other neurodegenerative disorders Trinucleotide diseases show genetic anticipation— An earlier age of onset and increasingly severe symptoms in cases where repeat lengths increase from generation to generationSee Chapter 4 for a discussion of genetic anticipation

Apotosis

Hypermethylated genes include those involved in DNA repair, differentiation, drug resistance, and apoptosis •Apoptosis = programmed cell death of certain cells such as those predisposed to becoming cancerous

Merozygotes 2

IS mutant: was discovered wherein the operon, as predicted, is "superrepressed" An additional I+ gene does not effectively relieve repression of gene activity These observations are consistent with the idea that the repressor contains separate DNA-binding domains and inducer-binding domains

Phenotypes in Twins

Identical twins (monozygotic)• Phenotypic variance equals environmental variance; there is no genotypic variance Fraternal twins (dizygotic)• Phenotypic differences represent environmental variance and approximately half genotypic variance

G W A S: Genome-wide associated studies of e Q T Ls

Identified loci for asthma susceptibility, pulmonary diseases, cleft lip, Type 2 diabetes, and coronary artery disease

Nucleic Acid Blotting

If you are wondering, the name has to do with the last name of its inventor, not the geographical direction • Used to identify which clones in library contain given D N A sequence • Used to characterize size of fragments from restriction digest • Methodology: Hybridization between complementary nucleic acid (D N A or R N A) molecules

Developing Targeted Drugs

Immunohistochemistry (I H C) and fluorescence in situ hybridization (F I S H) are molecular assays that can be used to determine the gene / protein status of breast cancer cells Herceptin used in combination with chemotherapy increases survival by 25 to 50 percent versus chemotherapy alone• There are dozens of drugs whose prescription and use depend on the genetic status of the target cells• Approximately 10 percent of F D A-approved drugs have labels that include pharmacogenomic information

Personalized T-Cell-Based Therapies

Immunotherapy with genetically engineered T Cells Create recombinant T-cell receptors (T C R s) that specifically recognize antigens on cancer cells D N A sequences that encode these engineered T C R s are then introduced in vitro into a patient's normal naïve T cells which then express these T C R s on their surfaces The T C R-transduced T cells are then selected, amplified, and reinfused into the patient Chimeric antigen receptors (C A Rs) can directly recognize antigens on the tumor cell without requiring T-cell activation by antigen presenting cells• Normal T cells need activation by antigen C A R proteins are fusions of several proteins

Epigenetics and Imprinting

Imprinted genes show expression of only the maternal allele or the paternal allele This parent-specific pattern of allele expression occurs during gamete formation Differential methylation of CpG-rich regions produces allele-specific imprinting and subsequent gene silencing

Blue-White Screening

In blue-white screening, DNA inserted into the multiple cloning site of a plasmid disrupts the lacZ gene so that bacteria containing recombinant DNA are unable to metabolize X-gal, resulting in white colonies that allow direct identification of bacterial colonies carrying DNA inserts to be cloned. (Bottom) Photo of a Petri dish showing the growth of bacterial cells after uptake of plasmids. Cells in blue colonies contain vectors without DNA inserts (nonrecombinant plasmids), whereas cells in white colonies contain vectors carrying DNA inserts (recombinant plasmids). Nontransformed cells did not grow into colonies due to the adding of ampicillin in the plating medium (and because, if they were "not transformed" that means they did not successfully take up the plasmid with the resistance gene

Lactose metabolism in E. coli is regulated by an inducible system

In the presence of lactose, the concentration of enzymes responsible for its metabolism increases = inducible enzymes

Steps in the PCR amplification and analysis of one STR locus (D8S1179)

In this example, the person is heterozygous at the D8S1179 locus: One allele has 7 repeats and one has 10 repeats. Primers are specific for sequences flanking the STR locus and are labeled with a red fluorescent dye. The double-stranded DNA is denatured, the primers are annealed, and each allele is amplified by PCR in the presence of all four dNTPs and Taq DNA polymerase. After amplification, the labeled products are separated according to size by capillary electrophoresis, followed by fluorescence detection.

Dizygotic twins

Includes approximately half genetic variance

Interphase

Interphase Interval between mitotic division Cell grows and replicates its D N A (G1, S, G2) Cells that stop proliferating enter G0 Do not grow or divide but are still metabolically active (like neurons) Cancer cells are unable to enter G0 and thus will cycle continuously (even when the cell is not supposed to)

Types of Alternative Splicing: Intron retention

Introns are normally noncoding sequences, but sometimes are included in the mature m R N As and are translated, producing novel isoforms (intron retention) Can negatively regulate gene expression at the posttranscriptional level • Such mRNAs are degraded or are retained in the nucleusI

Summary of Operon Model

Invokes series of molecular interactions between proteins, inducers, and DNA • No lactose: Enzymes are not needed and expression of genes encoding enzymes is repressed • Lactose present: Indirectly induces activation of genes by binding repressor • If all lactose gets metabolized: None is available to bind to repressor and transcription is repressed

Fluorescent in situ hybridization (F I S H):

Involves hybridizing probe directly to chromosome or R N A without blotting • Carried out with isolated chromosomes on slide or in situ in tissue sections or entire organisms • Helpful when embryos are used for various studies in developmental genetics

Chromatin Remodeling

Involves repositioning or removal of nucleosomes on DNA • Repositioned nucleosomes may make chromosome regions accessible to: • Transcription regulatory proteins • Transcription activators • RNAPII (RNA polymerase II)

Vulval Development

Involves three rounds of cell-cell interactions that transmit or receive signals from other cells• In C. elegans:• Hermaphrodites lay eggs through the vulva• Interactions between cells Z1.p p p and Z4.a a a determine the gonadal anchor cell and precursor to uterus

C M L: Chronic myelogenous leukemia

Involves translocation of C-A B L gene on chromosome 9 into B C R gene on chromosome 22 Structure is known as Philadelphia chromosome

Cancer is a genetic disease at the level of somatic cells

It means it is genetic" (has to do with damaged DNA)... ...BUT it is not usually "inherited" (since somatic cells are not inherited...only sperm / eggs.)

Recombinant D N A involves...

Joining of D N A molecules • Produced by artificially joining D N A from different biological sources not found together in nature (i.e. different species)

Recent successful trials by conventional gene therapy approaches

LCA: Leber congenital amaurosis• Degenerative disease of retina caused by alterations to photoreceptor cells• RPE 65 gene has been a gene therapy target• Protein product of RPE 65 metabolizes retinol• Retinol allows rod and cone cells to detect light and transmit electrical signals• After injections of normal RPE 65 gene, patients were able to detect light and read eye chart

Crystal structure analysis of repressor complexes has confirmed the operon model

Lac operon and regulatory regions... Detailed structure of lac operon and its regulatory regions reveals three sites for repressor binding

Regulatory Mutations

Lactose stimulates transcription of lac operon and induces synthesis of enzymes Gratuitous inducers: Chemical analogs of lactose that also induce expression, such as this chemical used in the laboratory: • IPTG—isopropylthiogalactoside • Constitutive mutations: Genes with these mutations produce enzymes regardless of lactose presence/absence

After a period with no change (stasis), species 1 is transformed into species 2, a process called anagenesis.

Later, species 2 splits into two new species (species 3 and 4), a process called cladogenesis.

Attenuation Mechanism

Leader region Antiterminator hairpin structure Terminator hairpin

RNA interference pathways

Left: Double-stranded RNA is processed into siRNAs by Dicer. siRNAs then associate with RISC containing an Argonaute (AGO) family protein. RISC unwinds the siRNAs into single-stranded siRNAs and cleaves mRNAs complmentary to the siRNA. (Thus, protecting the genome against "likely viral" genes that had integrated before!) Right: miRNA genes are processed, then exported to the cytoplasm and processed by Dicer. These miRNAs then associate with RISC and mRNAs. If the miRNA and mRNA are perfectly complementary, the mRNA is destroyed; if there is a partial match, translation is inhibited

Specific Gene Recovery

Library screening • Used to sort through library and isolate specific genes of interest Probes • Used to screen library and recover clones of specific gene • A probe is any D N A or R N A sequence complementary to target gene of sequence being identified (hence, it will "stick to" / hybridize with the sequence you are looking for... and probes are designed to be detected by the researcher).

Technical and ethical issues surrounding DNA profiling

Limitations of DNA profiling:• Most criminal cases have no DNA evidence for analysis or have DNA evidence not informative to the case• Potentially DNA evidence remains unprocessed and backloggedTechnical issues• Human error results in wrongful convictions• Contamination of DNA samples becomes an issue• Degraded DNA yielding partial profiles is difficult to interpret Ethical issues• Deliberate tampering of DNA samples:• Introduction of biological material to crime scene• Artificially manufactured DNA fragments• Ethical questions related to DNA profiling involve collection and storage of biological samples and DNA profiles

mRNA Localization and Localized Translational Control

Localization and translational regulation of actin mRNA. The RNA-binding protein ZBP1 associates with actin mRNA in the nucleus and escorts it to the cytoplasm. ZBP1 blocks translation and binds cytoskeleton motor proteins (MP), which transport ZBP1 and actin mRNA to the cell periphery. At the cell periphery, ZBP1 is phosphorylated by Src and dissociates from actin mRNA, allowing it to be translated by a ribosome (40S/60S). Actin translation and polymerization at the leading edge direct cell movement.

lncRNA

Long noncoding RNA (lncRNA) share many properties with mRNAs - 5′ caps - 3′ poly-A tails - Splicing of introns • Unlike mRNA, lncRNAs lack frame that codes for the insertion of amino acids into a polypeptide • lncRNA binds to chromatin-modifying enzymes - Affects chromatin modification and gene expression

Loss of Heterozygosity

Loss of heterozygosity: Phenomenon where second wild-type allele is mutated in tumor This is the essential first step in expression of inherited cancers (again- that means IF THIS does NOT happen... cancer will not actually get expressed) Further mutations in proto-oncogenes and tumor-suppresser genes are necessary for full malignancy Those further mutations could be accomplished by exposure to harmful chemicals, etc.

Mad Cow Disease

Mad cow disease—Bovine spongiform encephalitis• Cattle were produced lacking prion protein P r P gene• Early results indicate animals are resistant to the disease• My thoughts...(and food for thought):• The WAY mad cow disease WAS being "caught" was via the cows' eating of OTHER COWS' neural tissues.• But how did THAT happen...?• At that time, it was allowed to feed [*HERBIVORE*] cows the low-quality animal byproducts (including neural tissue) of other cows.• MY QUESTION: Will the "new and improved, mad cow disease-resistant" cows... give producers a "free pass" to continue the above-described, overall still-unhealthy practice???

Promoter Structure

Made up of DNA sequence elements including: • Initiator (Inr) • TATA box • TFIIB recognition element (BRE) • Downstream promoter element (DPE) • Motif ten element (MTE)

Genetic Proof of Operon Model

Major predictions of operon model • I gene produces diffusible product (trans-acting) • O region is involved in regulation • O region must be adjacent to structural genes to regulate transcription Creation of partially diploid bacteria allows for determination of these predictions

Inherited Cancers (some are!)

Many inherited cancers happen to be caused by defects in DNA repair genes Xeroderma pigmentosum (X P) Defective nucleotide excision repair leading to skin cancer Hereditary nonpolyposis colorectal cancer (H N P C C) Autosomal dominant syndrome (200/1000 affected) Increased risk of colon, ovary, uterine, and kidney cancers Eight genes mutated / implicated; four involved in D N A mismatch repair

Ubiquitin-Mediated Protein Degradation

Many people comment: It makes sense that the proteasome is shaped like a "waste basket"! Ubiquitin ligase enzymes recognize substrate proteins and catalyze the addition of ubiquitin (Ub) residues to create a long chain. Ubiquitinated proteins are then recognized by the proteasome, which removes ubiquitin tags, unfolds the protein, and proteolytically cleaves it into small polypeptides

Cancer Stem Cell Hypothesis

Many scientists believe most cells within tumors actually do not proliferate... Cancer stem cell hypothesis: Tumor cells that do proliferate give rise to cancer stem cells that have capacity for self-renewal Stem cells: Undifferentiated cells with capacity for self-renewal This is in contrast to other hypotheses that predict that every cell in tumor has potential to form new tumor

Clone-by-clone approach

Map-based cloning• D N A fragments from restriction digests are aligned, creating a restriction map of the chromosome• Restriction fragments are ligated into vectors• Contigs are sequenced and aligned to assemble the entire chromosome

Q T L study:

Mapping and characterizing quantitative traits in the tomato, including fruit shape and weight • Identified more than 28 Q T Ls • A specific gene (O R F X) is responsible for about 30% of variation in fruit weight

Two different gene sets control embryonic development in Drosophila:

Maternal-effect genes• Products: m R N A and/or proteins • Zygotic genes• Transcribed in embryonic nuclei• Products differentially transcribe

Inbreeding

Mating among closely related individuals For a given allele......Inbreeding increases proportion of homozygotes in population Completely inbred population theoretically will consist only of homozygotes

Polygenic traits that do not show continuous variation:

Meristic traits = • Phenotype can be recorded by counting whole numbers• Examples• Number of seeds in pod• Number of eggs laid by chicken

Metagenomics applies genomics techniques to environmental samples

Metagenomics (environmental genomics)• Use of whole-genome shotgun approaches• Sequence genomes from entire communities of microbes in environmental samples of water, air, and soil• Oceans, glaciers, deserts, etc. are being sampled for metagenomics• Sequence genomes of all microbes in given environment• Involves isolating D N A from environmental sample without cultures of microbes and viruses

Methyl groups (highlighted in red)

Methyl groups (highlighted in red) occupy the major groove of DNA and prevent binding of transcription factors, silencing genes

Methylation

Methylation involves the addition of a methyl group catalyzed by methyltransferase enzymes Addition of a methyl group (-CH3) to cytosine on the 5-carbon of the cytosine nitrogenous base resulting in 5-methylcytosine (5mC) Reaction catalyzed by a family of enzymes called D N A methyltransferases (DNMTs)

Methylation occurs on cytosine bases

Methylation occurs on cytosine bases adjacent to guanine called CpG dinucleotides, which are clustered in regions called CpG islands CpG islands are located in and near promoter sequences adjacent to genes • CpG Islands adjacent to essential genes (housekeeping genes) and cell-specific genes are unmethylated and available for transcription • Other genes are methylated and transcriptionally silenced

Allelic variation in the CCR5 gene

Michel Samson and colleagues used polymerase chain reaction (PCR) to amplify a part of the CCR5 gene containing the site of the 32-bp deletion, cut the resulting DNA fragments with a restriction enzyme, and ran the fragments on an electrophoresis gel. Each lane reveals the genotype of a single individual. The 1 allele produces a 332-bp fragment and a 403-bp fragment; the 32 allele produces a 332-bp fragment and a 371-bp fragment. Heterozygotes produce three bands.

Genetic variation is present in most populations and species

Microevolution• Evolutionary change within populations of a species Macroevolution• Evolutionary change leading to emergence of new species and other taxonomic groups Population = • A group of individuals belonging to same species• Live in same geographic area• They do, or can potentially, interbreed

Migration and gene flow can alter allele frequencies

Migration: Occurs when individuals move between populations Species divides into populations that are separated geographically • Allele frequencies in new populations may differ over timeMigration and gene flow can alter allele frequencies Migration as a force in evolution. The IBallele of the ABO locus is present in a gradient from east to west. This allele shows the highest frequency in central Asia and the lowest in northeastern Spain. The gradient parallels the waves of Mongol migration into Europe following the fall of the Roman Empire and is a genetic relic of human histo

Mitochondrial D N A Profiling

Mitochondrial DNA (mtDNA) profiling:• Important addition to DNA profiling methods• Are passed from human egg to zygote during fertilization• Like Y chromosome, mtDNA undergoes little recombination Creating mtDNA profiles:• Amplify regions of mtDNA that show variability between unrelated individuals and populations Two commonly used regions• Hypervariable segment I (HVSI)• Hypervariable segment II (HVSII)

Transcription is Positively Regulated

Model for transcriptional regulation of GAL structural genes. Although all four structural genes are regulated by a similar mechanism, only GAL1 and GAL10 are shown here. The GAL10 and GAL1 gene UASG region has four binding sites for Gal4p homodimers. (a) In the absence of galactose, Gal80p homodimers bind Gal4p and mask the activation domain. (b) In the presence of galactose, Gal3p binds to Gal80p and dissociates dimers and/or prevents dimerization leading to exposure of the Gal4p activation domain and transcriptional activation

Crystal Structure Analysis

Models of the lac repressor and its binding to operator sites in DNA, as generated from crystal structure analysis. (a) The repressor monomer, showing the inducer-binding site. The DNA-binding region is shown in red. (b) The repressor tetramer bound to two 21-base-pair segments of operator DNA (shown in dark blue). (c) The repressor (shown in pink) and CAP (shown in dark blue) bound to the lac DNA. Binding to operator regions O1 and O3 creates a 93-base-pair repression loop of promoter DNA

Histone Modification

Modifications occurs at conserved amino acid sequences in the N-terminal histone tails, which protrude from the nucleosome Chemical modification of histones alters the structure of chromatin, making genes accessible or inaccessible for transcription

Golden Rice

Most G M crops are made to increase yield, but:• Golden Rice was engineered to combat vitamin A deficiency in developing countries• Golden Rice was genetically engineered to synthesize beta-carotene (precursor to vitamin A)

Variations in Nucleotide Sequence

Most direct way to estimate genetic variation:• Compare nucleotide sequences of genes carried by individuals in population• Example: Alcohol dehydrogenase Alcohol dehydrogenase (A d h):• Analyzed gene (A d h) in Drosophila melanogaster• Gene has two alleles, A d h-f and A d h-s• Encoded proteins differ by a single amino acid (threonine versus lysine at codon 192)• A d h genes from five natural populations were cloned and sequenced

Additive and Nonadditive Alleles

Multiple-gene hypothesis—Additive alleles and nonadditive alleles • Various grain color phenotypes due to additive and nonadditive alleles• Additive allele: Contributes equally to red grain color• Nonadditive allele: Fails to produce red pigment Greater number of additive alleles in genotype = more intense red color expressed in phenotype

The H T T Gene and Its Protein Product

Mutant HTT alleles (mHTT ) have expanded cytosine-adenine-guanine (CAG) repeats on exon 1 ...Gain of function mutation• HD is one of several behavioral disorders associated with progressive accumulation of misfolded proteins and protein aggregates• Disrupts cellular functions and leads to neurodegeneration and cell death

Organ Development

Mutations in homeotic genes in plants cause organs to develop in abnormal locations• Example: In Arabadopsis:• Wildtype: sepal, petal, stamen, and carpel• Apetala2 mutants: carpel, stamen, stamen, and carpel• Pistillata mutants have sepals, sepals, carpels, and carpels• Agamous mutants have petals and sepals at places where stamens and carpels should form

GAL Genes are Inducible 2

Mutations in the regulatory gene GAL4 prevent activation GAL4 encodes the Gal4 activator protein (Gal4p) required for transcription of the GAL structural genes • Gal4p consists of 881 amino acids and functions as a homodimer • A DNA-binding domain (DBD) that recognizes and binds specific DNA sequences • An activation domain (AD) that activates transcription

Natural selection is a major force driving allele frequency change

Natural selection:• Major force driving allele frequency change• Chief mechanism for transforming populations• Principal force that shifts allele frequencies within large populations• Discovered independently by Charles Darwin and Alfred Russel Wallace Wallace-Darwin concept of natural selection:• Individuals exhibit variations in phenotype• Variations are heritable (passed on)• Organisms tend to reproduce in exponential fashion• More offspring are produced than can survive• Some phenotypes are more successful at survival and they reproduce at higher rates

Reverse Transcriptase

Naturally involved in extending telomeres (the telomerase enzyme), especially for egg / sperm cells to make the new generation(!) • Some reverse transcriptases are harmful (i.e. those in HIV) • In the laboratory, they are used to generate c D N A from m R N A • Extends oligo(d T) primer and synthesizes complementary D N A copy of m R N A • Produces m R N A-D N A double-stranded hybrid molecule

Neanderthals and Denisovans

Neanderthals (Homo neanderthalensis)• Lived in Europe and western Asia about 300,000 years ago• Disappeared about 30,000 years ago• Coexisted with anatomically modern humans (Homo sapiens) for about 30,000 years in several regions Denisovans• Genome sequence shows that they are more closely related to Neanderthals than to Homo sapiens Denisovans and Neanderthals are separated from a [likely] common ancestral species more than 430,000 years ago Denisovan genome contains sequences from another, as yet unknown, archaic group that made no contribution to the Neanderthal genome

Small Noncoding RNAs (sRNAs)

Negative Regulation: sRNA pairing inhibits ribosome binding Positive Regulation: Translation repressed -sRNA pairing unmasks the RBS - translation proceeds

Regulation of inducible or repressible type system under positive control or negative control:

Negative control: Genetic expression occurs unless shut off by regulator molecule Positive controL: Transcription occurs only when regulator molecule directly stimulates RNA production Either type of system, or both combined, can induce or repress systems

Positive and Negative Selection

Negative selection• Involves a marker gene: An antibiotic-resistant marker• Example: Hygromycin-resistance gene• The product from this gene kills bacteria in addition to some other groups of organisms• The marker gene and a promoter are introduced into plant cells along with the gene of interest• Cells are grown in medium containing hygromycin• Only cells that express the resistant gene survive

Gene editing approaches to gene therapy

New approaches are being developed in gene therapy• DNA-editing nucleases for gene targeting (see first special topic for CRISPR-Cas discussion)• RNA silencing for gene inhibition• Antisense oligonucleotides to inhibit translation of mRNAs from defective genes• Blocking/silencing gene expression• Not a reliable method for gene therapy• RNA interference (RNAi) is a form of gene expression regulation• Main challenge of RNAi-based therapeutics:• In vivo delivery of dsRNA or siRNA• RNA s degrade quickly in body• Two approaches to RNA delivery• Inject siRNA directly• Deliver via DNA plasmid vector

Nutrigenomics

New field that focuses on understanding interactions between diet and genes • Analyze genome for genes associated with medical conditions or nutrient metabolism (people with certain genes may benefit from specific diets more than other people, with different genes)

The future of GM foods

New genome information may allow us to more accurately edit a plant's endogenous genes, decreasing, increasing, or eliminating expression to obtain desired phenotypes• New techniques can devise creative ways to protect plants from insects and disease• Transgenic salmon is likely to receive marketing approval soon• The AquAdvantage salmon grows twice as fast as a non-GM Atlantic salmon, reaching market size in half the time (and yes, there is farmed salmon whose species name is "Atlantic salmon" - that name does NOT mean it is wild. Any wild fish will say "wild" on the package only

Dividing the Embryo...

New patterns of gene expression can be generated by overlapping regions containing two different gene products. (a) Transcription factors 1 and 2 are present in an overlapping region of expression. If both transcription factors must bind to the promoter of a target gene to trigger expression, the gene will be active only in cells containing both factors (most likely in the zone of overlap). (b) The expression of the target gene in the restricted region of the embryo.

Controversy Regarding Golden Rice

Non-GM and Golden Rice 2. Golden Rice 2 contains high levels of beta-carotene, giving the rice endosperm a yellow color. The intensity of the color reflects the amount of beta-carotene in the endosperm. Fun fact: On the more "organic" side of agriculture, my own calculations show that certain brands of organic carrots are the most frugal source of vitamin A (in terms of % RDA vitamin A per dollar). For more, see the PPTs in the Supplemental Module)

Nonrandom mating changes genotype frequency but not allele frequency

Nonrandom mating • Can change frequencies of genotypes in population (but remember, genotypes are simply certain combinations of alleles, not new alleles) • Subsequent selection for or against certain genotypes can then possibly affect overall allele frequencies • But nonrandom mating itself does not directly change allele frequencies

Northern Blots

Northern blot analysis of dFmr1 gene expression in Drosophila during embryogenesis. A prominent dfmr1 RNA transcript of approximately 2.8 kb is present in ovaries and 0- to 3-hour-old embryos (blue asterisks). At 3 to 6 hours and beyond, the major transcript detected is about 4.0 kb and peaks in abundance between 9 and 12 hours of embryonic development (red asterisks). Note, transcripts of different lengths (4.0, 2.8 kb) are produced from the same gene, and that the levels of expression for each transcript vary during different stages of development. These data suggest that dFmr1 gene expression may be regulated at the levels of transcription or transcript processing during embryogenesis. The dFmr1 gene is a homolog of the human FMR1 gene. Loss-of-function mutations in FMR1 result in human Fragile-X mental retardation

Nucleosomal Modification

Nucleosomes get modified via change in composition: • Important step in gene regulation; involves changes to either nucleosome or DNA • Histones contain normal histone H2A • Variant histones (H2A.Z) affect nucleosome mobility and positioning on DNA • Nucleosome position may repress or activate transcription via gene promoter

Promoters (core and proximal) =

Nucleotide sequences that serve as recognition sites for transcription machinery • Located immediately adjacent to regulatory genes • Critical for transcription initiation • Core promoter: Determines accurate initiation of transcription • Proximal-promoter elements: Modulate efficiency of basal levels of transcription

Mutation rates =

Number of new mutant alleles per given number of gametes• If mutation rate is known, the extent of change to allele frequency from one generation to next can be estimated

X Chromosome Inactivation

Occurs early in development at random All cancer cells within a tumor, both primary and metastatic, within one female individual contain the same inactivated X chromosome (demonstrates the cancer cells are "clonal")

X chromosome inactivation:

Occurs early in development at random All cancer cells within a tumor, both primary and metastatic, within one female individual contain the same inactivated X chromosome (demonstrates the cancer cells are "clonal")

Optimizing Drug Therapies

On average, a drug will be effective in about 50 percent of patients Trial and error is used until the correct drug is found for a patient This is a waste of time and can be dangerous

Metastasis

Once cancer cells have disengaged, they enter blood or lymphatic system 0.01% of metastatic cells become metastatic tumors Metastasis is controlled by a large number of gene products: Cell-adhesion molecules, cytoskeleton regulators, and proteolytic enzymes

Chimeric antigen receptor (CAR) T-cell therapy

Once the D N A has been cloned it is introduced into normal T cells that have been purified from the patient's blood Screening and amplifying the C A R T cells occurs Then the C A R T cells are reinfused into patients who have been treated with chemotherapy or radiation therapies to reduce the numbers of endogenous immune system cells (I could definitely foresee potentially harmful side effects of this...) C A R T-cell therapy promising results: Clinical trials for B cell tumors known as leukemia and lymphoma have resulted in some long-term remissions 2 F D A approved drugs for large B cell lymphoma therapy

Oncogenes

Oncogene: Is a proto-oncogene mutated or aberrantly expressed Has a gain-of-function alteration Contributes to development of cancer Only one allele needs to be mutated or misexpressed to contribute to cancer Confers a dominant cancer phenotype

SWI/SNF

One of the best-studied remodeling complexes • Loosens attachment between histone and DNA • Thus, loosens DNA strand from nucleosome core • Causes reorganization of internal nucleosome component

Methylation

One of the three major epigenetic mechanisms is the reversible modification of DNA by the addition or removal of methyl groups In mammals, methylation of the DNA takes place after replication and during differentiation of adult cells...... this is what prevents a skin cell from expressing "bone cell proteins" even though those genes are still in the cell

An overview of whole-genome sequencing and assembly:

One strategy involves using restriction enzymes to digest genomic DNA into contigs, which are then sequenced and aligned using bioinformatics to identify overlapping fragments based on sequence identity. EcoRI digestion produces two fragments (contigs 1 and 2-4), whereas digestion with BamHI produces three fragments (contigs 1-2, 3, and 4).

Monoallelic expression (MAE):

Only one allele is transcribed, while the other allele is transcriptionally silent

Lac operon: Negative control

Operon subject to negative control: Transcription occurs only when repressor fails to bind operator region • Repressor normally binds D N A sequence in operator region • Inhibits RNA polymerase • Represses transcription of structural genes • Why? Probably because this way, bacteria don't waste energy making gene products for a "food molecule" that is not around anyway!

Alternative splicing of the Drosophila Dscam gene mRNA

Organization of the Dscam gene and the pre-mRNA. The Dscam gene encodes a protein that guides axon growth during development. Each mRNA will contain one of the 12 possible exons for exon 4 (red), one of the 48 possible exons for exon 6 (blue), one of the 33 possible exons for exon 9 (green), and one of the 2 possible exons for exon 17 (yellow)

The genes toy, ey (and perhaps eyg) are master control genes and are at the top of the hierarchy of gene action in eye development

Other genes, so, eya/cli and dac, all of which encode transcription factors, are second-level genes that are regulated by the master control genes. This complex program is a network with genes interconnected via feedback loops, and not a linear system

Metalloproteinase gene M M P1:

Overexpressed in breast cancer cells that metastasize to bone M M P1 and M M P2 genes that are overexpressed in cancer cells spread to the lungs Level of aggressiveness of correlates positively with levels of proteolytic enzyme activity expressed by tumor

Pair-Rule Genes

Pair-rule genes• Are expressed as series of seven narrow bands or stripes that extend around circumference of embryo• Expression of pair-rule genes• Establishes boundaries of segments• Establishes developmental fate of cells within each segment by controlling expression of segment polarity genes

Technical, social and ethical challenges

Personalized genome analysis needs to be used with caution until the technology becomes highly accurate and reliable A greater challenge lies in the ability to store the vast amount of emerging sequence data; each personal genome generates the equivalent of 200 large phone books Personalized medicine will also need to integrate information about environmental, personal lifestyle, and epigenetic factors Personalized medicine will also require additional education and training by physicians

Developing Targeted Drugs

Personalized medicine was successfully used in the HER-2 gene and the use of the drug Herceptin in breast cancer The human epidermal growth factor receptor 2 (HER-2) gene is located on chromosome 17 and codes for a transmembrane tyrosine kinase receptor protein called HER-2 In about 25 percent of invasive breast cancers, the H E R-2 gene is amplified and the protein is overexpressed on the cell surface In some breast cancers, the H E R-2 gene may have as many as 100 copies per cell This amplification is associated with increased tumor invasiveness, metastasis, and cell proliferation as well as poorer patient prognosis

Developing Targeted Drugs

Pharmacogenomics increases the efficacy of drugs by matching those drugs to subpopulations of patients who will benefit Personalized pharmacogenomics is widely practiced in the diagnosis and treatment of cancers

Phenotypic Variance (Vp)

Phenotypic variance (Vp) components: • Genotypic variance• Environmental variance• Genotype-by-environment interaction variance• Heritability estimates are obtained using experimental and statistical techniques• Partitions Vp into genotypic variance (VG) and environmental variance (VE)

Monozygotic twins

Phenotypic variance equals environmental variance (no genotypic variance or very little)

Posttranslational modifications regulate protein activity

Phosphorylation is the most common type of posttranslational modification • Kinases catalyze the addition of a phosphate group to serine, tyrosine, or threonine amino acid side chains. • Phosphatases are enzymes that remove phosphates Phosphorylation usually induces conformational changes and therefore functional changes in proteins

Phylogenetic Trees from D N A Sequences

Phylogenetic reconstruction of specifically mitochondrial D N A can be done Differences in D N A sequence between species are approximately proportional to evolutionary distance One popular question: What is the common ancestor to vertebrates?• [Ancestors that likely resembled] the lungfish versus coelacanth• Fossil record, living species, and mitochondrial D N A cannot rule out [an ancestor more similar to] either one

Phylogeny can be used to analyze evolutionary history

Phylogeny (evolutionary history)• Genetic differences among present-day species can be used to reconstruct their [likely] evolutionary histories (phylogenies)• I add the word "likely" because these reconstructed histories are not perfect / "set in stone" and could always change based on newer evidence! Phylogenetic trees:• Branches represent lineages over time• Monophyletic groups consist of an ancestral species and all its descendants

Plants have evolved developmental regulatory systems that parallel those of animals

Plants and animals appear to have diverged from common unicellular ancestor 1.6 billion years ago• Development patterns evolved independently in plants and animals• Plant pattern formation is studied using model organism Arabidopsis thaliana

Plasmid

Plasmid = Extrachromosomal double-stranded D N A molecule • Replicates independently from chromosomes within bacterial cells (hence, is one type of "vector")

Method for creating Roundup-Ready soybeans

Plasmids were loaded into the gene gun and fired at high pressure into cells growing in tissue cultures. Cells were grown in the presence of glyphosate to select those that had integrated and expressed the epsps gene. Surviving cells were stimulated to form calluses and to grow into plantlets.

Polygenes

Polygenes:- Genes contributing to quantitative trait- Number of polygenes (n) contributing to quantitative trait is estimated based on ratio of F2 individuals resembling either of two extreme P1 phenotypes (see next slide's table)- 1/4n = ratio of F2 individuals expressing either extreme phenotype

The study of polygenic traits relies on statistical analysis

Polygenic traits:• Measured in large sample and representative individuals of population• Data form a normal distribution: A characteristic bell-shaped curve when plotted as a frequency histogram

The polymerase chain reaction is a powerful technique for copying DNA

Polymerase chain reaction (P C R): • Rapid method of D N A cloning that eliminates the need to use host cells for cloning (but the trade-off is that more errors / mutations are introduced) • Copies specific D N A sequence via in vitro reactions; can amplify target D N A sequences present in very small quantities (that is the benefit of this technique- sometimes, tiny quantities is all we have to start with!)

There are different forms of nonrandom mating

Positive assortive mating:• Similar genotypes more likely to mate than dissimilar ones (not necessarily directly related though) Negative assortive mating:• Dissimilar genotypes are more likely to mate than similar one Inbreeding:• Mating individuals are related genetically

Selectable Markers: Positive Selection

Positive selection• One example involves the marker gene encoding P M I: Phosphomannose isomerase enzyme• Catalyzes interconversion of mannose 6-phosphate and fructose 6-phosphate• This enzyme is common in animals but not plants• Plant cells that express p m i gene survive• Can be positively selected by growing cells on mannose-containing medium

Introduction: Posttranscriptional Regulation

Posttranscriptional regulation plays an equal, if not more significant, role compared to transcriptional control • Perhaps the major type of regulation in eukaryotes

Abnormal regulation of the epigenome leads to human genetic disorders such as

Prader-Willi syndrome (in SOME cases): • NOT a change in gene sequence, but "loss of function of" particular genes: • • Angelman syndrome • Beckwith-Weidemann syndrome The loss or alteration of other epigenetic states can result in cancer

Gene therapy setbacks

Problems arise when viral vectors transfer therapeutic genes into cells:1. Integration of retroviral genome occurs only if host cells are replicating their D N A2. Injection of viral vectors can cause an adverse immune response• Patients died3. Insertion of viral genome into host chromosome activates or mutates genes Patients developed leukemia and some died4. Possibility that a fully functional infectious virus could be created by recombination of an inactive gene therapy virus with a functional virus in the patient

Gap Gene Mutations

Produce large gaps in embryo's segmentation pattern• Proper transcription of gap genes normally divides embryo into head, thorax, and abdomen• BUT for (for example) the Hunchback mutants lose the head and thorax structures

Protein Product

Product of hMTIIA = • Protein that binds to heavy metals and protects cells from toxic effects • Protects cells from oxidative stress • Expressed in low levels in all cells • Transcribed at high levels when exposed to heavy metals Think about this: What do you think would happen if there were a lot of random mutations from carcinogens... in the DNA for this very important gene...?

Epigenetics has been implicated in:

Progressive restriction of gene expression during development • Allele-specific expression in gene imprinting • Environment-genome interactions during prenatal development that affect adult phenotypes

Prokaryote vs. eukaryotic genome

Prokaryotic genomes sequenced are from organisms causing human diseases: • Cholera, tuberculosis, leprosy, and lyme diseases • Genome size and gene number in various prokaryotes are highly variable Two generalizations about prokaryotes • Gene density is high • Bacterial genomes contain operons Genome size in eukaryotes is highly variable• Range: 10 M b in fungi to 100,000 M b in plants Eukaryotic genomes have features not found in prokaryotes: • Low gene density, introns, repetitive sequence

Enhancer and promoter elements:

Promoter elements: 30,000 promoter regions found within protein-coding genes • ENCODE data show enhancer elements influence transcription of several different promoters • Enhancer elements: Contain promoter elements that initiate transcription

Proximal-Promoter Elements

Promoters contain proximal-promoter elements: • Located upstream of TATA and BRE motifs • Enhance levels of basal transcription • Examples: CAAT and GC boxes

Western blot

Protein

Proteomics identifies and analyzes the protein composition of cells

Proteomics• Identification, characterization, and quantitative analysis of all proteins (proteome) encoded by genome of cell, tissue, or organism (remember, not all genes actually code for protein)• Used to reconcile differences between number of genes in genome and number of different proteins produced• Allows comparison of proteins in normal and diseased tissue

Proto-oncogenes and tumor-suppressor genes are altered in cancer cells

Proto-oncogenes Genes whose products promote cell growth and division Proto-oncogenes encode: Transcription factors that stimulate expression of other genes Signal transduction molecules that stimulate cell division Cell-cycle regulators that move cell through cell cycle So... abnormal upregulation of these can be harmful.

Expression Q T Ls (e Q T Ls)

Q T Ls that affect fruit size in tomatoes work by controlling at least two developmental processes Cell division early in development (O R F X) and the determination of the number of seed compartments (l c and f a s)

Quantitative Loci

Quantitative loci:• Numerous loci can function in similar fashion• Control various quantitative phenotypes• Greater and greater numbers of classes appear in F2 generation in more complex ratios

Quantitative traits can be explained in Mendelian terms

Quantitative patterns• Mendelian factors were not thought to account for the range of phenotypes seen in quantitative patterns of inheritance Multiple-gene hypothesis• Many genes, individually behaving in Mendelian fashion, contribute to phenotype in a cumulative/quantitative way• Example: Grain color in whea

Quantitative trait loci are useful in studying multifactorial phenotypes

Quantitative trait loci (Q T Ls):• Multiple genes contributing to quantitative trait• Involves finding associations between D N A markers and phenotypes • Q T L mapping:• Artificial selection over many generations—highly divergent lines created

Polygenic Traits

Quantitative traits display continuous variation• Measured and described in quantitative terms (quantitative inheritance) Polygenic = Varying phenotypes result from input of many genes (so, not all of these are continuously varying) Multifactorial or complex traits• Result of both gene action and environmental influences

Noncoding RNAs play diverse roles in posttranscriptional regulation

RNA interference (RNAi): • Sequence specific posttranscriptional regulation • Short RNA molecules regulate gene expression in cytoplasm of plants, animals, and fungi; repress translation and trigger mRNA degradation Phenomena known as RNA-induced gene silencing

RNA plays diverse roles in regulating gene expression in bacteria

RNA's secondary structure provides regulation: Attenuation and riboswitches: • Attenuation: trp structural genes are preceded by leader sequence containing regulatory site called an attenuator mRNA of the leader sequence has the potential to fold into two different stem-loops (hairpins) Antiterminator hairpin: Forms in absence of tryptophan (forms because the ribosome "stalls" waiting for tryptophan to insert into a growing peptide but tryptophan is not there) Terminator hairpin: Forms in presence of tryptophan...ribosome has tryptophan to do translation with, at the LEADER sequence, BUT that causes a DIFFERENT hairpin to form elsewhere which prevents the structural genes from getting translate

RNAi in biotechnology

RNAi studied in lab • Developed as pharmaceutical agent • Therapeutic RNAi attacks diseases caused by overexpression of specific gene or normal expression of abnormal gene product • Potential use of RNAi in diagnosis of cancers • RNAi reduces severity of infections by viruses such as HIV, influenza, and polio

Epigenetics and Random Inactivation of Autosomal Genes

Random inactivation of genes are scattered throughout the genome ●(1) Expression of both alleles (biallelic expression) ●(2) Expression of only the maternal allele ●(3) Expression of only the paternal allele ●(4) Expression of neither allele

Genomic Techniques

Range of techniques valuable for functional genomic studies • Example:• Chromatin immunoprecipitation (C h I P) • Designed to map protein-D N A interactions • Useful for identifying genes regulated by D N A-binding transcription factors

Rate of Macroevolution and Speciation

Rate of macroevolution and speciation • Average time for speciation 100,000-10 million years • But... rapid speciation events have occurred• Fishes in East African lakes• Marine salmon• Palm trees on isolated islands• Polyploid plants (can happen in only 1 generation!)• Brown algae in Baltic Sea

Realized Heritability

Realized heritability• Selective breeding estimates potential success for artificial selection• Example:• Artificial selection for increased oil content in corn

Genetic engineering, genomics, and biotechnology raise ethical, social, and legal questions

Recombinant D N A and genomic technologies• Identify genes• Diagnose and treat genetic disorders• Produce commercial and pharmaceutical products• Solve crimes Applications of knowledge gained raise ethical, social, and legal issues

Clones

Recovered copies of recombinant D N A molecules (in this case- clones generally mean several identical copies of something in general, too) • Used to study structure and orientation of D N A Recombinant D N A technology is used to isolate, replicate, and analyze genes

Sex Determination in Drosophila

Regulation of pre-mRNA splicing that determines male and female sexual development in Drosophila. The ratio of X chromosomes to autosomes (AA) leads to transcription of the Sxl gene in females. The presence of SXL protein begins a cascade of pre-mRNA splicing events that culminate in female-specific gene expression and production of the DSX-F transcription factor. In the absence of SXL protein, a male-specific pattern of pre-mRNA splicing results in male-specific patterns of gene expression induced by the DSX-M protein.

Cis- and trans-acting sites:

Regulatory site events determine if genes are transcribed into mRNA • Binding of trans-acting element at cis-acting site regulates genes cluster negatively or positively • Negatively by turning off transcription • Positively by turning on transcription

H I V-1 Infection Resistance

Resistance to HIV-1 • Homozygous individuals are resistant to HIV-1 infection • Heterozygotes susceptible to infection but progress more slowly to AIDS Genotypes determined by direct DNA analysis using P C R and restriction-enzyme digest analysis

Recombinant DNA technology began with two key terms: Restriction enzymes and DNA cloning vectors

Restriction enzymes = Produced naturally by bacteria as a defense mechanism against bacteriophage (viruses that infect them)... ...OR they may respond to any "strange pattern of sequences" including (speculatively), GMO sequences (for more on that hypothesis, see pages 5-9 of the "Theory of..." paper in the Supplemental Module!• They are DNA-cutting enzymes• Bind to D N A at specific recognition sequence (restriction site) and cleaves D N A to produce restriction fragments• Enzyme cleaves both strands of D N A (digestion)

Molecular techniques for analyzing DNA and RNA

Restriction map: • Establishes number of, order of, and distances between restriction enzyme cleavage sites on cloned segment of D N A • Provides information on length of cloned insert and location of restriction sites within clone • Created by cutting D N A with different restriction enzymes and separating D N A fragments by gel electrophoresis, which separates fragments by size • Smallest fragments move farthest down on gel • Fragments visualized on gel by staining with ethidium bromide and illuminated by U V light

Cancer: Genetic Disease at Level of Somatic Cells

Results from mutated gene products or abnormally expressed genes Mutations affect multiple cellular functions Cancer cells share two fundamental properties: Abnormal cell growth and division: unregulated cell proliferation Metastatic spread (if "malignant")

Retroviruses and Human Cancers

Retroviruses associated with human cancers: Human immunodeficiency virus (H I V) Human T-cell leukemia virus (H T L V-1)

Retroviruses can Cause Cancer

Retroviruses can cause cancer in three ways: Proviral D N A may integrate near proto-oncogenes, which stimulates high levels of transcription Acute transforming retroviruses: Normal retrovirus with normal viral gene products can stimulate inappropriate cell growth

Viruses contribute to cancer in both humans and animals

Retroviruses: Are certain types of RNA viruses They are animal viruses that cause cancer Reverse transcriptase enzyme copies R N A to D N A D N A copy enters nucleus of infected cell Retroviruses integrate into host genome as provirus, which is replicated with host's D N A during normal cell cycle

Reverse transcription P C R (R T-P C R)

Reverse transcription P C R (R T-P C R) • Methodology for studying gene expression (m R N A production by cells or tissues) • Reverse transcriptase is used to generate d s-c D N A Quantitative real-time P C R (q P C R)• Real-time P C R allows researchers to quantify amplification reactions as they occur in real time

Alternate forms of mRNA structure, which can create either a terminator OR an "anti-terminator" structure, are called ________. These contain both "aptamers" and expression "platorms" which have different purposes.

Riboswitches

Roundup-Ready Soybeans

Roundup-ready soybeans:• G M plant with resistance to the herbicide glyphosate, the main ingredient in Roundup• Utilizing Agrobacterium, an epsps gene was cloned into soybean plant to produce a glyphosate-resistant soybean plant• Biolistic technique introduced the plasmid• Farmers can spray Roundup on crops and kill only weeds, not kill their crop of soybeans. But unfortunately regarding the now-well-known toxicity of glyphosate to human mitochondria and DNA etc...glyphosate can thus be sprayed more heavily on food crops (and it also gets inside the plant) without killing the crops, but then of course, those crops get eaten by humans).

Saccharomyces Cerevisiae

Saccharomyces cerevisiae (yeast) is widely used as host for D N A cloning and expression of eukaryotic genes (because... remember, yeasts are fungi which are eukaryotes, NOT bacteria) • It can be grown easily and manipulated • Its genetics have been studied intensively • Its entire genome has been sequenced • It can posttranslationally modify eukaryotic proteins • This yeast itself is considered to be safe (it is "baker's yeast", a different species than many opportunistic infectious fungi)

Selective breeding is one of the oldest methods

Selective breeding is one of the oldest methods of genetic alteration of plants. Shown here is the likely ancestor of corn- teosinte (Zea canina, top), a selectively bred hybrid (center), and modern corn (Zea mays, bottom)

Despite comparisons often made between the techniques, genetic engineering is quite different from selective breedinG

Selective breeding• Selection and breeding of naturally occurring (or mutagen-induced) variants• Technique utilized by farmers for thousands of years (i.e. 10,000 years ago. Contrast that with genetic engineering for only the last ~20+ years!)• Manipulates genetic makeup (but ONLY via meiosis, crossing over and random fertilization) of plant and animal to enhance food production• Example: Selective breeding in corn; even though selective breeding has recently been criticized to be "too slow" by some... if patience CAN be spared, the results can still be quite remarkable(

O R F s: Open reading frames

Sequences of triplet nucleotides translated into amino acid sequence of a protein • Suggestive of protein-encoding gene• Typically begin with initiation sequence A T G • End with termination sequence T A A, T A G, or T G A

Human genome sequence explosion.

Sequencing costs have steadily declined since 2000 due to innovations in sequencing technology. As a result, notice that the number of individual genomes sequenced has dramatically increased

Structural Genomics

Sequencing genomes• Analyzing nucleotide sequences to identify genes and sequences such as gene-regulatory elements Characteristics of a protein-coding gene that can be used to identify a gene in an unknown sequence of genomic DNA. Most eukaryotic genes are organized into coding segments (exons) and noncoding segments (introns). To determine whether a sequence contains a gene, it is necessary to distinguish between introns and exons, gene-regulatory sequences, such as promoters and enhancers, untranslated regions (UTRs), and gene termination sequences.

Epigenome projects and databases

Several projects are underway to map the human epigenome Helps to explain how environmental settings in early life can affect predisposition to adulthood disease

The first successful gene therapy trial

Severe combined immunodeficiency disease (SCID) was the first disease treated with gene therapy• SCIDs individuals do not have a functional immune system so can die from minor infections• Autosomal SCID is caused by a mutation in adenosine deaminase (ADA) enzyme• Example: Gene therapy of Ashanti DeSilva• Isolated white blood cells (T cells) were mixed with a retrovirus carrying inserted copy of normal ADA gene• The virus infected T cells, and a copy of normal ADA was inserted into genome of normal T cells• A billion cells expressing the ADA gene were injected into the bloodstream• Gene therapy has successfully restored the health of about 20 children affected by SCID

Sex Determination in Drosophila

Sex determination in Drosophila—Alternative splicing Three major genes in pathway • Sex lethal (Sxl) • Transformer (tra) • Doublesex (dsx) Product of sxl gene selects pathway of sexual development by controlling splicing

Signal Transduction

Signal transduction Cells in G0 are stimulated to reenter cell cycle by external growth signals Signal transduction initiates gene expression that propels cell out of G0 and back into cell cycle Cancer cells often have defects in signal transduction pathways (they may always be turned "on" for example)

Enhancers and Silencer

Silencers regulate transcription of eukaryotic genes • Also are cis-acting transcription regulatory elements Silencers: Repress the level of transcription initiation

Narrow-Sense Heritability

Single heritability estimate• Only provides information about one population in specific environment Narrow-sense heritability• A more valuable predictor of response to selection• Estimates calculated for many populations and environments—shows trends

Genetic analysis from single cells

Single-cell sequencing:• We can now sequence the genome from a single cell• Involves isolating D N A from single cell and executing whole genome amplification• Produces sufficient D N A to be sequenced

Single-Nucleotide Polymorphism Profiling

Single-nucleotide polymorphisms (S N Ps)• Single-nucleotide differences between two D N A molecules• Base-pair differences or small insertions or deletions • Occur randomly throughout genome and on m t D N A, every 500 to 1000 nucleotides Forensic S N P profiling• Has one major advantage over S T R profiling• Involves only one nucleotide of a D N A molecule• Makes S N P analysis suitable for analyzing D N A samples that are severely degraded

miRNA

Small, noncoding RNA molecules also participate in epigenetic regulation of gene expression After transcription, these miRNA (microRNA) molecules associate with protein complexes to form RNA-induced silencing complexes (RISCs)

Genetic engineering of plants has greatly changed agriculture

Some groups focus on the benefits of specific GMO plants (GMO = genetically modified organism), such as: Additional nutrient in Golden Rice (vitamin A) Pesticide resistance (i.e. Bt toxin-producing corn) Lack of need to "apply pesticide" to such Bt toxin-producing cropsBut others focus on the drawbacks, such as: Potential triggering of inflammation by "inter-species gene patterns" if they get detected by our gut bacteria or even immune cells Potential toxicity of Bt toxin (especially to some individuals, like those who may not make enough stomach acid to denature the protein toxin The fact that even though pesticide does not "need to be applied", that is because it is being produced directly by the plant itself

Limitation of P C R

Some information about nucleotide sequence of target D N A is required to synthesize primer • Minor contamination from other sources can cause problems (e.g., skin cells from researcher) • P C R cannot amplify long segments of D N A

Long Noncoding R N As and Posttranscriptional Regulation

Some lncRNAs function as competing endogenous RNAs (ceRNAs) A ceRNA is important for the differentiation of muscle cells in mice and humans • Undifferentiated muscle cells, known as myoblasts, express an lnc R N A called long intergenic noncoding RNA muscle differentiation 1 (linc-MD1) • linc-MD1 was introduced into cultured myoblasts from DMD patients, muscle cell differentiation was partially restored

Steps in the development of colorectal cancers

Some of the genes that acquire driver mutations and cause the progressive development of colorectal cancer are shown above the photographs. These driver mutations accumulate over time and can take 40 years or more to result in the formation of a malignant tumor.

EnviroPig

Some sources suggest the funding for this actually has been discontinued...• The concept: Pig with salivary glands that expresses gene encoding enzyme phytase (naturally expressed by fungi)• Allows pig to break down dietary phosphorus, a major pollutant in pig farms, reducing its excretion• But here is one thing to think about, regarding the physics "First Law of Thermodynamics" (matter cannot get created nor destroyed)... if the pigs are not excreting the phosphorus... where IS it going?• The excessive levels are staying in their body (then get into OUR BODY when we EAT them!), likely with effects such as kidney dysfunction as described in this paper:

Speciation can occur through reproductive isolation

Species = Group of actually or potentially interbreeding organisms that is reproductively isolated in nature from all other such groups Speciation = making of different species...• Associated with changes in genetic structure of populations with genetic divergence • Genetic divergence of populations can reflect action of natural selection, genetic drift, or both

Lac Y

Specifies primary structure of permease, an enzyme that facilitates entry of lactose into bacterial cell

Types of Alternative Splicing Alternative polyadenylation

Spliceforms with different 3′ ends are produced When an exon containing a polyadenylation signal is skipped, downstream exons are included and a downstream polyadenylation signal will be used

Human Disease

Spliceopathies: Mutations that affect regulation of splicing and contribute to several genetic disorders • Examples:• Myotonic dystrophy • Spinomuscular atrophy (SMA) • Involves loss of motor neurons muscle atrophy breathing cessation • Caused by mutation in the SMN1 (survival of motor neurons 1) gene normally encodes SMN (RNA binding protein involved in assembly of the spliceosome) • The resulting splicing defects suggest SMN is needed for proper splicing" (as per Klug et. al., Concepts of Genetics, 12th editio

Regulation of Alternative Splicing

Splicing enhancers and splicing silencers: • These are cis-acting sequences that regulate alternative splicing SR proteins bind to splicing enhancers and activate splicing by recruiting spliceosome components • FYI, "SR" stands for the symbols for the amino acids "serine" and "arginine". Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a class of proteins that bind splicing silencers and inhibit splicing

Standard Deviation

Standard deviation (s)- Square root of the variance- > 95% of all values are found within two standard deviations to either side of mean- Standard deviation can be interpreted in the form of probability

Standard Error of Mean

Standard error of the mean• Describes errors between means determined for multiple samples• Value is always smaller than standard deviation

Gene expression is regulated by mRNA stability and degradation

Steady-state level of mRNA • Amount of mRNA in cell available for translation • Determined by combination of transcription and mRNA degradation rates Half-life (t1/2)• MRNA is degraded at some point after synthesis• Lifetime of mRNA varies; regulated by cell need

Cellular and molecular approaches to therapy

Stem cells for transplantation• Stem cells are undifferentiated somatic cells with two properties:1. The ability to renew their numbers by mitosis2. The ability to differentiate and form tissue-specific specialized cell typesCellular and molecular approaches to therapy• H D research uses:- Human embryonic stem cells (h E S C s)▪ Derived from the inner cell mass of early embryos- Induced pluripotent stem cells (i P S C s)▪ Produced through genetic reprogramming of adult skin cells

Steps of Apoptosis

Steps in apoptosis Fragmentation of nuclear envelope Disruption of internal cellular structures Dissolution of cell into small, spherical apoptotic bodies Engulfing of apoptotic bodies by phagocytic cells Caspases: Series of proteases responsible for initiating apoptosis and digesting intracellular components

Stress-Induced Behavior is Heritabl

Stress-induced epigenetic changes that occur prenatally or early in life can influence behavior Example:• Newborn rats raised with low levels of maternal nurturing (low-M N)• Had low glucocorticoid receptor (G )R) expression• As adults did not adapt well to stress (glucocorticoids help an animal or human adapt to stress)• Newborn rats exposed to high levels of maternal nurturing care early in life (high-M N)• G R expression is increased• As adults adapt well to stres

Structural Genes of trp Operon

Structural genes of trp operon: Five contiguous structural genes transcribed as polycistronic message • Involved in tryptophan production • trpE, D, C, B, A • Enzymes catalyze biosynthesis of tryptophan trpP: promoter—binding site for RNA Pol trpO: operator—binds repressor

GAL Genes are Inducible

Structure and function of the Gal4p activator. (a) Gal4p contains a DNA-binding domain, shown in dark blue, and two transcriptional activation regions within the activation domain, shown in light blue. (b) Effects of various deletions on the activity of Gal4p

Assigning the H D Gene to Chromosome 4

Studies narrowed the H D gene to a region containing a few million base pairs at the tip of the short arm of chromosome 4A physical map of the short arm of chromosome 4 showing the location of D4S10 near the tip of the chromosome in the 4p16.3 region, an area that encompasses about 3 percent of the length of the chromosome.

Identifying Regulatory Genes via Mutations

Study of mutagenized flies done to identify all genes controlling development in DrosophilaGradients of maternal-effect gene products along anterior-posterior axis of embryo regulate expression of:• Segmentation genes (gap, pair-rule, and segment polarity genes)• Homeotic selector (Hox) genes, which specify the fate of each segment

The molecular and cellular alterations in Huntington Disease

Studying the functions of the normal & mutant versions of HTT is extremely difficult because HTT interacts with more than 180 different proteins...The molecular and cellular alterations in Huntington DiseaseThe major pathways of cellular functions disrupted in HD. The letters (a-e) within the neuron correspond to the details of the disrupted processes within the nucleus and cytoplasm shown in parts a-e of the figure. (a) Transcriptional disruption occurs by blocking access of transcription factors and interfering with histone acetylation. (b) Impaired protein degradation is caused by blocking the loading of large protein aggregates into vesicles called autophagosomes for transport to lysosomes and by hindering transport of ubiquitin-tagged proteins to proteasomes. (c) Altered protein folding caused by expanded copies of glutamines that destabilize normal protein conformation and by a lack of chaperones to direct proper refolding along with reduced proteasome activity leads to accumulation of mHTT. (d) Altered synaptic function results from blockage of synaptic vesicle transport by mHTT. (e) Disruption of mitochondrial function by binding of mHTT to the outer membrane triggers a cascade of problems with function, transport, biogenesis, and maintenance.

Synpolydactyly (S P D)

Synpolydactyly (S P D)• Caused by mutations in HOX D 13• Characterized by:• Extra fingers and toes• Bone abnormalities in hands and fee

Synthetic genomes and the emergence of synthetic biology

Synthetic genome• An artificially constructed genome for artificial cells or designer organisms Bacterium Mycoplasma genitalium• Simple self-replicating bacteria• Serves as model for understanding minimal elements of genome necessary for self-replicating cel

Northern blot analysis

THIS technique was not named after its inventor... but merely meant to be a contrasting name to the first technique!• Used to determine whether gene is actively being expressed in given cell or tissue• Used to study patterns of gene expression in embryonic tissues, cancer, and genetic disorders

Precision oncology

Targeted Cancer Immunotherapies:• Therapies that harness the patient's own immune system to kill tumors• Some have brought remarkable therapeutic effects in clinical trials• Two of the most promising precision cancer immunotherapies• Adoptive cell transfer• Engineered T-cell methods

Ubiquitin-Mediated Protein Degradation

Targets a protein for degradation by covalently modifying it with ubiquitin Ubiquitinated proteins are recognized by the proteasome, a multi-subunit protein complex with protease (protein cleaving) activity The proteasome unwinds target proteins, removes their ubiquitin tags, and breaks the protein into small peptides about 7-8 amino acids long

C R I S P R-Cas is an Adaptive Immune System in Prokaryotes

The C R I S P R-Cas Mechanism for R N A-Guided Destruction of Invading D N A: Adaptive immunity is also dependent on a set of adjacent C R I S P R-associated (cas) genes Cas genes encode Cas proteins that function as D Nases and R NasesThe C R I S P R-Cas Mechanism:1. Spacer acquisition2. c r R N A biogenesis (acronym explained in later slide)3. Target interference

C R I S P R-Cas is an Adaptive Immune System in Prokaryotes

The C R I S P R-Cas Mechanism:1. Spacer acquisition:- Invading phage D N A is cleaved into smaller fragments known as protospacers, which are then inserted into C R I S P R loci to become new spacers- When new spacers are added to the C R I S P R locus, repeat sequences are duplicated such that each spacer is flanked by repeats on each side

C R I S P R-Cas is an Adaptive Immune System in Prokaryotes 3

The C R I S P R-Cas Mechanism:3. Target interference:- Mature c r R N As associate with Cas nucleases, or nuclease complexes, and recruit them to complementary sequences in invading phage D N A- Cas nucleases then cleave the viral D N A, thus neutralizing infection

Genomic Techniques

The ChIPSeq method screens for specific transcription factor binding sites across a whole genome. In this method, formaldehyde is added to tissues or cultured cells to crosslink DNA-binding proteins currently attached to chromatin. Then the chromatin is extracted from cells and sheared into small fragments. An antibody or antibodies that recognize specific DNA-binding proteins of interest (POI), such as a transcription factor, are added to the mixture, and the antibodies attach to the POI. Then the antibody, together with its protein-DNA fragment, is pulled out of the mixture (immunoprecipitated) by centrifugation. The immunoprecipitated DNA fragments are released from crosslinked proteins and attached antibodies and are then sequenced. Sequence data reveal the DNA-binding site for the POI and these sequences can be mapped to specific locations in the genome.

qPCR

The Taqman approach to quantitative real-time PCR (qPCR) involves a pair of PCR primers along with a probe sequence complementary to the target gene. The probe contains a reporter dye (R) at one end and a quencher dye (Q) at the other end. While the quencher dye is close to the reporter dye, it interferes with fluorescence released by the reporter dye. When Taq DNA polymerase extends a primer to synthesize a strand of DNA, it cleaves the reporter dye off of the probe allowing the reporter to give off energy. Each subsequent PCR cycle releases more reporter dyes. A readout of the resulting increase in fluorescence is produced by a computer

Introduction

The ability to specifically and efficiently edit a genome has broad implications for research, biotechnology, and medicine C R I S P R-Cas has been developed into an efficient, cost-effective molecular tool that can introduce precise and specific edits to a genome• The target effects may be "precise" but as this paper shows, there are mechanistic consequences that may lead to harmful effects such as cancer: The discovery of C R I S P R-Cas has impacted genetics and other related fields at an unprecedented pace

Methylation

The added methyl groups occupy the major groove of DNA and silence genes by blocking the binding of transcription factors and other proteins necessary to form transcription complexes

Core Promoter TFIID 2

The assembly of general transcription factors (TFIIA, TFIIB, etc.; abbreviated as IIA, IIB etc.) required for the initiation of transcription by RNAP II: Note: TFIID (as shown in step 1) is a combination of TAFs and TBP). What does TAF stand for? "TBF Associated Factor"

Methylation

The bulk of methylated CpG dinucleotides are found in repetitive DNA sequences located in heterochromatic regions of the genome including the centromere Heterochromatic methylation: • Maintains chromosome stability by preventing translocations and other chromosomal abnormalities

Cancer cells contain genetic defects affecting cell-cycle regulation

The cell cycle: Cellular events in sequence from one division to another Phases of cell cycle: Interphase G1 S phase G2 M phase

Lethal Recessive Allele

The change in the frequency of a lethal recessive allele, a. The frequency of a is halved in two generations and halved again by the sixth generation. Subsequent reductions occur slowly because the majority of a alleles are carried by heterozygotes.

Negative Control

The components of the wild-type lac operon and the response in the absence and presence of lactose.

Cytoplasmic Polyadenylation

The control of translation by cytoplasmic polyadenylation: When it is "ready" to get translated, a kinase allows a polymerase to add "A's" to the poly-A tail... enough for translation to start

development and maintenance of malignant growth in cancer

The development and maintenance of malignant growth in cancer involves the interaction of gene mutations, hypomethylation, hypermethylation, overexpression of oncogenes, and the silencing of tumor-suppressor genes.

Disruptive Selection

The effect of disruptive selection on bristle number in Drosophila. When individuals with the highest and lowest bristle numbers were selected, the population showed a nonoverlapping divergence in only 12 generations

Degrees of Selection

The effect of selection on allele frequency. The rate at which a deleterious allele is removed from a population depends heavily on the strength of selection.

Genetic and Epigenetic Regulation of Development

The epigenetic process begins early in embryogenesis with a global D N A demethylation that erases parental epigenetic marks Contributes to establishment of pluripotency By the blastula (ball of identical-looking cells) stage, methylation gradually resumes and is associated with the initial steps in specification and determination of embryonic stem cells (E S C s) and embryonic germ cells (E G C s)

lac Z

The gene that encodes β-galactosidase, which is an enzyme that converts disaccharide lactose to monosaccharides glucose and galactose (so it basically, "digests" it) Conversion is necessary for lactose to serve as primary energy source in glycolysis (the entire disaccharide can't go right into glycolysis, but glucose can!)

Quantitative Loci

The genetic ratios (on the x-axis) resulting from crossing two heterozygotes when polygenic inheritance is in operation with 1-5 gene pairs. The histogram bars indicate the distinct F2 phenotypic classes, ranging from one extreme (left end) to the other extreme (right end). Each phenotype results from a different number of additive alleles.

human metallothionein 2A gene promoter

The human metallothionein 2A gene promoter and enhancer regions, containing multiple cis-acting regulatory sites. The transcription factors controlling both basal and induced levels of MT2A transcription are indicated below the gene, with arrows pointing to their binding sites. Notice how the glucocorticoid receptor regulates this "damage control" gene. Cortisol is one example of a glucocorticoid (and cortisol is a "stress hormone"), so this all makes sense!

The Adaptive System Has Two Branches

The humoral branch involves B lymphocytes (B cells) that synthesize antibodies directed at specific antigens The cell-mediated branch consists of T lymphocytes (T cells) including cytotoxic T cells and helper T cells• Recognize specific antigens on the surface of or inside cells that are infected or cancerous• Cytotoxic T cells then contact the cell, release cytotoxic molecules, and trigger apoptosis of the target cell

Precision Oncology

The immune system Consists of cell types and chemical signals constituting the innate and the adaptive systems

Immune System

The innate system acts rapidly and nonspecifically to external and internal "nonself " entities (i.e. bacteria, viruses, toxins, and tumor cells) The adaptive (or acquired) system Destroys pathogens, tumor cells, and molecules such as toxins by recognizing and acting specifically against each entity• It does this by recognizing specific "nonself " molecules called antigens Cells of the adaptive system develop a memory of previous contact with nonself antigen,• allows them to quickly replicate and respond to a subsequent appearance of the antigen

Epigenetic traits are heritable

The irony with this phrase ("epigenetic traits are heritable") is... ...the "old dogma of genetics" is [generally speaking] only mutations in DNA (and specifically, only the DNA of sperm or egg cells) can result in heritable changes for the next generation. But with the discovery of epigenetics, we learn of this exception: The effects of agents causing epigenetic changes are heritable as well. And many such agents...are from the environment

A phylogenetic tree showing the relationships among modern humans, Neanderthals, and Denisovans

The latter two groups branched off from our last common ancestor before our species left Africa. Genomic analysis shows that there was interbreeding between members of our species with Neanderthals and Denisovans, making our genome a mosaic with contributions from at least two other human species.

Epigenetics and Imprinting

The pattern of imprinting in mammals is reprogrammed every generation ●When gamete formation begins in female/male germ cells, both chromosome sets have their imprints erased and are each reprogrammed by changing the pattern of methylation to carry a female/male imprint that is transmitted to the next generation through the egg/sperm ●This is one factor that humans have not yet learned to fine-tune in the process of cloning (which is likely why cloned animals tend to have respiratory problems and age-related disorders)

Alternative splicing of the CT/CGRP gene transcript

The primary transcript, which is shown in the middle of the diagram, can be spliced into two different mRNAs, both containing the first three exons but differing in their final exons.

Role of epimutations

The role of epimutations versus genetic mutations in the initiation of cancer. (a) An inherited genetic mutation causes the loss of a tumor-suppressor allele. Several mechanisms can cause the loss or silencing of the second allele: mutation, chromosomal aberration, or an epimutation. (b) An epimutation silences one allele of a tumor-suppressor gene. The 2nd allele can be lost through mutation, chromosomal aberration, or silencing by an epigenetic event.

The histone code =

The sum of the complex patterns and interactions of histone modifications that change chromatin organization and gene expression

benign tumor

The term for a tumor that results from unregulated cell growth but which often causes no serious harm and can be more easily removed via surgery is "benign ", while the term for cells that have traveled and invaded other tissues is "malignant .

Herbicide-Resistant G M Crops

There was a demand for herbicide-resistant G M crops because:• Weed infestations destroy 10 percent of crops worldwide• Herbicides applied before seeding can only be so efficient; they kill crop plants

Insect-Resistant G M Crops

There was also a demand for insect-resistant G M crops:• Insect resistance is the second most prevalent G M modification• Makes plants resistant to agricultural pests• Insect damage is a serious threat to food production• Farmers use insecticides to combat issue

What is true of restriction enzymes?

They are found naturally in bacteria and can be used in the laboratory to make a GMO

Homeotic Genes

Three classes of floral homeotic genes control development of organs (see next slide's Table)• Sepal development controlled by class A genes alone• Petal development controlled by class A and class B genes expressed together• Stamen development controlled by class B and class C genes• Carpel development controlled by class C genes alone

Three Steps of PCR

Three steps of P C R • Denaturation • Primer annealing (hybridization/annealing) • Extension • Steps are repeated over and over using thermocycler to amplify D N A exponentially D N A strand is doubled in each cycle New strands along with old strand serve as templates in next cycle

Polygenic TraitsPolygenic traits that do not show continuous variation:

Threshold traits:• Polygenic and often multifactorial• Have a small number of discrete phenotypic classes• An increasing number of diseases show this pattern of polygenic inheritance:• Type II diabetes (adult-onset diabetes

Environmental Carcinogens

Tobacco smoke One of the best-known environmental carcinogens Contains at least 60 mutagenic chemicals, giving smokers a 20-fold increased risk of developing lung cancer HISTORICAL NOTE: It actually took a WHILE to "prove" this and expose these facts. Alcohol may cause inflammation and lead to liver cancer

Not all polygenic traits show continuous variation

Traits with variation often show a continuous range of phenotypes• Examples of continuous variation in phenotype:• Height in humans• Milk and meat production in animals• Yield in seed protein• Quantitative inheritance:• Continuous variation across range of phenotypes measured and described in quantitative terms

Mechanism of Transcription Activation and Repression

Transcription activators and repressors bring changes to RNA Pol II transcription: • DNA looping delivers activators, repressors, and general transcription factors to promoter vicinity • Recruitment model: enhancers and silencer elements act as donors; affect regulatory proteins at gene promoters

Functional Domains

Transcription factors (proteins): • Have two functional domains (clusters of amino acids with a specific function): • DNA-binding domain: • Binds to specific DNA sequences in the cis-acting regulatory site • Trans-activating domain: • Activates or represses transcription by binding to other transcription factors or RNA polymerase

Eukaryotic transcription initiation is regulated by transcription factors that bind to cis-acting sites

Transcription factors = • Transcription regulatory proteins • Target cis-acting sites of genes regulating expression • Activators increase transcription initiation • Repressors decrease transcription initiation - • Don't get activators and repressors confused with enhancers and silencers. • Enhances & silencers = DNA sequences (cis-acting elements) • Activators & repressors = proteins (trans-acting factors) Multiple transcription factors bind to several different enhancers and promoter elements and fine-tune the level of transcription initiation

Transcriptome analysis reveals profiles of expressed genes in cells and tissues

Transcriptome analysis (global analysis of gene expression)• Studies expression of genes by genome qualitatively and quantitatively• Qualitatively: Identifies which genes are expressed and which are not• Quantitatively: Measures varying levels of expression of different genes

Genetically modified animals serve specific roles in biotechnology

Transgenic animals• Can be for research purposes to study gene function Examples of transgenic animals• Mice with human growth hormone transgene• Transgenic Atlantic salmon...• One more example has a lot of irony to it, in my opinion...! Examples of transgenic animals• "Transgenic cows to battle mastitis (infection of mammary glands that reduces output and contaminates milk"• Ironically (the textbook does not mention this!) one contributing factor of mastitis may be the treatment with extra, recombinant bovine growth hormone (in addition to what their body makes). If so, it would be ironic that one GMO application is being used to "solve a problem"...caused by another / different GMO application (!)• The "extra growth hormone" application principle as well as the mastitis symptom can be seen in this video starting at 41 minutes into the video:Genetically modified animals serve specific roles in biotechnology

Transgenic Cows

Transgenic cows developed for battling mastitis... so how do they "work" anyway?They use an incorporated gene whose protein product destroys S. aureus bacterium.Thus, [somewhat] mastitis-resistant cattle get produced:Transgenic cows were generated that possessed lysostaphin gene from Staphylococcus simulans; lysostaphin cleaves S. aureus cell wall.However, our textbook (p. 527) cautions: "The mastitis problem is not completely solved in these transgenic cows, however, because lysostaphin is not effective against other microbes such as E. coli and Streptococcus uberis that occasionally cause mastitis; moreover, there is also the potential that S. aureus may develop resistance to lysostaphin

Transgenic Organisms

Transgenic organisms• Genetic engineering allows genes to be cloned and transferred from one organism to another• When genes are transferred between unrelated species, a transgenic organism is created Cigenic: Used to describe genes transferred within a species/This is still an artificial type of gene manipulation, BUT unlike the classic part of the "GMO" definition, this process would not end up producing "inter-species" gene sequences. The ligation properties and process may still be quite different compared to natural meiosis processes such as crossing over, however.

Transplantation of Synthetic Genome

Transplantation of "synthetic genome" has been done: Mycoplasma mycoides• Genome transplantation is the true test of functionality of synthetic genome• A functional synthetic genome of M. mycoides was created in 2010 (had all essential genes for life, for that species, but not actually all the DNA)• It was successfully transplanted into existing genome of M. capricolum

The tryptophan (trp) operon in E. coli is a repressible gene system

Tryptophan (trp) operon: • Repressible gene system in E. coli • Wild-type E. coli produce enzymes for biosynthesis of amino acids and essential macromolecules • If tryptophan present: Enzymes necessary for synthesis of tryptophan are not produced (since it does not need to waste energy "making it from scratch" if it is provided in its environment)

Concordance

Twins are concordant for a given trait IF either both express the trait... OR if neither express the trait• Discordant: One expresses while the other does not• Comparison of concordant values (M Z versus. D Z) gives potential value for heritability assessment

Computing frequencies

Two methods for computing frequencies of alleles in population surveyeda) Counting allelesb) From genotype frequencies

Eukaryotic gene expression is influenced by chromatin modifications

Two structural features of eukaryotes distinguish them from prokaryotes: Eukaryotic genes are situated on different chromosomes that occupy a distinct location • Eukaryotic DNA is combined with histones and nonhistone proteins to form chromatin • Compact chromatin structure inhibits transcription, replication, and DNA repair (one way of regulating whether or not the gene "does something" / gets expressed)

2 D G E

Two-dimensional gel electrophoresis (2DGE) is a useful method for separating proteins in a protein extract from cells or tissues that contains a complex mixture of proteins with different biochemical properties. The two-dimensional gel photo shows separations of human platelet proteins. Each spot represents a different polypeptide separated by isoelectric point, pH (x-axis), and molecular weight (y-axis). In this photo, some protein spots have been identified by name based on comparison to a reference gel or by determination of a protein sequence using mass spectrometry. Notice that many spots on the gel are unlabeled, indicating proteins of unknown identit

Western blot (named for same reason as the "Northern")

Used for analyzing proteins

Adenovirus Vectors

Used in early gene therapy trials• Capable of carrying large number of genes• Disadvantage: Humans produce antibodies against adenoviruses that renders therapeutic genes ineffective• Adeno-associated virus is now widely used

Applications of P C R

Useful tool; the most widely used technique in genetics and molecular biology • Allows for screening of mutations involved in genetic disorders • Location and nature of mutation can be determined quickly • Allele-specific probes for genetic testing can be synthesized, making P C R important for diagnosing genetic disorders

Stone-Age Genomics

Uses small amounts of ancient D N A from bone and other tissue (there are limits to the oldest DNA we can find... the oldest so far is million-year-old mammoth DNA but no 65-million-year-old dinosaur DNA!• Data used to study evolutionary relatedness of various extinct and present-day species

What is DNA phenotyping?

Using DNA sequence information to reveal a person's [likely] physical features and ancestral origins

Finding Linkage between Huntington Disease and an R F L P Marker

Using pedigrees and R F L P s from Venezuelan families linkage was discovered to H D Haplotype C of the G8 marker is coinherited with H D in all cases• Haplotypes are specific patterns of D N A fragments form R F L P analysis Indicated that the R F L P marker and the mutant H D allele are on the same chromosome

Developing Targeted Drugs

Using recombinant D N A technology, Genentech Corporation in California developed a monoclonal antibody known as trastuzumab (or Herceptin®) In cancer cells that overexpress H E R-2, Herceptin treatment causes cell-cycle arrests and, in some cases, death of cancer cells Herceptin only acts on breast cancer cells that have amplified H E R-2 genes; therefore, it is important to know the H E R-2 phenotype of each cancer Herceptin has potentially serious side-effects; therefore, its use must be limited to those who could benefit from the treatment

Transgenic animal models of Huntington disease

Using transgenic mice to study Huntington disease• Mice carried mutant alleles with different numbers of CAG repeats• Behavioral changes and degeneration of specific brain regions in transgenic mice parallel the progression of HD in humansCAG repeats• Normal alleles carry 7-34 repeats• Addition of 40 CAG repeats• HTT becomes toxic• Causes cell death in certain regions of brain• Leads to behavioral abnormalities

V N T Rs: Benefits and Limitations

V N T R s are useful in D N A profiling• 30 different possible alleles at any V N T R in population• Creates large number of possible genotypes• If four different V N T R loci were examined, this would mean more than 2 billion possible genotypes to examine (420) V N T R s require large samples of D N A; therefore, autosomal S T R D N A profiling is now being utilized

DNA fingerprint at two VNTR loci for two individuals.

VNTR alleles at two loci (A and B) are shown for two different individuals. Arrows mark restriction-enzyme cutting sites that flank the VNTRs. Restriction-enzyme digestion produces a series of fragments that can be separated by gel electrophoresis and detected as bands on a Southern blot (bottom). The number of repeats at each locus is variable, so the overall pattern of bands is distinct for each individual. The DNA fingerprint profile shows that these individuals share one allele (B2).

DNA profiling methods

VNTR-based DNA fingerprinting:Depends on variable number tandem repeats (V N T R), or minisatellites• Located in noncoding regions of genome• D N A sequences between 15 and 100 b p long• Number of repeats at V N T R locus varies in individuals• D N A is extracted from tissue sample and digested with restriction enzyme• Restriction enzyme cleaves on either side of V N T R• Digested D N A is separated on gel and Southern blot analysis is done

Stem Cells for Delivering Therapeutic Genes

Viral and nonviral vectors are being used to deliver therapeutic genes into stem cells in vitro Then the stem cells are:• Introduced directly into the patient• Differentiated in vitro into mature cell types then transplanted into the correct organ of a patient Hematopoietic stem cells (HSCs) are found in bone marrow and make blood cells• Easily accessible from the bone marrow of the patient• No immune rejection• Replicate quickly in vitro• Fairly long lived• Differentiate into both red blood cells and white blood cells (leukocytes)

Protein Domains and Motifs

When a gene sequence is used to predict polypeptide sequence, the polypeptide can be analyzed for specific protein domains and motifs • Protein domains: Ion channels, membrane-spanning regions, secretion, and export signals • Motifs: Helix-turn-helix, leucine zipper, or zinc-finger motifs

D N A-Editing Nucleases for Gene Targeting

While there have been some successful animal studies etc... When C R I S P R-Cas9 was used to edit the H B B gene in [very early] human embryos:• Out of the 86 human embryos donated• Two days after C R I S P R-Cas treatment:• 71 embryos (of 86) had survived• Only 4 (of 71) carried the intended change to the H B B gene.• Many other embryos had acquired mutations in other genes

Whole-genome sequencing is a widely used method for sequencing and assembling entire genomes

Whole-genome sequencing (shotgun cloning):The most widely used strategy for sequencing and assembling an entire genome1) Genomic D N A is cut into fragments and contigs (continuous fragments) made (= overlapping fragments adjoining segments that collectively form one continuous D N A molecule within chromosome2) Entire chromosome is assembled by computer program3) Fragments are aligned based on identical D N A sequences

Biotechnology:

Wide range of methods (NOT limited to the more recent "genetic engineering" techniques) that manipulate organisms or their components, such as simply isolating enzymes via physical processes, or producing wine, cheese, or yogurt using older methods that have been used for thousands of years. • Genetically modified plants and animals are only one [quite recent] aspect of [newer] biotechnology

Phylogenetic Trees from D N A Sequences

With that being said... Phylogenetic reconstruction of genomic D N A was used to answer the popular question: What was the [likely] common ancestor to vertebrates?• [Ancestor most resembling] Lungfish...or " " Coelacanth? Genomic data [most recently] suggests Lungfish!

Methylation

X chromosomes in mammalian females are inactivated by converting them into heterochromatin (dosage compensation) CpG methylation in euchromatic regions causes a parent-specific pattern of gene transcription

Nutrition connection: What do zinc fingers DO, and what do they require, to function?

Zinc fingers: Again, they are important domains of transcription factors, found in about 3% of genes in the human genome: Stabilized by a zinc ion (hence their function is dependent on this important essential dietary mineral) Examples of what some of these transcription factors, and their functions include: • Histone acetyltransferases (as we went over, these generally "let genes get appropriately expressed") • A protein for developing neurons :• AND EVEN a breast tumor-suppressor protein (ST18)

What is the "operator" in an operon?

a DNA sequence to which a repressor protein can bind

totipotent

a cell (such as a zygote) that can give rise to any cell type including the placenta

Lactose

a disaccharide composed of galactose and glucose Lactose is the "inducer"

What is a "zinc finger"?

a domain of some transcription factors, which is stabilized by a zinc ion

ubiquitin

a molecule that gets added to a protein group to target it to a proteosome

In gene cloning, an antibiotic resistance gene could serve as ______

a selectable marker

Founder effect =

a sub-type of genetic drift • Occurs when population originates from small number of individuals• Gene pool may not reflect larger population from which founders are drawn:• i.e. the few "founders" may just happen, by chance, to have certain genetics that are different from the larger population from which they came

D N A Cloning with Plasmids

a) Color-enhanced electron micrograph of plasmids isolated from E. coli. (b) Diagram of a typical DNA cloning plasmid. "Apply your knowledge" question: Find the "selectable gene marker" in this image of the plasmid, and describe why it has been inserted as part of the plasmid

Gap Proteins

a) Progressive restriction of cell fate during development in Drosophila. Gradients of maternal proteins are established along the anterior-posterior axis of the embryo. (b-d) Three groups of segmentation genes progressively define the body segments. (e) Individual segments are given identity by the homeotic genes

Transgenic mice incorporating the GFP gene (a popular reporter gene) from jellyfish enable scientists to tag particular genes with green fluo

a) Transgenic mice incorporating the GFP gene (a popular reporter gene) from jellyfish enable scientists to tag particular genes with green fluorescent protein. Thanks to the expression of GFP, which makes the transgenic mice glow green under ultraviolet light, scientists can track activity of the tagged genes, including activity in subsequent generations of mice generated from these transgenics.(b)The mouse on the left is transgenic for a rat growth hormone gene, cloned downstream from a mouse metallothionein promoter. When the transgenic mouse was fed zinc, the metallothionein promoter induced the transcription of the growth hormone gene, stimulating the growth of the transgenic mouse.a) Transgenic mice incorporating the GFP gene (a popular reporter gene) from jellyfish enable scientists to tag particular genes with green fluorescent protein. Thanks to the expression of GFP, which makes the transgenic mice glow green under ultraviolet light, scientists can track activity of the tagged genes, including activity in subsequent generations of mice generated from these transgenics.(b)The mouse on the left is transgenic for a rat growth hormone gene, cloned downstream from a mouse metallothionein promoter. When the transgenic mouse was fed zinc, the metallothionein promoter induced the transcription of the growth hormone gene, stimulating the growth of the transgenic mouse.

Methylation involves _______

addition of -CH3 groups, which transcriptionally silences genes

Proteomics is the study of ______

all proteins encoded by the genome of a cell

Formation of a DNA loop

allows factors that bind to an enhancer or silencer at a distance from a promoter to interact with general transcription factors in the pre-initiation complex and to regulate the level of transcription

Which mechanism of posttranscriptional regulation would allow many proteins to get made from one gene?

alternative splicing

kinase

an enzyme that adds phosphate group into certain amino acids

What is a proteosome?

an organelle that digests proteins

Which process would be most likely to help prevent cancer?

apoptosis

A comparison of gene regulation in bacteria (left) and eukaryotes (right

bacteria: transcription translation protein modification Eukaryotes: chromatin remodeling transcription nuclear export mRNA stability mRNA localization translation- cytoplasm protein modification

One type of GMO crop called "RoundUp Ready" is designed to _______

be tolerant to the herbicide glyphosate

What is / are the quality or qualities of a concordant trait in twin studies?

both twins OR neither twins could express the trait

Glucose Inhibits CAP Binding

cAMP: Cyclic adenosine monophosphate • To bind to promoter, CAP must be bound to cyclic adenosine monophosphate (cAMP) • Glucose inhibits activity of adenylyl cyclase, which catalyzes conversion of ATP to cAMP This is what prevents CAP from binding when glucose is present

The formation of cAMP from ATP

catalyzed by adenyl cyclase

Which technique would be useful for finding genes that interact with proteins such as transcription factors

chromatin immunoprecipitation

Which technique would be useful for finding genes that interact with proteins such as transcription factors?

chromatin immunoprecipitation

Promotors, enhancers and silencers are all _____

cis-acting sequences

A specific type of DNA sequence could be called a ________

cis-acting site

A specific type of DNA sequence could be called a ________

cis-acting site CORRECT trans-acting factor transcription factor all of the other answers are correc

Lac Operon

cluster of genes controlling lactose use in bacteria

CRISPR stands for _______

clustered regularly interspaced short palindromic repeats

CRISPR-Cas as a Tool for Basic Genetic Research

dCas9: Mutations to the HNH and RuvC nuclease domains create a "dead" version of Cas9 = can bind but cannot cut target DNA Genetically fusing the activation domain of a transcriptional activator to dCas9 activate the transcription of any gene by designing an appropriate targeting sgRNA Genetically attaching the repression domain of a transcriptional repressor to dCas9 enables a researcher to turn off any given target gene Genetically attaching the catalytic domain of an epigenetic modifier enzyme, such as a histone deacetylase or a DNA methyltransferase, to dCas9 enables a researcher to add or remove epigenetic tags Genetically attaching fluorescent proteins, such as the green fluorescent protein (G F P),are attached to d Cas9 enables a researcher to see where a specific sequence is located within a cell using fluorescence microscopy

Which of the following are common features of cancer cells?

disrupted [epigenetic] histone modifications defects in DNA repair genes defects in genes for cell cycle control defects in signal transduction pathways

Which of the following would qualify as "macroevolution"?

emergence of new species or broader taxonomic groups

Expression of the trp operon would allow _________ to get made.

enzymes capable of synthesizing tryptophan "from scratch"

The type of GMO called "Bt crops" are designed to _______

express an insecticidal toxin within their own cells

What would be an example of an expected result if a homeotic selector (Hox) gene was mutated?

fly legs may grow out of its head (instead of antenna)

Restriction enzymes are ______

found naturally in bacteria

What is / are examples or causes of "genetic drift"?

founder effect or bottleneck effect

p

frequency of allele A

2pq

frequency of the genotype Aa

Drosophila melanogaster

fruit fly

Adenovirus vectors were used in early gene therapy trials, but one disadvantage is that humans produce antibodies against such vectors.

gene therapy antibodies

hypomethylation of DNA

general increase in gene expression

Acetylation of histones results in ______

genes becoming available for transcription

Metagenomics focuses on studying ______

genomes from environmental microbes

passenger mutation

has no direct contribution to cancer cell phenotype

Relative size ranges and fluorescent dye labeling colors of 24 STR products generated by a commercially available DNA profiling kit.

he scale at the bottom of the diagram indicates DNA fragment sizes in base pairs.

Which of the following can play a role in early embryonic development?

hedgehog pathway notch signaling pathway receptor tyrosine kinase pathway

Maskin

helps makes mRNA dormant until it is ready to be translated

Pharmacogenomics

is the study of how an individual's entire genetic makeup determines the body's response to drugs (goal is to optimize drug therapies) There are many interactions that occur between a drug and proteins within the patient, many genes and different genetic polymorphisms can affect a person's response to a drug Important to note: We still do not 100% understand the genetics of antibody production, including autoantibodies that could cause life-threatening anaphylactic allergies or autoimmune disease to antigens such as drugs or vaccine particles- that is why there is always a risk of such severe side effects that should not be downplayed

What is NOT true of the Lac Operon?

it is found in eukaryotes

What is NOT true of the Lac Operon?

it is found in eukaryotes CORRECT it controls the use of lactose the regulation of its structural genes depends on the presence or absense of lactose it includes a gene whose enzymes yields the products glucose and galactose

Visualization of active sites of transcription

known as transcription factories (green), in mouse cell nuclei. Transcription of one specific gene (at both alleles), encoding the cell-cycle regulatory protein Myc, is visualized with a sequence-specific probe (red; red and green colocalization is yellow).

The response of the lac operon in the presence of lactose in a cell bearing the IS mutation.

lactose- binding region is altered, no binding to lactose repressor always bound to operator, blocking transcription

Maternal-Effect Gene

m R N A and proteins deposited in egg cytoplasm (by mother fly)• Products distributed in gradient or concentrated specific regions of cell• Encode transcription factors and proteins• Regulate gene expression• Products activate or repress expression of zygotic genome

mRNA Surveillance and Nonsense-Mediated Decay

mRNA surveillance: mRNAs with a premature stop codon result from a nonsense mutation in the gene, or due to an RNA polymerase error during transcription Are degraded

Levels of gene expression regulation

mRNAs have wide range of half-lives (t1/2) There is extensive modulation of mRNA translation, protein processing, modification, and degradation

What field of genomics specializes in sequencing genomes from environmental microbes (usually ones that are difficult or impossible to culture in the laboratory)? (Pick the most fitting answer).

metagenomics

Addition of -CH3 groups, which transcriptionally silences genes, is called ______.

methylation

Epigenetics often involves _______

methylation of DNA changes in gene expression histone modifications

Which of the following are true of cancer genetics?

most cancers are not inherited Correct! all cancers involve genetic anomalies Correct! cancer usually involves genetic mutations in somatic cells Correct! certain cancers may be caused by viruses, harmful chemicals, or radiation, and some cases are inherited

What is the term for a type of trait that is classically known for being the result of both gene action and environmental influences?

multifactorial trait / complex trait

Caenorhabditis elegans

nematode / roundworm

What is / are the quality or qualities of a discordant trait in twin studies?

one twin expresses the trait while the other does not

p53 Tumor-Suppressor Gene

p53 tumor-suppressor gene: Most frequently mutated gene (50% of all cancers) Encodes transcription factor that represses or stimulates transcription different genes Is continuously synthesized but is rapidly degraded, so is present at low levels Increased levels due to increases in protein phosphorylation, acetylation, and other posttranslational modifications

pharmacogenomics is the study of how an individual's entire genetic makeup determines the body's response to drugs, so that drug therapies can be optimized. This is particularly important since, currently on average, any given drug will only be effective in about 50 of patients.

pharmacogenomics 50

directional selection

phenotypes at one end of a spectrum get selected for or against

Which of the following is a method of gene expression regulation for eukaryotes, but not prokaryotes?

polyadenylation of mRNA acetylation of histones chromatin remodeling

hypermethylation of a DNA repair gene

predisposition to nucleus

Specific parts of a ______ in eukaryotes include a TATA box, Initiator and TFIIB recognition elements (BRE).

promoter

Over 90% of disease-associated variations in the human genome are located in _______

regulatory regions

Alternate forms of mRNA structure, which can create either a terminator OR an "anti-terminator" structure, are called ________. These contain both "aptamers" and expression "platorms" which have different purposes.

riboswitches

Transcription factories can actually involve many genes in separate chromosomes

rnp transcription factor protein rich core RNA polymerase II

runt Homolog in Mice

runt homolog gene (in mice):• One mutant copy: Have skeletal abnormalities as seen with humans• Two mutant copies: Have complete absence of bone formation, with skeletons containing only cartilage• runt gene is important in controlling initiation of bone formation

Flower organs establish the following order, from the outermost concentric ring, to the innermost:

sepals, petals, stamens, carpals

Molecular mechanisms of RNA-induced gene silencing:

siRNA (small interfering RNAs) and micro RNAs • Short, double-stranded ribonucleotides • siRNAs: Arise in cell due to virus infection—produce double-stranded RNA, which is recognized and cleaved by Dicer (double stranded RNA means "virus"!) • micro RNAs: Noncoding RNAs that negatively regulate gene expression

What field of genomics specializes in identifying DNA sequences that actually are genes, as well as sequences such as regulatory sequences? (Pick the most fitting answer)

structural genomics

Comparative Genomics

studies model organisms, whose genomes revealed interesting elements of the human genome: • Sea urchin genome, dog genome• Chimpanzee genome, rhesus monkey genome• Neanderthal genome

I know this isn't "nutrition",

t it's also important in everyday life, that fact (you can have too little OR too much of an otherwise-very-essential nutrient) might beg the question: HOW does an animal in the wild (OR a human on a healthy (organic and whole-food) diet) know how much of which types of foods to eat...? In short, sticking to "healthy cravings" can help an animal (or a human with a healthy diet) reliably seek appropriate levels of this important nutrient, without "overdosing" on it.

a) In normal imprinting

the ICR (imprinting control region) on the paternal chromosome is methylated (filled circles); the IGF2 allele is active and the H19 allele is silent. The ICR on the maternal chromosome is not methylated (open circles), and the IGF2 allele is silent while the H19 allele is active. (b) In one form of BWS (Beckwith-Wiedemann syndrome), both the maternal and paternal ICRs are methylated (filled circles), both IGF2 alleles are active, and both H19 alleles are silent. The result is dysregulation of cell growth, resulting in the overgrowth of structures.

After mRNA gets made, ________

the half life of mRNA varies, which depends on cell need

The mechanism by which certain transgenic cows are made resistant to mastitis is:

they express lysostaphin which destroys S. aureus bacteria

What is the role of the human metallothionein gene?

to make a protein that protects cells from the toxic effects of excessive heavy metals by binding to them

A transcription factor could be considered a ______

trans-acting factor

Presence of ______ will ______ expression of the trp operon.

tryptophan ; repress

segment polarity genes

type of zygotic genes that divide EACH segment into anterior and posterior halves.

pair-rule genes

type of zygotic genes that divide the embryo into stripes about two segments wide, and help define segment borders

gap genes

type of zygotic genes that divide the embryo into the broadest divisions (head, thorax, abdominal regions)

______ is a molecule that gets added to a protein to target it to a proteosome.

ubiquitin

Population's gene pool =

• All alleles present in population;• Genetic information carried by members of population• Most populations contain high degree of heterozygosity

Gene Therapy: Early Trials

• Almost all early trials focused on monogenic diseases: Conditions caused by a single gene (not always the case for diseases!)• Identify mutant gene• Replace or supplement with normal copy of gene• Examples• Blood disorders (leukemia, hemophilia)• Respiratory system issues• Cystic fibrosis

Genetic variation

• Can be detected using artificial selection• If genetic variation does exist, then phenotype will change over generations

Nonviral Delivery Methods

• Chemically assisted transfer of genes across membranes• Nanoparticle delivery• Fusion of cells with artificial lipid vesicles (liposomes)• Short-term expression with "gene pills" is being explored

Mechanisms of posttranscriptional gene regulation:

• Control of alternative splicing • mRNA stability • Translation • RNA silencing

The Significance of D N A Profile Match

• Demonstrates method of probability calculations• Probabilities of S T R loci alleles calculated to obtain genotype frequencies Genotype frequency from five-locus profile calculated by multiplying genotype probabilities: 9 × 107• Means that 9 people out of 10 million (approximately 1 in a million) chosen at random would share this five-locus D N A profile

Heritability values estimate the genetic contribution to phenotypic variability

• Describes proportion of total phenotypic variation in population due to genetic factors (so yes, this concept acknowledges there are non-genetic factors at work too) • Does not indicate how much of a trait is genetically determined or the extent to which an individual's phenotype is due to genotype (i.e. in one individual- this concept just focuses on VARIATION between individuals in a population)

The Hardy-Weinberg Law describes allele frequencies and genotype frequencies in population gene pools

• Describes what happens to allele and genotype frequencies in "ideal" populations• "Ideal" population =• infinitely large with random mating; not subject to evolutionary forces (mutation, migration, or selection)... so this type of population rarely exists! But it is still good as a model for doing certain calculations...

Y-Chromosome S T R Profiling

• Differentiates profiles of 2 or more people in mixed sample• More than 200 STR loci on Y chromosome, but less than 20 are used for forensic analysis PCR amplification uses specific primers that do not amplify DNA on X chromosome Limitations of Y chromosome profiling• Cannot differentiate between DNA from fathers and sons, or from male siblings• Y chromosome does NOT undergo recombination, so less genetic diversity exists• Two unrelated males may have the same Y profile if they share a distant male ancestor Y-chromosome profiles useful in genealogy studies and for missing persons

Hardy-Weinberg law—three additional consequences:

• Dominant traits do not necessarily increase from one generation to the next • Genetic variability can be maintained • By knowing frequency of one genotype, frequencies of other genotypes can be calculated (by this equation):p2 + 2pq + q2 = 1 Since genotypes (AA, Aa and aa) = p2, 2pq, and q2 Also remember there is a difference between "genotype frequency" and "allele frequency":• p = frequency of allele A• q = frequency of allele a

Hardy-Weinberg model assumes

• Equal rate of survival and reproduction (no selection)• No new alleles arise or created by mutation• No migration into or out of population• Infinitely large population• Random mating occurs Again, all of this rarely exists for a population.

The Prosecutor's Fallacy

• Equates guilt with numerical probability derived from one piece of evidence• Human error, contamination, or even deliberate tampering must be taken into account• D N A profiles must be interpreted in context of all evidence in case

Retroviruses as vectors:

• First viruses used for gene therapy were a family of murine retroviruses called Moloney murine leukemia virus• Replication and disease-causing genes are removed from retrovirus• Recombinant viral vector with therapeutic genes infects patient's cells

Health and Safety

• G M foods have been consumed for over 20 years• [Some] toxicity testing in animals shows no negative effects (quote as per textbook)... however there is much conflicting data to this statement in the documentary Genetic Roulette along with the reasons such data gets suppressed and often does not make it into textbooks as readily... see this link again, for example! )• In fact, OTHER textbooks (such as a more recent edition of Campbell's Biology) ARE INCLUDING the newer-unveiled data on RoundUp's carcinogenicity. I had personally found papers on that starting in 2013, but the data did not make it into a single textbook to my knowledge, until 2018!• [Some] short-term toxicity studies in animals are considered "well established methods" for detecting toxins

Homologous Genes

• Genes that are evolutionarily related• Similarity searches are able to identify homologous genes • Orthologs• Genes from different species thought to have descended from common ancestor • Paralogs• Homologous genes in same species

Heritability Estimate

• Gives proportion of phenotypic variation attributed to genetic variation within a certain population in particular environment • Example: Heritability mean of 0.65 human height• 65% of overall variation in height is due to genotypic difference in individuals• Not "65% of the trait in one individual is due to genes"

Floral Meristem

• Group of undifferentiated cells that gives rise to flowers• Each flower consists of four organs that develop from concentric rings of cells within meristem:• Sepals, petals, stamens, carpe

H I V is a lentivirus

• H I V could be used as a gene therapy vector• Any vector must be genetically engineered to be inactive• H I V infects T lymphocytes so would be a good vector for delivery of therapeutic genes into the bloodstream

Huntington disease (HD)

• Inherited as autosomal dominant• Affects development, structure, function of brain and nervous system• Results in uncontrolled movements (chorea), cognitive decline, and psychiatric disturbances• Affects 1 in 10,000 people; symptoms appear ages 30 - 50• Death occurs within 10 years of onset Monogenic and 100 percent penetrant Nearly all analytical approaches developed in molecular genetics have been successfully applied to the study of HD• Validates its significance as a model for all the neurodegenerative disorders

Stabilizing Selection

• Intermediate types are favored• both extreme phenotypes are selected against • Reduces population variance over time but not the mean • Example: Human birth weight study over an 11-year period

Adoptive cell transfer (A C T)

• Involves removing TIL s from a patient's tumor• Selecting those that specifically recognize tumor antigens• Amplifying these specific T I L s in vitro• Reintroducing them back into the patient• Promising results of clinical trials on: • Metastatic melanoma, Breast cancer, Some blood cancers

Nonlethal Recessive Alleles:

• Outcome of different degrees of selection against nonlethal recessive allele a • Intensity of selection varies (see next slide):• Strong (red curve) to weak (blue curve)• Intermediate values (yellow, purple, and green curves)

Biolistic Method

• Physical method of introducing D N A to cells• Particles of heavy metals (i.e. gold) are coated with the D N A that will transform the cells• These are introduced to cells in vitro using a gene gun• Plants with gene are selected for the desired phenotype

Selectable Markers

• The rates of successful T-D N A integration and expression are low (lower than laypeople usually assume)• Only 1 in 1000 cells is successfully transformed• So... HOW do genetic engineers PICK OUT the desired cells...? Especially if they do not "look different"??• Selectable markers allow scientists to distinguish between transformed and nontransformed products (colonies, plants, tissues, etc.)......Very often, these markers are antibiotic resistance genes. And yes, this does mean antibiotic resistance genes are going to come WITH the "gene of interest." If this seems like an obvious source of controversy, it indeed is.

attenuation mechanism is common to several operons... for the "synthesis from scratch" for amino acids such as:

• Threonine • Histidine • Leucine • Phenylalanine

D N A Phenotyping

• Uses D N A sequence information to reveal a person's physical features and ancestral origins• Can predict a person's eye, hair, and skin colors based on their D N A S N P patterns• Some algorithms claim to predict 3-dimensional facial structures which allow them to compile full-color photographic representations of a person's face, based only on their D N A sample

Agrobacterium-Mediated Technology

• Utilizes bacteria Agrobacterium tumefaciens, a soil microbe that infects plant cells and causes tumors• These characteristics are conferred by Ti plasmid• After Agrobacterium infection, the Ti plasmid integrates a segment of D N A (T-D N A) into plant genome• Scientists remove T-D N A and replace with cloned D N A of genes desired Ti plasmid of Agrobacterium Agrobacterium tumefaciens inserts T-D N A portion of plasmid into host cell's nuclear genome and induces tumors

Bacterial artificial chromosomes (B A Cs) and yeast artificial chromosomes (Y A Cs)

• Vectors used to clone large fragments of D N A • B A Cs are generally very large but low copy number (one to two copies/bacterial cell) plasmids • Y A Cs have telomeres at each end, O R I and centromere

Mutation creates new alleles in a gene pool

• Within a population, the gene pool is reshuffled each generation • Mutation is the only process that creates new alleles in gene pool • Most mutations are recessive• Indirect methods using probability and statistics or large-scale screening programs are often employed to estimate mutation rates

Epigenetics and cancer

●DNA hypomethylation turns on genes, leading to high transcription of many gene sets including oncogenes (genes that predispose to cancerous processes) ●Hypermethylation at CpG islands and inactivation of certain genes are also found in many cancers (but of course the specific genes affected are different than those affected by HYPOmethylation) •Including tumor-suppressor genes (genes that would protect against cancerous processes)

Epigenetics and cancer

●Hypomethylation is a property of all cancers examined to date (it makes sense because... overall, hypomethylation means "too much gene activity") ●In the 1980s Feinberg and Vogelstein observed that colon cancer cells had much lower levels of methylation than normal cells derived from the same tissue ●But keep in mind, for some complex diseases, there are strong links to some environmental factors, such as smoking and lung cancer

Epigenetics and Assisted Reproductive Technologies (ART)

●Imprinted genes play major roles in controlling growth during embryonic and prenatal development ●External or internal factors that disturb the epigenetic pattern of imprinting or the expression of imprinted genes can have serious phenotypic consequences •In vitro fertilization (IVF) in humans can cause problems with imprinted genes •Children born after IVF and other ART procedures are at risk of very low birth weight

Epigenetics and Imprinting

●Most imprinted genes encode growth factors or other growth-regulating genes ●Most imprinting disorders have their origins during fetal growth and development •Prader-Willi syndrome, Angelman syndrome, and Beckwith-Weidemann syndrome ●BWS is a disorder of imprinting •Caused by abnormal patterns of DNA methylation resulting in altered patterns of gene expression

Epigenetics and Random Inactivation of the X Chromosome

●Random inactivation of an X chromosome happens in cells of female mammals ●About half of embryonic cells randomly inactivate the maternal X chromosome and the other half inactivate the paternal X chromosome •Effectively silencing almost all the 900 or so genes on whichever homolog is inactivated ●Once inactivated, the same X chromosome remains silenced in all cells descended from this progenitor cell

Epigenetics and Imprinting

●Reprogramming occurs in the parental germ line and in the developing embryo just before implantation ●After implantation, differential genomic remethylation recalibrates which maternal and paternal alleles will be inactivated

Environmental Induction of Epigenetic Change

●The clearest evidence for the role of environmental factors comes from studies in experimental animals: ●A reduced protein diet fed to rats during pregnancy results in permanent changes in the expression of several genes in the F1 and F2 offspring •Variable expression of yellow phenotype (and associated obesity) in mice caused by diet-related epigenetic changes in the genome

Four classes of lncRNA loci

(a) Antisense lncRNA genes usually partially overlap protein-coding genes, have their own promoter, and are transcribed in the opposite direction. (b) Intronic lncRNA genes are contained in introns within protein-coding genes. (c) Bidirectional lncRNA genes have no sequence overlap with protein-coding genes, but use the promoter of an adjacent protein-coding gene and transcribe in the opposite direction. (d) Intergenic lncRNA genes are completely independent loci, do not overlap with protein-coding genes, and use their own promoters.

Cas9-mediated cleavage of target DNA

(a) Cas9 is guided by the crRNA/tracrRNA duplex to target sequences in the viral genome based on complementarity to the crRNA and the presence of a PAM sequence on the noncomplementary target DNA strand. The HNH domain of Cas9 cuts the complementary strand, while the RuvC domain cuts the other strand. (b) The CRISPR-Cas9 system has been simplified for use as a genome-editing tool by linking together a target-specific crRNA and a portion of the tracrRNA into a single guide RNA (sgRNA).

Steps in the regulation of p53 levels and activity

(a) In normal unstressed cells, p53 is kept inactive and at low abundance. The presence of ubiquitin promotes p53 degradation. (b) After various types of cellular stress, cellular kinases add phosphates (P's) to serines and threonines in the TAD, leading to dissociation of MDM2 and subsequent loss of ubiquitin. As the levels of p53 increase in the nucleus, acetyl transferases add acetyl groups (A's) to lysines in the CTD, which increases p53 stability and affinity for specific DNA sequences within the promoter regions of target genes. Examples of genes that are transcriptionally stimulated by p53 are p21 (leading to G1/S cell-cycle arrest), BAX (stimulating apoptosis), GADD45 (contributing to DNA repair), and MDM2 (returning p53 to an inactive and low abundance state).

QTLs

(a) Individuals from highly divergent lines created by artificial selection are chosen from generation 25 as parents. (b) The thick bars represent the genomes of individuals selected from the divergent lines as parents. These individuals are crossed to produce an F1 generation (not shown). An F2 generation is produced by crossing members of the F1. As a result of crossing over, individual members of the F2 generation carry different portions of the P1 genome, as shown by the colored segments of the thick bars. DNA markers and phenotypes in individuals of the F2 generation are analyzed. (c) Statistical methods are used to determine the probability that a DNA marker is associated with a QTL that affects the phenotype. The results are plotted as the likelihood of association against chromosomal location. Units on genetic maps are measured in centimorgans (cM), determined by crossover frequencies. Peaks above the horizontal line represent significant results. The data show five possible QTLs, with the most significant findings at about 10 cM and 60 cM.

(a) Microinjecting DNA into a fertilized egg to create a knockout (or

(a) Microinjecting DNA into a fertilized egg to create a knockout (or a transgenic) mouse. A fertilized egg is held by a suction or holding pipette, and a microinjection needle delivers cloned DNA into the nucleus. (b) On the left is a null mouse (-/-) for both copies of the obese (ob) gene, which normally produces a peptide hormone called leptin (which suppresses appetite when it is functional). The mouse on the right is wild type (+/+) for the gene. The knockout mouse weighs almost five times as much as its wild-type sibling. Mutations in the human LEP gene can create weight disorders for affected individuals BUT it is SO important to note that this does not account for the majority of cases of weight disorders! This is just one "clearer" cause to us (out of the many, MANY total causes)

Four models for lncRNA mechanisms of action

(a) decoy model. lncRNA-binding sites compete to prevent transcription. (b) adapter model. lncRNAs act as a platform proteins to form active DNA-binding protein complexes. (c) guide model. lncRNAs recruit protein complexes (e.g., chromatin modifiers) and guide them to specific loci. (d) enhancer model. lncRNAs form enhancer-like loops to regulate gene action

Histone Modification 2

(c) Shows just one way chromatin can get remodeled (components of the core nucleosome particle may be rearranged, such as by swapping in and out variant histone proteins). (a) The DNA-histone contacts may be loosened, allowing the nucleosomes to slide along the DNA, exposing DNA regulatory regions. (b) The path of the DNA around a nucleosome core particle may be altered.

Expression Platform

(the terminator structure)

Analysis of Quantitative Trait

- Example: Tomato weight- Distribution of F1 and F2 derived from theoretical cross

When exposed to carcinogens, it most / more often takes ______ to develop cancer.

5+ years or even as long as 40+ years

Expression Q T Ls (eQTLs)

Expression Q T Ls (e Q T Ls)• Important tool in identifying genes that cause complex diseases

Discoveries by ENCODE include:

More than 80 percent of human genome contains regulatory elements• (once considered to be "junk DNA")... but we do not believe this anymore! • (Just one example of how "conclusions in science can change")

Molecular Clocks

Molecular clocks• Measure rate of evolutionary change• Measured in terms of amino acid or nucleotide sequences; evolutionary changes accumulate at a [relatively] constant rate over time• These rates are not exact however, as more recent evidence has shown• Example: Molecular clock showing divergence times for humans and other vertebrates compared with fossil record

T. rex samples

Molecules extracted from tissue• Liquid chromatography and mass spectrometry used to determine protein samples contain collagen protein• This was HIGHLY controversial when discovered, as it was (until then) commonly accepted that proteins are too fragile to withstand fossilization processes.• After the discovery, contamination was highly suspected as a cause for the finding of collagen in the samples.• Subsequent studies such as the below attempted to find out if / how preservation mechanisms may have been possible

What specific nutrient must be ABSENT in order for the Lac operon to be expressed optimally?

glucose

Nonsense-mediated decay (NMD)

(NMD), the most thoroughly studied mRNA surveillance pathway, efficiently eliminates mRNAs with premature stop codons Are degraded

Lentivirus Vectors Used for Gene Therapy Delivery

Lentivirus vectors are used for gene therapy delivery• Lentivirus is a retrovirus that accepts large pieces of genetic material• Can infect nondividing cells• Insertional mutation rate is lower because the virus does not gravitate toward gene-regulatory regions

Repressible System

Presence of specific molecule inhibits gene expression • Abundance of end product in environment represses gene expression (a type of negative feedback)

Isolating Mechanisms

Prezygotic isolating mechanisms• Prevent individuals from mating in the first place Postzygotic isolating mechanisms• Create reproductive isolation even when members of two population mate with each other (i.e. non-compatible egg/sperm receptors, non-viable embryo etc)

Model Organisms

Primary model organisms used for study of development:• Drosophila melonogaster (fruit fly)• Caenorhabditis elegans (nematode)• Arabidopsis thaliana (flowering plant)

P C R requires two primers:

Primers: Short, single-stranded sequences, one complementary to 5′ end and another complementary to 3′ end • dsDNA to be cloned is put in tube with D N A polymerase, Mg2+,(this is one of the functions of dietary magnesium, too- making new DNA!) and d N T Ps • Primers anneal to denatured D N A • Complementary strands are synthesized by heat-stable D N A polymerase

Northern blot

RNA sequence

The Mean

The mean: • Provides information on where central point lies along range of measurements• Gives quantitative trait • Mean (X) is the arithmetic average of set of measurements • Variance—average squared distance of all measurements from mean; provides information about spread of data around mean

The C R I S P R-Cas Mechanism

The mechanism of CRISPR-Cas adaptive immunity in bacteria. CRISPR loci contain spacer sequences derived from viral DNA separated by repeats, and a leader sequence. Nearby cas genes encode proteins that are also involved. The CRISPR-Cas mechanism has three steps: (1) spacer acquisition (viral DNA is inserted into CRISPR loci), (2) crRNA biogenesis (CRISPR loci are transcribed and processed into short crRNAs), and (3) target interference (viral DNA is cleaved by crRNA-guided Cas proteins

Alternative Splicing

The number of proteins cell can make (proteome) is not directly related to number of genes in genome • At least two-thirds of protein-coding genes in humans undergo alternative splicing.

Mass spectrometry (MS) fragmentation patterns for a peptide sequence from T. rex.

The peptide sequence, contains a charged hydroxyl group characteristic of collagen. Mass spectrometry of a synthetic version of collagen peptide shows good alignment with the m/z spectra for fragmented ions from the T. rex peptide, thus confirming the T. rex sequence as collagen and demonstrating the value of MS techniques.

c D N A Library

c D N A library is constructed by: • Isolating m R N A from cells • Synthesizing complementary D N A using reverse transcriptases • Cloning c D N A molecules into vector

qPCR

can quantify amplification reactions as they happen in real time

Classical regulatory mechanisms

cannot fully explain how some phenotypes arise Epigenetic changes lead to phenotypic changes throughout the organism's life cycle The phenotype of an organism is the product of interactions between the genome and the epigenome (hatched areas). The genome is constant from fertilization throughout life, but cells, tissues, and the organism develop different epigenomes as a result of epigenetic reprogramming of gene activity in response to environmental stimuli. These reprogramming events lead to phenotypic changes throughout the life cycle

pluripotent

cells that can form the different cell types that will be present in an adult

Epigenetics often involves _______

changes in gene expression Correct! methylation of DNA Correct! histone modifications

Normal frequency distribution,

characterized by a bell-shaped curve.

This is a multiple-answer question: You must pick all correct answers, and no incorrect answers, for full credit: Pick all examples of postzygotic mechanisms of reproductive isolation:

hybrid nonviability hybrid sterility

D N A ligase

joins restriction fragments covalently to produce intact D N A molecules

A Profile Probability Calculation Based on

of Five STR Loci

What is true of two different species?

the two species are reproductively isolated

A pangenome attempt acknowledges that ______

there are gene variations within a species

Western blot

protein

Activators and repressors are examples of _______

proteins

q2

q2 frequency of the genotype aa

Ras Proto-Oncogene

ras genes: Examples of proto-oncogenes Some of the most frequently mutated genes in human tumors Encode signal transduction molecules associated with cell membrane Regulate cell growth and division

80% of the human genome is composed of sequences that code for ______

regulatory elements

The technique of selective / natural breeding falls within the definition of ______

biotechnology

Aptamer

(ligand binding site)

Hox genes

Action of maternal and segmentation genes defined by action of homeotic (Hox) genes

Aptamers and Expression Platform

All riboswitches posses metabolite-sensing RNA sequence • Allows transcription of RNA to proceed or not

Cancer now viewed as

Cancer is now viewed as a disease that involves both epigenetic and genetic changes that lead to alterations in gene expression

CRISPR-Cas9 genome editing in cultured mammalian cells.

Cas9, with nuclear localization signals (NLSs), and an sgRNA are expressed from plasmids in mammalian cells. The sgRNA guides Cas9 to cleave a target site adjacent to a PAM sequence. The double-stranded DNA break can be repaired by NHEJ, which introduces insertions or deletions (indels), or by HRR. The latter pathway can make specific edits using an introduced donor template.

Domains

Characteristic domains of DNA-binding proteins: • Helix-turn-helix (HTH) • Present in both eukaryotic and prokaryotic transcription factors • Basic leucine zipper (bZIP) • Allows for protein-protein dimerization • Zinc-finger- • Found in wide range of transcription factors that regulate gene expression

Human Genome Project (H G P)

Coordinated effort to sequence and identify all genes of human genome• Illustrated that humans and all other species share a common set of genes essential for cellular function and reproduction

Southern blot

DNA sequence

Fraternal Twins

Dizygotic twins

Overview of Drosophila Development

Early stages of development in Drosophila:• Egg is fertilized• After tenth division, nuclei migrate to periphery of egg• Germ cells are formed• Nuclei become enclosed in membrane (blastoderm)• Segmentation pattern established

A B O Blood Type

Example of genotype frequency calculations for multiple alleles:A B O blood type

Eyeless Gene

Eyeless gene is part of a seven-gene network• Is the master regulator of eye formation• Eyeless gene expression in cells normally destined to form a leg will result in eye formation on the leg of the fly (see next picture!)

Macroevolution

Genetic changes that result in reproductive isolation between or among populations • Leads to formation of new species (or even larger groups / taxons)

Human Genetic Disorders

Humans and most vertebrates have four clusters of Hox genes• HOX A, HOX B, HOX C, and HOX D, containing 39 genes• Cluster of two to four Hox genes is involved in forming specific structures

Posttranscriptional regulation:

In eukaryotes there are many opportunities for the regulation of gene expression after transcription

Polygenes

Low numbers of polygenes (n)• Number of additive loci can be estimated from total number of possible phenotypes (2n + 1) = number of distinct phenotypic categories observed

Trans-acting elements:

Molecules (like proteins) that bind the cis-acting sites

Epigenetics and Imprinting

Once a gene has been methylated and imprinted, it remains transcriptionally silent during embryogenesis and development Most imprinted genes direct aspects of growth during prenatal development • In mice, genes on the X chromosome are expressed in the placenta, but genes on the paternal X chromosome are silenced

Transformation

Plasmids may be introduced into bacteria via transformation; Two main techniques: 1. Using calcium ions and brief heat shock to pulse D N A into cells 2. Electroporation: A brief but high-intensity pulse of electricity to move D N A into bacterial cells

Types of Selection

Selection for traits classified as:• Directional selection• Stabilizing selection• Disruptive selection Directional selection:• Phenotypes at one end of a spectrum become selected for or against • Usually as a result of changes in environment • Example: Beak size in finches during dry years increased due to strong selection

Animal Models

Successful RNAi treatment in animal models • Viral infection • Eye diseases • Cancers • Inflammatory bowel disease Science of RNAi holds powerful promise for molecular medicine

Palindrome

Symmetry exhibited by the recognized sequences• Nucleotide sequence reads same on both strands • Restriction enzymes cut D N A in a characteristic cleavage pattern • Sticky ends (cohesive ends) often get produced: Fragments produced with overhangs • Blunt ends: Some fragments are produced with double-stranded ends

Environmental Induction of Epigenetic Change

To evaluate the role of environmental factors in modifying the epigenome, the diet of pregnant mice was supplemented with methylation precursors including folic acid, vitamin B12, and choline Variation in coat color in the offspring was reduced and shifted toward the pseudoagouti phenotype

Zygotic Genes

Transcribed in nuclei of developing embryo• Transcribed in specific regions in response to distribution of maternal-effect proteins• Deleterious recessive mutations in homozygotes lead to embryonic lethality

p53 Protein and Ubiquitin

Translational and posttranslational regulation :p53 protein- • p53 levels increase if cell suffers DNA damage or metabolic stress • p53 is a transcription factor that induces transcription of Mdm2 gene (ubiquitin ligase, blocks transcription) • Ubiquitin tags proteins for degradation by enzyme

By the time they are born, identical twins do not necessarily have identical genomes.

True

Vaccines

Vaccines = Stimulate immune system to produce its own antibodies against disease-causing organisms • Inactivated vaccines: Prepared from killed samples of infectious virus or bacteria • Attenuated vaccines: Live viruses or bacteria that can no longer reproduce but can cause mild form of disease Subunit vaccines• Surface proteins from virus or bacterium developed by genetic engineering• Hepatitis B virus• Causes liver damage and cancer• Surface protein cloned into yeast expression vector → purified from yeast → packaged for use as vaccine• Gardasil: Vaccine that provides immunity against human papillomavirus (HPV

Lethal Recessive Allele

When a a genotype is lethal and eliminated by death• In this case, "a" allele declines quickly at first in population• As time passes, the frequency of allele a declines ever more slowly as heterozygotes "A "a" are not selected against • So, it is difficult for selection to completely eliminate a recessive allele from a population

Predisposition to some cancers can be inherited

While most cancers result from somatic cell mutations... There do exist 50 forms of hereditary cancer (still, they account for only about 5% of all cancers) Also... (quite important): Remember that the following chart on the next slide demonstrates known inherited mutations that merely predispose to cancer: Even though it increases the risk of cancer, it does not 100% mean the person will get cancer

What is a "population" as defined in biological terms?

a group belonging to the same species in a particular geographic area

CRISPR loci help with adaptive immunity in bacteria.

adaptive immunity

Which of the following may be mutations / mutation results implicated in cancers?

aneuploidy Correct! translocations

Which of the following is a method of gene expression regulation for eukaryotes, but not prokaryotes?

chromatin remodeling acetylation of histones polyadenylation of mRNA all of the other answers are true - CORRECT

In Huntington disease, the HTT gene, which is mutated, has ______ compared to the normal gene.

extra CAG repeats

Arabidopsis thaliana

flowering plant

What is the term associated with the acknowledgement that there are gene variations within a species?

pangenome

RT-PCR

uses reverse transcriptase to make cDNA to study gene expression

Antiterminator hairpin:

Forms in absence of tryptophan (forms because the ribosome "stalls" waiting for tryptophan to insert into a growing peptide but tryptophan is not there)

Familial adenomatous polyposis (F A P):

Genetic disposition to cancer One mutant copy inherited of A P C gene on long arm of chromosome 5 Mutations include deletions, frameshift, and point mutations

Additive variance

Genotypic variance due to additive action of alleles at quantitative trait loci

C C D

Human disorder• Result of mutation in human C B F A gene• Homolog of runt (a pair-rule gene in Drosophila)• Characterized by numerous skeletal defec

Lac Operon Structural Genes

Lac operon structural genes = lac Z, lac Y, and lac A

Identical twins

Monozygotic twins

For which species has a synthetic genome been accomplished?

Mycoplasma genitalium (bacteria)

response of the lac

The response of the lac operon in the absence of lactose when a cell bears either the I− or the OC mutation

stabilizing selection

both extremes are selected against

Huntington disease is ____

autosomal dominant

What process creates new alleles?

mutation