15:282 Principles of Genetics: Term Test #2

Operon

• Is a genetic unit of coordinated expression and consists of the structural genes as well as the promoter and operator

Prokaryotic Regulation of Lac Operon

• Is negatively regulated by a repressor protein:

  • lac repressor binds to the operator to block transcription

  • In the presence of lactose, an inducer molecule binds to the repressor protein →

  • Repressor can no longer bind to operator

  • Transcription proceeds

Negative Control

• Regulation of gene expression in which mRNA is not transcribed until a repressor is removed from the DNA of the gene

Smart Cells

• In the presence of both glucose and lactose, bacterial cells prefer to use glucose

• Glucose prevents induction of the lac operon

Positive Control

• Mechanism of gene regulation in which an element must be bound to DNA in an active form to allow transcription

Prokaryotic Positive Control

• In the presence of both glucose and lactose, bacterial cells prefer to use glucose

• Glucose prevents induction of the lac operon

  • Binding of CAP - cAMP complex to the CAP binding site is required for induction of the lac operon

  • High glucose levels cause low cAMP levels

  • High glucose → low cAMP → no induction

Tryptophan (trp) Operon in E.coli

• Is a repressible gene system

Tryptophan

• Enzymes for the production of this amino acid form an operon

• In the presence of this amino acid, the operon is repressed

• Functions as a corepressor

  • which is required for the repressor to bind to the operator

Tryptophan Operon Components of the Regulatory Region

• Promoter

• Operator

• Leader

• Attenuator

Prokaryotic Regulation

• Tryptophan operon is negatively regulated by the trp repressor protein

  • trp repressor binds to the operator to block transcription

  • Binding of repressor to the operator requires a corerepressor which is tryptophan

  • Low levels of tryptophan prevent the repressor from binding to the operator

Tryptophan Attenuates Transcription of the trp Operon - High Tryptophan Level

• Leader region is completely translated

• Formation of stem and small loop results in the termination of transcription

Tryptophan Attenuates Transcription of the trp Operon - Low Tryptophan Level

• Ribosome is stalled at trp codons

• Transcription continues

Overview of Transcriptional Regulation - Eukaryotic

• Ground State: Off

• Active State: On

Gene Regulation in Eukaryotes

• More complex than it is in prokaryotes because of the larger amount of DNA, larger number of chromosomes, spatial separation of transcription and translation mRNA processing, RNA stability, and cellular differentiation

Levels of Gene Regulation in Eukaryotes

• Transcriptional control

• Processing control

• Transport control

  • mRNA degradation control

    • Inactive mRNA

  • Translational control by ribosome selection among mRNAs

    • Protein

      • Protein degradation control

        • Degraded proteins

Eukaryotic Gene Expression Features

• 1. RNA polymerases

• 2. Monocistronic gene structure

• 3. RNA processing

• 4. Split gene structure

• 5. Role of chromatin (Most Important)

Eukaryotic Gene Expression Features - RNA Polymerases

• Possess 3 nuclear enzymes

  • RNA pol I synthesizes rRNA

  • RNA pol II synthesizes mRNA

  • RNA pol III synthesizes tRNA and other small RNAs

Eukaryotic Gene Expression Features - Monocistronic Gene Structure

• Most eukaryotic mRNAs encode single gene product

  • While many prokaryotic genes are polycistronic, multiple gene products per transcription unit (e.g operon)

Eukaryotic Gene Expression Features - RNA Processing

• Messages are “capped” at 5’ end with 7-methyl guanosine

• Messages are polyadenylated at 3’ end

• Internal portions of primary transcript are spliced of intervening sequences

  • This RNA splicing is due to their split gene structure

Eukaryotic Gene Expression Features - Split Gene Structure

• Many genes contain introns, whose RNA product is spliced out before mRNA transport to the cytoplasm. Leaving only exon sequences in mature mRNA

Eukaryotic Gene Expression Features - Role of Chromatin

• To relieve repression by chromatin, much more must happen at eukaryotic promoters

• Key to the process is controlling access to promoters

Transcript Initiation

• Is a major form of gene regulation

Transcription in Eukaryotes

• Eukaryotic chromosomal DNA is complexed with histones to form chromatin

• Nucleosomes in the chromatin can inhibit transcription, and chromatin remodelling is required for transcription to occur

Chromatin Remodelling

• Exposes regulatory sequences

  • Nucleosomes block the binding of RNA polymerase II to the promoter

  • Addition of acetyl groups to histone tails remodel the solenoid so that DNA is accessible for transcription

Histone Code Hypothesis

• Proposes that specific combinations of modifications, as well as the order in which they occur, help determine chromatin configuration and influence transcription

Histone Modifications

• In histone acetylation, acetyl groups are attached to positively charged lysines in histone tails

• This loosens chromatin structure, thereby promoting the initiation of transcription

• The addition of methyl groups (methylation) can condense chromatin; the addition of phosphate groups (phosphorylation) next to a methylated amino acid can loosen chromatin

Eukaryotic Regulation

• Controlling the expression of eukaryotic genes requires transcription factors

• General transcription factors are required for transcription initiation

  • Required for proper binding of RNA polymerase to the DNA

• Specific transcription factors increase transcription in certain cells or in response to signals

General Transcription Factors

• Required for transcription initiation

• Required for proper binding of RNA polymerase to the DNA

Specific Transcription Factors

• increase transcription in certain cells or in response to signals

Promoter-Proximal Elements

• Necessary for efficient transcription

• The TATA box is the region to which RNA polymerase II binds

• The CAAT box and GC box are elements that bind transcription factors

Eukaryotic Transcription

• General transcription factors bind to the promoter region of the gene

• RNA polymerase II then binds to the promoter to begin transcription at the start site (+1)

Enhancers

• Are DNA sequences to which specific transcription factors (activators) bind to increase the rate of transcription

Properties of Enhancers

• Can greatly increase transcription rates from promoters on same DNA molecule

• May act up to several thousand base pairs away

• Function in either orientation (can flip’em around) and can function upstream or downstream of the promoter they are enhancing

Properties of Enhancers - Considerations

• They are for trans-acting factors

• Action at a distance reflects conformation of the gene in chromatin - its protein-bound form, sites that are distant in linear DNA molecule may be adjacent in chromatin

• Hence, distance and orientation are independent

Eukaryotic Transcription

• Coactivators and mediators are also required for the function of transcription factors

  • Coactivators and mediators bind to transcription factors and bind to other parts of the transcription apparatus

Activators

• Regulatory proteins bind to DNA at distant sites known as enhancers. When DNA folds so that the enhancer is brought into proximity with the initiation complex to increase the rate of transcription

• Is a protein that binds to an enhancer and stimulates transcription of the gene

• Have two domains, one that binds DNA and a second that’s activated transcription

• Bound version of these facilitate a sequence of protein-protein interactions that result in transcription of a given gene

Coactivators

• Transcript factors that transmit signals from activator proteins to the general factors

Functional Domains Possessed by Eukaryotic Transcriptional Regulatory Proteins

• Domain that recognizes a DNA sequence

• Domain that interacts with one or more proteins of the transcriptional apparatus

• Domain that interacts with proteins bound to nearby regulatory sites (cooperatively)

• Domain that influences chromatin structure (directly or indirectly)

• Domain that acts as a sensor of conditions within the cell

How do Activators Work?

• Some recruit the transcriptional machinery

• Some recruit proteins that modify chromatin structure and allow RNA polymerase II and other proteins access to DNA

• Some do both

Gene Expression Via Steroid Hormone

• 1. Hormone enters its target cell and combines with a receptor protein

• 2. Hormone-receptor complex binds to a hormone response element in the DNA

• 3. Bound complex stimulates transcription

• 4. Transcript is processed and transported to the cytoplasm

• 5. mRNA is translated into proteins

Methylation & Gene Regulation

• In eukaryotes, several observations suggest that this process in DNA plays a role in gene regulation

• An inverse relationship exists between the degree of methylation and the degree of gene expression; methylation patterns are tissue specific and heritable for all cells in that tissue

Posttranscriptional Regulation

• Control of gene expression usually involves the control of transcription initiation

• But gene expression can be controlled after transcription, with mechanisms such as:

  • RNA interference

  • Alternative splicing

  • RNA editing

  • mRNA degradation

RNA Interference

• Involves the use of small RNA molecules

• The enzyme Dicer chops double stranded RNA into small pieces of RNA

  • Micro-RNAs bind to complementary RNA to prevent translation

  • Small interfering RNAs degrade particular mRNAs before translation

RNA Editing

• Creates mature mRNA that are not truly encoded by the genome

• E.g →

  • Apolipoprotein B exists in 2 isoforms

  • One isoform is produced by editing the mRNA to create a stop codon

  • This RNA editing is tissue-specific

Posttranscriptional Regulation

• Mature mRNA molecules have various half-lives depending on the gene and the location (tissue) of expression

• The amount of polypeptide produced from a particular gene can be influenced by the half-life of the mRNA molecules

Post-translational Control

• Proteins to be degraded are tagged with ubiquitin (76 aa long)

Eukaryotic Gene Regulation - Summary

• 1. & 2. Transcription

  • DNA packing

  • transcription factors

• 3. & 4. Post-transcription

  • mRNA processing

  • splicing

  • 5’ cap & poly-A tail

  • breakdown by siRNA

• 5. Translation

  • block start of translation

• 6. & 7. Post-translation

  • protein processing

  • protein degradation

Epigenetics

• Simply means “on top of genetics”

• All cells contain the same gene, but gene expression patterns are different in different cells

• Chemical modifications of chromosomal DNA and/or structures that change the pattern of gene expression without altering the DNA sequence

Epigenome Changeability

• Cells are constantly listening for signals to change what they are doing

• Signals come from inside the cell, neighbouring cells or the environment

• Environmental signals may be direct (diet) or indirect (stress)

Mechanism of Epigenetics

• RNA Interference

• Histone Modifications

• DNA Methylation

  • (All lead to Gene expression)

Epigenetic Modifications

• Histone modifications act to tighten or loosen DNA coils, expose or hide genes from the cell

The Epigenetic Therapy

• Turning genes on and off is easier than changing the DNA sequence

• Many drugs have been approved for use or are under development

• Even selective diet can erase the epigenetic tags

Nucleosome

• DNA wrapped around a core of 8 histone proteins

• Are spaced 200 nucleotides apart along the DNA

Eukaryotic Chromsomes

• Linear chromosomes

• Every species has a different number of chromosomes

• Composed of chromatin

• Heterochromatin - not expressed

• Euchromatin - expressed regions

Chromatin

• A complex of DNA and proteins

Karyotype

• The particular array of chromosomes of an organism

Chromosomes

• Must be replicated before cell division

Satellited Chromosome

• Nucleolar organising region

Eukaryotic Cell Cycle

• 5 main phases:

  • 1. G1 (gap phase 1)

  • 2. S (synthesis)

  • 3. G2 (gap phase 2)

  • 4. M (mitosis)

  • 5. C (cytokinesis)

• Length of a complete cell cycle varies greatly among cell types

Mitosis

1. Early Prophase

   • Middle Prophase

   • Late Prophase

2. Metaphase

3. Early Anaphase

   • Late Anaphase

4. Telophase

5. Interphase

Features of Meiosis

• Includes two rounds of division:

  • Meiosis I (reductional) and Meiosis II (equational) with no replication of genetic material between them

• Results in a reduction of the chromosome number from diploid to haploid

• During meiosis I, homologous chromosomes (homologues) become closely associated with each other → this is synapsis

• Proteins between the homologues hold them in a synaptonemal complex

Meiosis I

• Metaphase I - Chiasmata hold homologues together. The kinetochores of sister chromatids fuse and function as one. Microtubules can attach to only one side of each centromere

• Anaphase I - Microtubules pull the homologous chromosomes apart, but sister chromatids are held together

Mitosis

• Metaphase - Homologues do not pair; kinetochores of sister chromatids remain separate; microtubules attach to both kinetochores on opposite sides if the centromere

• Anaphase - Microtubules pull sister chromatids apart

Wild-Type Sequence

• Loss of genetic material

  • Deletion

  • Missing chromosomes

• Gain of genetic material

  • Extra chromosomes

  • Duplication

• Relocation of genetic material

  • Translocation (genetic material from another chromosome)

  • Inversion

Meaning of 1. “n” and 2. “x”

• 1. Haploid number = number of chromosome in a gamete

• 2. Basic number = number of chromosomes in a basic set of a diploid

Diploidy - Diploid

• 2n=2x=8

Monoploidy - Monoploid

• 2n=x=4

Polyploidy - Triploid & Tetraploid

• 2n=3x=12

• 2n=4x=16

Deficiency

• Can change chromosome size, Cl and banding pattern

• If large: deficiency loop

• Pollen less tolerant than sperm

• Genetic consequences

Deficiency - Genetic Consequences

• Homozygous: often lethal

• Heterozygous: effect less severe (del 5p, del 15q)

• Hemizygosity: unmasks deleterious alleles

• Pseudodominance (same as sex linked ineritance)

• Suppression of crossing over : supergenes?

• Position effect

Deficiency - Usages

• Linkage identification

• Genome mapping/deletion mapping

• E.g. Ae sq

Duplications

• Origin

• Not as severe as deficiency

• Changes in chromosome size, Cl and banding pattern

• Formation of duplication loop

Duplications - Genetic Consequences

• Dosage effect: increased expression

• CO suppressor

• Permanent heterozygosity

• Variability enhancing: genomes amplification : evolutionary role

• Genomic imbalance

Types of Inversions

• Pericentric (includes centromere)

• Paracentric (does not include centromere)

Inversions - Phenotypic Effects

• No major effect on bearer, but possibly position effects

Inversions - Cytological Effects

• Cl may change if pericentric

• Reasonably large: Inversion loop

• Small: no loop, no pairing, no CO

• Supergenes?

Inversions : What Happens at Meiosis?

• Dependence on Crossing Over in loop - No crossing over … all gametes are functional

• If Crossing Over in loop? -

  • Paracentric: acentric, dicentric bridge. Gametes: ¼ normal, ¼ inverted, 2/4 def-dup, Recombinant gametes not viable, CO suppressor

  • Pericentric: no acentric, no bridge. Gametes and rest : same as paracentric

Types of Translocations

• Nonreciprocal intrachromosomal

• Nonreciprocal interchromosomal

• Reciprocal interchromosomal

Genetic Effects of Translocations

• Chromosome restructuring

• Position effects: e.g. Philadelphia chromosome in human … Trans between “q” of 9 & 22 (oncogenes from 9 to 22 … Leukemia)

• Problem in pairing … Quadrivalent, Crossing Over suppressors

• Effect on gametes: No Crossing Over in quad … 50/50 sterility

  • (alt/asj); 33-50% sterility if Crossing Over in quad

Philadelphia Chromosome

• Origin in chronic myelogenous leukemia by a reciprocal translocation involving chromosomes 9 and 22

Significance of Translocations

• Identifying linkage groups

• Prenatal diagnostics

• Alien gene introgressions

• Speciation

• Controlling insect pests

Robertsonian Translocation

• Two acrocentric chromosomes fuse at their centric ends

Polyploidy Comparison - Plants & Animals

• Mainly viable in plants

• Certain insects (wasps, ants, male bees) are monoploids … parthenogenetic

• Lower animals (flatworms, leeches), some fishes (salmons, trouts), frogs, salamanders, lizards

• Triploid oysters

Monoploids - Production

• Naturally (rarely in some, regularly in others)

• Can be artificially produced

  • Anther culture

  • Microspore culture

  • In vitro haploid/monoploid production

    • e.g. Diploid plant → immature pollen cells plated → monoploid embryoids grow → monoploid plantlet → monoploid plant

Monoploids - Fertility & Cytology

• When fertile, gametes produce mitotically

Monoploids - Uses

• Hemizygosity … recessive mutations expressed

• No Crossing Over … favourable combinations retained

• Instant homozygosity … speeding variety development

• In vitro gametic selection possible … various situations

Triploids - Production

• In nature → low frequency

• Fusion of n and 2n gametes (rather x and 2x gametes)

• Artificially → Diploid x tetraploid cross

Triploids - Fertility & Cytology

• All odd number of euploids → highly sterile

• III or II + I; genome imbalance

Triploids - Uses

• Seedlessness

• Occasionally larger size → gene amplification (watermelon, wine sap, apples)

• Triploid oysters

• As a starting point for generating aneuploids

Triploids - Drawback

• Need for vegetative propagation

Autotetraploids - Production

• Naturally - Meiotic disturbances

• Artificially - Using spindle poison (e.g. colchicine)

  • ‘Dipoidization of polyploid’ (e.g. chickpea)

• Larger in size (gene amplification) → potatoes, McIntosh apples

Pairing Possibilities for Autoetraploids

• Two bivalents

• One quadrivalent

• Univalent + trivalent

Endopolyploidy

• 2n=4x, 8x, 16x

• Vertebrate liver cells and apical parenchyma cells in plants

• High levels of gene products (detox, growth factors)

Aneuploids

• Changes in chromosome number within a set

• Origin → nondisjunction

• Man-made in plants, naturally occurring in humans

• Survival in plants is higher than animals → genome imbalance

• Survival higher in polyploids than diploids

• Typically distorted segregation ratios

• Used for mapping genes to chromosomes

• Used for creating alien addition and alien substitution lines

Monosomics

• Quite deleterious → large scale heterozygous deletions

• For autosomes in humans die in utero

• XO → Turner syndrome survives (1/5,000 birth)

  • High incidence of X-linked recessives → hemizygous

Trisomics

• More tolerable than monosomics → less deleterious

• In the case of humans → most common for sex chromosomes

  • XXX … 1/700 births … metafemale

    • Gamete formation → NORMAL

  • XYY … 1/1000 births

    • Gamete formation → NORMAL

  • Tri-13 … 1/20,000 … Patau’s syndrome … < 6 months

  • Tri-18 … 1/8,000 … Edward’s syndrome … 4 weeks

  • Tri-21 … 1/700 … Down’s syndrome … 20 years

• Increased incidence with maternal age

• Gene mapping with this

Locating Recessive Allele

• Using Trisomic analysis

• Critical Cross

Somatic Aneuploidy

• XO/XX Sexual mosaics/gynandromorph in Drosophila (Fly)

• XO/XYY sexual mosaics in humans

Euploids: Autopolyploidy

• Diploid →

  • Triploid

  • Tetraploid

Euploids: Allopolyploids

• Diploid (orange) x Diploid (blue) →

  • Tetraploid (orange and blue)