MCB 250 Exam 4 Learning Objectives

Understand how changes to enhancer regions can lead to changes in form.

Changes in enhancer sequences can lead to changes in gene expression, which can in turn lead to changes in form and function of an organism. For example, changes to enhancers of genes involved in limb development can lead to the evolution of different limb structures in different animal species.

Be able to describe the fruit fly life cycle

The fruit fly life cycle involves a series of developmental stages: egg, larva, pupa, and adult.

Know why one gene therapy trial went awry

A patient named Jesse Gelsinger died from an immune reaction to the viral vector that was used to deliver the therapeutic gene. This tragic event led to increased safety regulations and more rigorous clinical trials for gene therapy.

Know how to make transgenic Drosophilia

1. Design a plasmid containing the gene of interest, a promoter to drive expression, and sequences recognized by the transposase enzyme. 2. Inject the plasmid DNA into early-stage Drosophila embryos. 3. Allow the injected embryos to develop into adult flies and screen them for the presence of the transgene. 4. Cross transgenic flies with wild-type flies to generate a stable line of transgenic flies that carry the gene of interest in their genome. Transposons, such as P elements, can increase the efficiency and specificity of transgene integration in the fly genome. Transgenic Drosophila are useful for studying gene function and regulation.

Be able to name three examples of genetically modified crops/livestock that benefit the food supply/human health and how those modifications were made

1. Golden Rice: Scientists inserted genes from daffodils and a bacterium into rice to enable it to produce beta-carotene, which the body can convert to vitamin A. 2. Bt Cotton: Scientists inserted a gene from the bacterium Bacillus thuringiensis (Bt) into cotton, which produces a protein toxic to certain pests that feed on the plant. 3. AquAdvantage Salmon: Scientists inserted a growth hormone gene from Chinook salmon and a promoter from ocean pout into Atlantic salmon, which results in faster growth rates and reduced feed requirements.

Be able to name two medical conditions that can be corrected with gene therapy, and know how they are corrected•

1. Severe combined immunodeficiency (SCID): a genetic disorder that affects the immune system. Gene therapy can be used to replace the defective gene that causes SCID with a functional copy, which can restore the immune system. 2. Leber congenital amaurosis (LCA): a genetic disorder that causes blindness. Gene therapy can be used to deliver a functional copy of the defective gene that causes LCA to the retina, which can restore vision.

Know what a reporter gene is and how to use reporter transgenes to identify tissue-specific enhancers

A reporter gene is a gene that encodes a protein whose activity or expression can be easily monitored, such as GFP or LacZ. Reporter transgenes can be used to identify tissue-specific enhancers by fusing them to a minimal promoter and inserting them into the genome, and then analyzing the expression pattern of the reporter gene in transgenic animals.

Know what a transposon is and how it's different from a virus

A transposon, or transposable element, is a DNA sequence that can move from one location in the genome to another. It differs from a virus in that it does not require a host cell to replicate and is not typically associated with disease.

why are mRNAs selectively spliced?

Alternative splicing allows a single gene to produce multiple mRNA isoforms by including or excluding certain exons during RNA splicing. This allows for greater diversity in the proteome, as different protein isoforms may have different functions or properties.

Know how the trp operon is regulated by attenuation

Attenuation involves a leader sequence in the mRNA. If tryptophan levels are low, the ribosome translating the leader sequence slows down, allowing a region of the mRNA called the attenuator to form a hairpin loop. This loop acts as a terminator, preventing transcription of the rest of the operon. If tryptophan levels are high, the ribosome doesn't slow down, and the attenuator doesn't form the hairpin loop, allowing transcription to continue.

Know the structure of the CRISPR locus and what it encodes

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a type of regulatory RNA found in bacteria that encodes for a system of adaptive immunity against invading viruses or plasmids. The CRISPR locus is composed of a series of short repeats separated by variable spacers. The CRISPR locus also encodes for associated proteins, such as Cas (CRISPR-associated) proteins, that are involved in the defense mechanism.

Know what the crRNAs target, and how that targeting occurs

CRISPR RNAs (crRNAs) are regulatory RNAs that target invading viral or plasmid DNA for destruction. The crRNAs are generated from the variable spacers in the CRISPR locus and are loaded onto Cas proteins to form a ribonucleoprotein complex. This complex recognizes the invading DNA and cleaves it, rendering it inactive.

Know what Dicer does to foreign dsRNA

Dicer is an RNA endonuclease that cleaves dsRNA into small interfering RNAs (siRNAs), which are typically 21-25 nucleotides in length.

Know what Drosha and Dicer do to pri-miRNAs and pre-miRNAs, and how they recognize their targets

Drosha and Dicer are endonucleases that process primary (pri-) and precursor (pre-) microRNAs (miRNAs) into mature miRNAs, which are typically 20-22 nucleotides in length. These mature miRNAs then associate with the RISC complex to target and downregulate mRNA expression. Pri-miRNAs are cleaved by Drosha in the nucleus to generate pre-miRNAs, which are exported to the cytoplasm where Dicer cleaves them to generate mature miRNAs. Drosha and Dicer recognize the hairpin-like structure of pri- and pre-miRNAs, respectively.

Know that tissue-specific enhancers can independently regulate the same gene in different cell types:

Enhancers are regulatory elements in the DNA that can activate or repress gene expression in a tissue-specific manner. Different tissues can have different enhancers that regulate the same gene, allowing for tissue-specific gene expression.

Be able to give examples of transgenes and knockouts that have contributed to our understanding of basic biology

Examples of transgenes and knockouts include the green fluorescent protein (GFP) transgene, which revolutionized cell biology and bioimaging, and the knockout of the p53 tumor suppressor gene, which revealed its critical role in preventing cancer.

Know that different cell types express different genes:

For example, liver cells express different genes than muscle cells, because they have different jobs to do. The expression of different genes is controlled by regulatory elements in the DNA, such as enhancers and promoters, as well as by transcription factors that activate or repress gene expression.

Know what the RISC complex/Argonaut does

The RNA-induced silencing complex (RISC) is a multiprotein complex that includes the Argonaute protein family. RISC binds to siRNAs and uses them to target and cleave complementary mRNA sequences, leading to their degradation.

Know the structure and function of an IgG protein

IgG is an immunoglobulin protein that is produced by plasma cells in response to an antigen. It consists of four polypeptide chains, two heavy chains, and two light chains, and it can neutralize or eliminate pathogens in a variety of ways, including by blocking their entry into host cells, tagging them for destruction, or directly killing them.

Know the difference between maternal effect and zygotic genes

Maternal effect genes are genes that are expressed in the mother and deposited in the egg, where they play a critical role in early development. Zygotic genes, on the other hand, are genes that are expressed in the embryo itself, after fertilization. Maternal effect genes are important for establishing the initial body axes in the embryo, while zygotic genes are important for further differentiation and patterning of the embryo.

Explain one example of how mis-regulation of signaling can lead to aberrant development:

Mis-regulation of signaling pathways can lead to developmental abnormalities, such as achondroplasia, which is caused by a mutated allele of Fibroblast Growth Factor Receptor 3 (FGFR3). (Peter Dinklage has this).

How quickly do mutations accumulate over time? Are there differences in homologous genes in different species?

Mutations occur naturally and randomly in DNA sequences slowly over time. As a result, homologous genes in different species can accumulate differences over time, which can result in differences in the traits and functions of those genes.

Know that many of these genes retain the same function despite these differences (name one example)

One example of a gene that retains the same function despite differences in different species is the p53 gene, which is a tumor suppressor gene found in humans and other vertebrates. Despite some differences in the p53 gene between different species, its fundamental function of suppressing tumors remains the same

Know what P elements are

P elements are a type of transposable element found in Drosophila that can be used as a tool for creating transgenic flies. Transgenic flies can be created by introducing a transposon carrying a gene of interest into the genome of a fly using the transposase enzyme.

Know how RNAi was discovered

RNA interference (RNAi) was first discovered in the nematode Caenorhabditis elegans by Fire and Mello in 1998, who found that double-stranded RNA (dsRNA) could trigger the degradation of mRNA in a sequence-specific manner.

Know how using RNAi is experimentally different from making a knockout or generating mutants

RNAi allows for the specific silencing of a single gene or a subset of genes without affecting the entire organism or cell. Knockouts and mutants, on the other hand, typically result in the complete loss of function of the targeted gene, which can have broad-ranging effects.

Know what Rag-1 and Rag-2 do

Rag-1 and Rag-2 are enzymes that are required for V(D)J recombination. They form a complex that recognizes specific DNA sequences and introduces double-strand breaks between gene segments, which are then joined by non-homologous end joining or other repair mechanisms.

Know how scientists have harnessed CRISPR to intentionally edit genomes

Scientists have harnessed the CRISPR system to intentionally edit genomes by designing a guide RNA (gRNA) that directs the Cas enzyme to the desired target site in the DNA, where it cleaves the DNA and initiates DNA repair mechanisms. This process can be used for gene knockouts, gene insertions, or precise gene editing.

Know the distinction among siRNAs, miRNAs, and piRNAs and what their different roles are

SiRNAs and miRNAs are both involved in post-transcriptional gene regulation through the RNAi pathway, but they differ in their biogenesis and mechanisms of action. SiRNAs are derived from exogenous dsRNA, whereas miRNAs are endogenous and are generated from pri- and pre-miRNAs. PiRNAs are a distinct class of small RNAs that function in germline development to silence transposable elements.

Understand JAK/STAT signaling:

The JAK/STAT pathway is activated by cytokines and growth factors, which are extracellular signaling molecules that bind to cell surface receptors. This triggers a cascade of events that results in the activation of the transcription factor STAT, which can then enter the nucleus and activate or repress the expression of specific genes. The JAK/STAT pathway plays an important role in immune responses, cell proliferation, and cell differentiation.

Understand MAPK signaling

The MAPK pathway is activated by a variety of extracellular signals, including growth factors and stress signals. This pathway involves a cascade of protein kinases that ultimately lead to the activation of transcription factors in the nucleus. The MAPK pathway regulates a wide range of cellular processes, including cell differentiation, survival, and proliferation.

Know how the AP axis of a Drosophila embryo is first established - How were the key genes identified? - How was the mechanism of mRNA localization identified?

The anterior-posterior (AP) axis of a Drosophila embryo is first established through a series of genetic interactions involving key genes, including bicoid, nanos, hunchback, and caudal. -These genes were identified through genetic screens and molecular cloning techniques. -The mechanism of mRNA localization was also identified through experiments involving RNA labeling and imaging techniques.

Know the axes of an organism: dorsal, ventral, anterior, posterior

The axes of an organism refer to its major body planes: dorsal (back), ventral (belly), anterior (head), and posterior (tail).

Know how the DV axis of a Drosophila embryo is established -How is a TF gradient converted into bands of target gene expression? -What genetic and molecular experiments demonstrated the regulatory relationships among the genes?

The dorsal-ventral (DV) axis of a Drosophila embryo is established through the formation of a morphogen gradient of a transcription factor called Dorsal. -This gradient is converted into discrete bands of target gene expression through the action of other transcription factors, such as Twist, Snail, and Decapentaplegic. -These regulatory relationships were demonstrated through genetic and molecular experiments, including loss-of-function and gain-of-function analyses.

Know how one genome generates an IgG population capable of recognizing up to billions of different antigens

The genome of a B-lymphocyte undergoes a process called V(D)J recombination, which involves the random recombination of V, D, and J gene segments that encode the variable region of the antibody. In addition, the insertion of nucleotides at the junctions between these segments can further increase the diversity of the antibody repertoire.

Understand the steps of transposition

The steps of transposition are as follows: transposase enzyme recognizes and binds to the transposon sequence, cleaves the DNA at the insertion site, the transposon is excised and moved to a new location in the genome, and the DNA is repaired at the original insertion site.

Know how to make a targeted gene knockout in mice

To make a targeted gene knockout in mice, scientists can use homologous recombination in embryonic stem cells to replace a specific gene with a selectable marker, select for cells that have undergone recombination, inject the cells into a blastocyst, and implant the blastocyst into a foster mother. The resulting chimeric mice can be bred to produce homozygous knockout mice that lack the target gene.

Know how to make a transgenic mouse when insertion site does not matter -Know how location of insertion might affect the phenotype

To make a transgenic mouse, scientists inject a DNA construct containing the gene of interest and a promoter into a fertilized mouse egg, implant the egg into a foster mother, and screen the offspring for the transgene.

Know that cell-specific gene expression is usually regulated by the specific transcription factors in those cells:

Transcription factors are proteins that bind to DNA and regulate the expression of specific genes. Different cell types express different transcription factors, which bind to specific regulatory elements in the DNA to activate or repress the expression of specific genes.

Know why scientists generate transgenic organisms

Transgenic organisms are generated to study gene function and regulation, model diseases, and produce beneficial traits.

Know how transposition is similar to and different from site-specific recombination

Transposition is similar to site-specific recombination in that both involve the movement of DNA sequences within the genome. However, site-specific recombination involves specific DNA sequences recognized by recombinase enzymes, while transposition involves the recognition and movement of transposon sequences by transposase enzymes.

Know how transposons have shaped the human genome

Transposons have played a significant role in shaping the human genome, accounting for roughly 45% of the total genome. They can cause mutations, alter gene expression, and even create new genes through the process of exon shuffling.

Who discovered transposons

Transposons were discovered by Barbara McClintock in her studies of maize, for which she was awarded the Nobel Prize in 1983.

Name and describe two ways in which a developing embryo can start turning on different genes in different cells; give one example of each:

Two ways a developing embryo can turn on different genes in different cells are spatial regulation (where different genes are activated in different regions of the embryo) and temporal regulation (where different genes are activated at different times during development).

Know how a pool of lymphocytes that recognizes millions-billions of antigens adapt to respond to an infection

When an antigen is encountered, B-lymphocytes that produce antibodies that recognize the antigen undergo clonal expansion and differentiation into plasma cells, which secrete large amounts of the antibody. In addition, T-lymphocytes that recognize the antigen are activated and differentiate into effector cells that directly kill infected or abnormal cells. This allows the immune system to mount a highly specific response against the pathogen.

Understand how the CRISPR locus changes to adapt to new threats to the bacterium

by acquiring new spacers through a process called spacer acquisition. During spacer acquisition, a small fragment of the invading DNA is integrated into the CRISPR locus as a new spacer. The CRISPR locus can then use this new spacer to recognize and destroy future invasions by the same or similar viruses or plasmids.

How is splicing regulated?

by the binding of splicing factors, such as splicing activators and repressors, to specific RNA sequences. Splicing activators promote exon inclusion, while splicing repressors inhibit exon inclusion. The balance between these factors determines the splicing outcome of a particular pre-mRNA.

How is splicing carried out?

by the spliceosome, which recognizes specific sequences called splice sites (donor and acceptor sites) at splice junctions and removes the intron as a lariat structure.

What do duplication and divergence of genes lead to? Specifically, understand Hox genes - what they are and what they do

changes in the form of an organism. Hox genes, are a group of homeobox-containing genes that regulate the development of body plans in animals. They control the identity and position of body segments along the anterior-posterior axis of the body.

Know why Nusslein-Volhard and Wieschaus won the Nobel Prize

for their discovery of genes that control the development of body axes in the fruit fly embryo.

Can genes have alternative start sites?

genes can also have alternative start sites that lead to the inclusion of alternative first exons in the mRNA. This can affect the translation efficiency or localization of the resulting protein isoforms.

Understand how sex determination occurs in Drosophila

sex determination is controlled by the ratio of X chromosomes to autosomes. The sex-lethal (Sxl) gene is a key regulator of sex determination in females. Sxl is activated by a splicing cascade that is initiated by the ratio of X chromosomes to autosomes. In females, Sxl is expressed and promotes the inclusion of a female-specific exon in its mRNA, which leads to the production of a functional Sxl protein. In males, Sxl is not expressed, and a different splicing pattern produces a non-functional Sxl protein. This leads to the development of male-specific traits in males and female-specific traits in females.

Know how the trp operon is regulated by trp repressor

the trp repressor is a protein that binds to the operator region of the operon in the presence of tryptophan, an amino acid. This prevents RNA polymerase from transcribing the genes in the operon, thus reducing the production of enzymes involved in tryptophan biosynthesis. When tryptophan levels are low, the trp repressor is not bound to the operator, allowing RNA polymerase to transcribe the operon and produce the enzymes necessary for tryptophan biosynthesis.

What are most genetically modified organisms used for?

they are used in laboratory research

Know why the choice of vector and promoter are critical to gene therapy

they determine the efficiency, specificity, and safety of gene delivery. The vector is the carrier that delivers the therapeutic gene to the target cells, while the promoter is the switch that turns on the expression of the therapeutic gene. The choice of vector and promoter depends on the specific disease and the target cells, and must be carefully optimized to avoid unintended side effects.

are B- and T-lymphocytes involved in adaptive immunity?

yes. B-lymphocytes produce antibodies that recognize foreign substances called antigens, while T-lymphocytes directly kill infected or abnormal cells.