Unit 1

Question 1

Bacterial gene expression and regulation

Answer 1

Bacterial genes occur in clusters controlled by a single promoter (aka operons) which are generally functionally related. Their polycistronic mRNA gives rise to multiple proteins. Organized with polarity (promoter proximal gene expression influences promoter distal genes).

Translation and transcriptional coupling exploited as a means of gene expression - ex: attenuation of trp operon. Low levels of tryptophan result in stalling at leader region, causing formation of a secondary structure that does not act as a terminator. High levels of tryptophan do not produce stalling, resulting in fromation of secondary structure that does act as a terminator.

Gene expression is regulated by sigma factors, which interact with DNA elements at -10 and -35 to define which sequences act as a promoter. Different sigma factors recognize different sequences allowing for differential expression under different conditions.

Positive and negative regulators control gene expression. Location of binding can inform you of activity. Pos regulators generally bind adjacent to promoter binding site to recruit RNA polymerase. Neg regulators generally bind between promoter binding site and genes to block RNA polymerase.

Gene regulation differences between prokaryotes and eukaryotes:

• Prokaryotes use sigma factors for promoter specificity
• Proteins have short half-life so bacteria can readily adapt to their environment
• Transcription and translational coupling is exploited for control of gene expression

Question 2

Common forward and reverse genetic techniques used in zebrafish

Answer 2

Mutagenesis

• chemical (ENU) to introduce point mutants
  
  • males mutagenized & crossed with female. Mutations passed on in heterozygous form. Can screen for dominant mutations in F1
• irradiation to introduce large deletions
• insertion via retrovirus or transposon to add small insertions
  
  • inject virus into blastula-stage embryos & raise/outcross founders. Identify transgenics in F1 pools by PCR and Southern blot. Can outcross to make F2 generation if need more fish with a given insertion. Inbreed fish with identical insertions and screen progeny

Morphological screens - Forward

• can screen behaviors like: startle/escape response, visually/autitory mediated behaviors, heart function, swimming/locomotion behavior, response to drugs
• Identify mutation location by linkage mapping (association of markers of genome that always segregate with phenotype)

Modifier screens: screen for interacting mutations in a mutant or ‘sensitized’ background

Reverse

• Loss-of-function: selected gene mutagenesis, anti-sense RNA interference, dominant negative gene induction
• Gain-of-function: overexpression of WT gene, misexpression/ectopic gene activation, constiutive active gene induction

Targeted double strand DNA break technology: ZFN, TALEN, CRISPR

Question 3

Bacterial horizontal gene transfer

Answer 3

Bacteria share DNA with eachother, which increases genetic variability.

1) Transformation: Naturally competent bacteria have a protein machine that can bind dsDNA from the environment and proccess it to ssDNA before internalizing and incorportaing into genome via homologous recombination. This process is regulated and only done when environmental conditions are favorable for their to be lysed bacteria. Ex: competence pheromone released when cell densities high (quorum sensing).

2) Conjugation: Requires donor cell to have machine apparatus carried on F plasmid, which allows DNA transfer through mating bridge. Donor cell makes a pilus that ssDNA is transferred through. Origin of transfier (oriT) binding site for nicking enzyme. Other plasmids also have oriT site and can be transfered via conjugation. If F plasmid is incorporated into chromsome via short regions of homology, the chromsome can be transferred via conjugation.

3) Transduction: Bacteriophages in lytic cycle sometimes package bacterial DNA into the capsid. When infect another cell, DNA injected and incorported into genome via homologus recombination.

Differences in DNA exchange between prokaryotes and eukaryotes:

• Bacteria exchange DNA readily to generate diversity (b/c asexual)
• Done via transformation, conjugation, & transduction
• Each increase organisms ability to surve an environemnt

Question 4

Linkage analysis

Answer 4

Linkage analysis: statistical method used to identify positions of human disease genes relative to known genetic markers

Syntenic genes: two genes located on the same chromosome

Linked genes: two genes located on the same chromosome and close enough together so that they do not segregate independetly

Crossing over/recombination: forms new combination of alleles on the same chromosome

• recombination rate is a function of physical distance between two genes
• degree of deviation from independent assortment is indicative of physical distance between two genes

Genetic markers: known DNA sequence at a known chromosomal location

Question 5

What is the mode of inheritance?

Answer 5

Autosomal-dominant

We know all genotypes in this case. Unaffected all are homozygous for normal allele. Affected are all heterozygous for mutant bc they all inherit a normal copy from unaffected parent.

Question 6

Discuss modern technologies for manipulating genomes

Answer 6

1) Zinc Finger Nucleases (ZFN)

• Artifical restriction enzyme that contains DNA-binding domain and DNA-cleaving domain. ZFN binds and cleaves DNA at specific positions.

2) Transcription activator-like effector nuclease (TALEN)

• Artifical restriction enzyme that contains Tal effector DNA-binding domain and DNA cleavage domain. Designed to bind targeted DNA sequence and cleave.

3) CRISPR/Cas9

• Engineered guide RNA targets region of DNA, where Cas9 cleaves.

Question 7

Mitochondrial inheritance

Answer 7

Mutation in mitochondrial DNA that is maternally inherited

Features:

• affected fathers produce no affected offspring
• offspring of affected mother are all affected

Question 8

Explain how to knockout a gene in specific tissues as well as all tissues of an adult mouse using conditional cre recombinase

Answer 8

Cre recombinase is fused to estrogen receptor ligand-binding domain (expressed in transgenic mouse with tissue-specific promoter).

Administration of tamoxifen binds the estrogen receptor and causes nuclear import of Cre

Once in the nucleus Cre deletes the target gene.

Question 9

Mitosis

Answer 9

Maintains chromosome number, produces identical daughter cells.

Conservative process: genotype of daughter cells is identival to parent cell

1) Interphase

• G1 phase: period prior to DNA synthesis. Cell increases in mass.
• S phase: DNA is synthesized
• G2 phase: Synthesis of proteins and cell increases size. Centrioles formed

2) Prophase
* Chromatin fibers coil into chromosomes (each chromsome has two chromatids jointed at centromere)
3) Metaphase
* Chromosomes line up at equator. Pulled by spindle fibers attached to centrioles
4) Anaphase
* Chromosomes seperate to opposite poles
5) Telophase
* Chromosomes begin to unravel back into chromatin
6) Cytokinesis
* division of parent cell into two daughter cells

Question 10

Explain monohbyrid crosses, dihybrid crosses, testcrosses, and backcrosses.

Answer 10

Monohybrid cross

• Breeding between two true-breeding parents with one contrasting trait
• Ex: spherical vs dented, green vs yellow
• Shows principle of independent segregation

Dihybrid cross

• Breeding between two true-breeding parents different by two traits
• Ex: yellow and spherical vs green and dented
• Showed independent assortment
• Will show as 9:3:3:1 if indepedently assort (unlinked)

Testcross - A- x aa

• Cross organsm wth a dominant phenotype (A-) with recessive phenotype (aa)
• Can determine if dominant organism is homozygous or heterozygous.

Backcross - F1 x P1

• Breeding of F1 organism with P1 organism
• Can be equivalent to a testcross

Question 11

What is the mode of inheritance?

Answer 11

Autosomal-recessive inheritance

We do not know the genotype of all individuals. Affected individuals must be homozygous for mutant. Unaffected individuals can be homozygous for normal or heterozygous carriers.

I1 and I2 must be heterozygous b/c they produce an affected offspring (II1, who is homozygous for mutant). II2-6 unknown but II5 more likely to be heterozygous than II6. IV1 and IV2 heterozygous.

Question 12

Bacterial DNA replication

Answer 12

Bacteria replicate their genome during the exponential growth phase via binary fission. The parent cell duplicates the genome into two chromosomes, which are separated to opposite sides before the bacterium is divided, forming two identical daughter cell clones.

Bacteria have one origin of replication containing a binding site for DnaA (encoded proximal to ori). DnaC is a helicase loader that binds at the ori. DnaB is the helicase that unwinds dsDNA, forming two replication forks advancing around circular DNA in opposite directions. The ssDNA is stabilized by Single Stranded Binding Protein (SSB). Multiple Ter regions 180º arond circle act as mechanisms of controlled termination. The replication forks stop after crossing the 180º mark - Ter regions have polarity.

Differences between prokaryotes & eukaryotes:

• Replication occurs in cytoplasm (no nucleus)
• Single origin of replication (vs many)
• Only 2 replication forks generated (vs many)
• Replication termination involves Ter sites and Tus proteins

Question 13

Reverse Genetic Strategies for the Mouse

Answer 13

1. Microinjection of DNA into male pronucleus of fertilized eggs results in multiple copies of transgene being randomly integregated throughout mouse genome
2. Homologous recombination of DNA within the genome of embryonic stem cell. Targets the alteration to very specific locus within genome
3. Targeted nuclease technologies such as zinc finger nucleases (ZFNs), transcriptional activator like effector nucleases (TALENs), and clustered regulatory interspaced plaindromic repeats (CRISPR)

Question 14

Mouse coat color genetics rediscover Mendel’s principles

Answer 14

Loci affecting coat color:

• A locus, agouti
  
  • AA, Aa = agouti; aa = nonagouti
• B locus black/brown
  
  • BB, Bb = black; bb = brown
• C locus albino
  
  • CC, Cc = color; cc = albino
• D locus depth of coat color
  
  • DD, Dd deep color; dd = light color

Study of inheritance of mouse coat color was instrumental in extending Mendel’s laws of inheritance to mammals

Question 15

Describe genetic markers that are used for the production of knockout mice

Question 16

Natural Transformation Experiment

Answer 16

S. pneumoniae have different types. R = rough and S = smooth. Smooth types have exopolysaccharide capsules that are virulence factors (kill mice), whereas rough type is nonvirulent.

Investigator mixed R and heat-killed S strain and inoculated a mouse. Mouse unexpectedly died. Bacteria isolated from dead mouse were type S. Type R was transformed into type S.

Was this due to DNA or protein? (DNA was not known to be genetic material. Refined version fractionated type S and isolated DNA and proteins. Seperate samples of DNA and protein were added to R cells. Only DNA samples transformed R cells to S cells. DNA = genetic material.

Question 17

Y-linked disorders

Answer 17

Affected gene is located on the Y-choromosome.

Features:

• observed only in males
• follows inheritance of the Y chromosomes (if a male has the disease, so does his father and paternal grandfather and his sons and their sons)

Question 18

What is the mode of inheritance?

Answer 18

X-linked recessive

All the dauthers in row II are carriers b/c get X chromosome from father. All affected feamles are homozygous for mutant allele.

Question 19

X-linked dominant inheritance

Answer 19

Affected gene is located on the X-chromsome and mutant dominant. Males condiered hemizygous for all X-linked traits

Features:

• trait is never passed from father to son
• daughters of an affected male and normal female are affected
• marriage of affected female and normal male produce 1/2 affected sons and 1/2 affected daughters
• females are more likely to be affected than males

Question 20

Classification of genetic disorders

Answer 20

1) Chromosomal

• Associated with addition/loss of chromosomes (aneuploidy), deletions/duplications of chromosomal material, and translocations
• Usually sporatic event during gamete formation but can be inherited
• Deletions/duplications can be identified via whole genome copy number assays

2) Complex (polygenic/multifactorial) disorders:

• Caused by mutations at more than one gene locus or combo of genetic/environmental factors
• Appear as familial clustering or aggregation
• Lower recurrence risk than single gene disorders

3) Monogenic (single-gene), Mendelian

• Disorder determined primarily by a single mutant gene

Question 21

X-linked recessive inheritance

Answer 21

Mutant gene is located on X-chromosome and is recessive.

Features:

• disease is never passed from father to son
• passeded from an affected grandfather, through his carrier daughters, to 1/2 of his grandsons
• all affected males in a family are related through their mothers
• males are more likely to be affected than females

Question 22

3 models of replication and experimental evidence supporting one

Answer 22

1. Conservative replication: dsDNA serves as a template for entirely new daughter dsDNA.

2. Dispersive replication: new chunks of DNA randomely insert themselves leading to a mixture of new and old dsDNA in the parent and daughter cells.

3. Semiconservative replication: ssDNA serves as a template for a new strand of DNA. Daughter DNA consits of 1 parental strand and 1 new strand.

E. coli was grown in the presence of ¹⁵N, which was isotopically labeled DNA. DNA sample was isolated and fractionated - formed a band of ‘heavy’ ¹⁵N DNA. Original culture was washed of heavy medium and transfered to light ¹⁴N media. A single generation was grown. DNA sample was isolated and fractionated - formed an intermediate density thereby ruling out conservative replication. Another round of replication in ‘light’ media was allowed. DNA sample was isolated and fractionated - formed a light and intermediate band, thereby proving conservative replication.

Question 23

Advantages of zebrafish as model organisms

Answer 23

• Fast, transparent development
• Used to study vertebrate organogenesis, whole animal disease modeling, and in vivo cell biology

Question 24

Autosomal-dominant inheritance

Answer 24

Affected gene is located on one of the autosomes and mutant dominant.

Characteristics:

• Each affected individual has an affected parent
• Normal progeny of an affected individual will only have normal offspring
• Males and females are affected in equal proportions
• Each sex is equally likely to transmit the condition to male and female offspring
• Every generation tends to have an affected individual

Question 25

Mendel's Principles

Answer 25

1) Genetic traits are controlled by unit factors (alleles) that exist in pairs in individual organisms. 2) When two unlike unit factors (alleles) responsible for a single trait are present in a asingle individual, one is dominant and the other recessive. 3) **Principle of segregation:** In the formation of gametes, the paired unit factors (gene's alleles) segregate in such a way that each gamete is equally likely to contain either member of the pair. 4) **Indepdendent assortment:** During gamete formation, segregating pairs of unit factors (alleles of different genes) assor independently of each other. This allows new combinations of alleles that may not be present in either parent.

Question 26

Genetic material differences between bacteria and eukaryotes.

Answer 26

Question 27

Define the term transgenic mouse and describe how a transgenic mouse is created

Answer 27

Transgenesis: process of introducing an exogenous gene (transgene) into a living organism so that the organism will exhibit a new property and transmit that property to its offsrping. Transgenic organisms: able to express foreign genes because the genetic code is similar for all organisms. EXAMPLE OF REVERSE GENETICS 1. Production of transgenic mice via microinjection: * cDNA of interest expressed by cloning 3' to promoter element * Promoter defines tissue distribution and level of expression for transgene * Inject cDNA + promoter into male pronucleus of fertilized eggs * Causes multiple copies of transgene being randomly integrated throughout mouse genome 2. Viral infection of embryo 3. Transposons See image for how to establish a transgenic or gene knockout colony

Question 28

Define the term gene targeting and describe in detail how a knockout mouse is created

Answer 28

Gene targeting: disruption or alterations to known genes in the genome How to make a KO mouse 101: 1. Isolation and characterization of a genomic clone 2. Construction of targeting vector * Linear targeting vector constructed with positive and negative selectable markers & introduced into ES cells * Correct integration confirmed by Southern blot or PCR 3. Homologous recombination in embryonic stem (ES) cells 4. Contribution of altered allele to germ line 5. Analysis of homozygous knockout mice

Question 29

What is the mode of inheritance?

Answer 29

X-linked dominant We know all genotypes of the individuals. All unaffected feamles are homozygous for normal allele. Affected males hemizygous for mutant.

Question 30

Describe the advantages of using mice as a model genetic organism.

Answer 30

* Close evoltuionary relationship to humans as both are mammals and have conserved genomic organization * Relatively short generation time * Well adapted to lab life & easy to breed and maintain * Large litters * Tool to dissect molecular basis of embryonic development and cell function * Can be used as animal model system for human disease

Question 31

Genetic testing

Answer 31

**Genetic tests** * Single gene/region assays using Sanger sequencing or copy number analysis * Multi-gene next generation sequencing panels * Whole exome/genome sequencing analyses **Benefits** * specific genetic diagnosis * guide clinical management/monitoring recommendations * opportunity for early clinical intervention * opportunity for family planning by defining recurrence risk * facilitation of new treatment development

Question 32

Forward-Genetic Strategies for the Mouse

Answer 32

Randomly mutate genome and screen offspring for phenotype of interest. Chemical mutagens Insertional mutagenesis using viruses or transposons **Dominant Screens** * Screen G1 progeny of G0 mutagenized male for interesting phenotypes * Cross G1 heterozygous male with a WT female and determine if trait exhibits Mendelian genetics (50% of G2 mice would be heterozygous for the mutation) * Clone & identify gene of interest **Recessive Screens** * Need to mate G1 male with 3-6 of G2 daughters. Screen six progeny from each G2 female * Phenotype not present until G3 generation **Assays for mutant identification** * Want simple, rapid, and standardized assays to select the desired mutants * Can be physiological, behavioral, developmental, or regulatory process assays **Cloning gene of interest** * Can determine location via linkage analysis and more genetic crosses * Once focused on a region, sequence cDNA for genes * Should find mutation (maybe several) in these genes * To prove which mutation is responsible for the phenotype, have to switch to REVERSE genetics

Question 33

Additional factors of pedigree analysis

Answer 33

**1) Variable Expressivity** * Affected individuals may express all of the symptoms or only a few and the severity of the features may vary. AKA people with diesase associated genotypes can have variable phenotypes. **2) Incomplete Penetrance** * Person with disease-associated genotype may not express the disease phenotype (phenotype is normal). **3) Genetic anticipation** * Each generation has progressively earlier age of onset and increased severity of the disorder * Can be because expansion of trinucleotide repeats (ex: Huntington's) **4) Late onset** * Member of pedigree has late or variable age of onset. * Complicates analysis because cannot determine which indviduals of younger generation are affected. **5) Imprinting** * Certain genes are rexpressed in a parent-of-origin-specific manner. Allele of gene can be maternally imprinted or paternally imprinted and phenotype of offspring depends upon which parent transmits the mutated allele. **6) Mosaicism** * Mutation originates as a somatic mutation during embryogenesis of one parent. Two cell types are present - normal and mutant. Can be somatic (involving multiple cell types) or germline (involves only germline). **7) De novo mutations** * Mutation originates spontaneously in parental gamete. Risk to future offspring is the same as the risk of having a new mutation occur again. Trait can appear as an isolated event or a recessive characteristic but show dominant inheritance in next generation. **8) Genetic locus heterogeneity** * Existence of multiple genes which, when mutated, could cause the same phenotype. **9) Common recessive alleles/isolates** * Isolated populations have increased risk of recessive alleles/disorders because inbreeding * Pedigree can appear dominant, but if you know it is from an isolated population, could be a recessive disorder

Question 34

Explain the terms forward and reverse genetics

Answer 34

**Forward genetics:** phenotype-driven mutagenesis * Identification of heritable phenotype --\> mapping & positional cloning --\> discovery of causative gne **Reverse genetics:** gene-driven mutagenesis * Gene targeting --\> mating & phenotyping --\> discovery of biological function

Question 35

Meiosis

Answer 35

Reduces chromosome number x2 to produce haploid daughter cells. Daughter cells can have new combinations of chromosomes/alleles, which increases genetic variation in the offspring 1) Prophase I * chromosomes condense * pair of homologous chromosomes line up (synapsis) to form tetrads * crossover may occur 2) Metaphase I * homologous pairs lne up at the metaphase plate 3) Anaphase I * Homologous pairs pulled apart to opposite poles 4) Telophase I * each pole has haploid number of chromosomes * cytokinesis occurs at same time 5) Prophase II * chromosomes begin migration to cell's equator 6) Metaphase II * chromosomes line up at cell's equator 7) Anaphase II * sister chromatids seperate and migrate to opposite poles 8) Telophase & cytokinesis II

Question 36

Complementation analysis

Answer 36

The construction of merodiplid strains can be used to understand biological processes. Ex: Can determine which of the three genes required for *E. coli* growth on galactose contains a mutation. * gal-1/galK^- galT⁺ galE⁺ (Gal⁺ phenotype) * gal-1/galK⁺ galT^- galE⁺ (Gal⁺ phenotype) * gal-1/galK⁺ galT⁺ galE^- (Gal^- phenotype) \<-- mutation in galE Ex: Paper concludes a *yfgA^-* genotype results in defective yff phenotype and concludes YfgA is required for yff (your fav function). What's the next step? * Must perform complementation assay and make *yfgA^-/yfgA⁺* merodiploid. If function restored, conclusion supported. If function still defective, something else is involved and YfgA may or may not be involved (given polar nature of operon). Ex: (see image). A collection of mutants unable to synthesize histidine are isolated and complementation studies are performed. How many genes are represented in this set of mutants? * At least 2 genes - one in his-1 & his-4, one in his-2 & his-3

Question 37

Autosomal-recessive inheritance

Answer 37

Affected gene is located on one of the autosomes and mutant allele recessive. Features: * Parents of an affected individual are usually normal * Both parents of an affected individual are carriers * the more rare the mutant allele is in a population, the more likely that affected individuals are the product of a consanguineous marriage

Question 38

What is the mode of inheritance?

Answer 38

Y-linked inheritance