ASCI Genetics Exam 4

examples of cloning used as a genetic rescue of endangered animals

- Elizabeth Ann Black-Footed Ferret - Kurt Przewalski Horse - Wolly Mammoths

Przewalski Horses

- Endangered Species but once listed as extinct in the wild - Live in the Mongolia and China - Recovering from historic bottleneck - Captive breeding an reintroductions resulted in ~ 2,000 living Przewalski horses - But they are descendants of 12 individuals saved from extinction in the early 1900's - Kurt - Born August 6, 2020 - Cloned from cell line cryopreserved since 1980

biological considerations for sequencing a genome

- How will the genomic sequence of an organism inform our understanding of disease or hold the potential for improving health? - How will genomic sequence of a particular organism lead to a better understanding of biological function?

reproductive cloning

- Methods that produce two or more genetically identical individuals - Identical twins are genetic clones from one fertilized egg

strategic issues for sequencing a new genome

- What is the size of the research community that will use the data? Is the community enthusiastic about using the data? - Why would the complete sequence of this organism be more useful than just sequencing targeted regions?

hierarchical sequencing: why was this approach thought to be a good idea?

-By first generating a map of the genome, researchers had access to a genetic resource that could be used for downstream analysis. -Because only known regions are sequenced, it's pretty easy to keep track of the project -Assembled regions can be produced and released as you go -Assembly of shorter regions is thought to be more efficient

what were the two main medical genetics foci of the humane genome project?

-Identify variation in the genome that is disease causing -Determine how individual genes play a role in health and disease

disadvantages of shotgun sequencing

-Need vast amounts of computer power and sophisticated software to reassemble the genome -To Sequence the genome of a mammalian species, you need about 60,000,000 individual DNA sequence reads. -Reassembling these sequenced fragments required a HUGE investment in IT. -No assemblies can be released until the end.

advantages of shotgun sequencing

-No prior mapping required -Faster -Cheaper

why are animals genetically engineered?

-to study human disease -transgenic animals are generated to produce disease resistant animals -farm animals engineered to produce drugs or proteins that are useful in medicine

What does p+q equal?

1

Goals of genetic modification

1. Altering specific genes that are already present in an animal's genome 2. Introducing a cloned gene into the genome

shotgun sequencing approach steps

1. DNA is broken up into fragments 2. Fragments are sequenced randomly. 3. Sequenced fragments are assembled by looking for overlapping sequence

Why bring back woolly mammoths?

1. Ecosystems approach to confronting climate change 2. Ancient DNA holds secrets that impact modern biology and medicine 3. The future of large mammal conservation• Ultimate goal: Bring back mammoths so that healthy herds may one day re-populate the tundra and boreal forest in Eurasia and North America

approaches to gene modification

1. inactivate a gene by introducing a deletion 2. introduce a missense mutation into a gene

how do populations change from one generation to the next?

1. size 2. geographic location 3. genetic composition

hierarchical sequencing steps

1.Map first 2.Sequence Later

What are the certain conditions under which a population would be in equilibrium?

1.No new mutations (ultimate source of all genetic variation!) 2.No genetic drift. The population is so large allele frequencies do not change due to random sampling effects 3.No migration (i.e., no gene flow) 4.No natural selection 5.Random mating (i.e., no mate choice, no inbreeding)

Modern tenets of natural selection

1.Within a population there is allelic variation arising from various factors such as mutations causing differences in DNA sequences 2.Some alleles may encode proteins that enhance an individual's survival or reproductive capacity 3.Individuals with beneficial alleles are more likely to survive and reproduce 4.Over the course of many generations, allele frequencies of many different genes may change through natural selection 1.This significantly alters the characteristics of a species 2.The net result of natural selection is a population that is better adapted to its environment and/or more successful at reproduction

how long did the human genome project take to sequence?

10 years

average size of a mammalian genome

3 billion bps

example of gene addition

A gene from jellyfish that encodes a green fluorescent protein (GFP) was inserted into the zebrafish genome • Red fluorescent gene from a sea coral • Yellow fluorescent variant of jellyfish gene • In 2003, the GloFish became the first genetically modified organism to be sold as a pet

local populations vs large populations

A large population is usually composed of smaller groups called local populations

consensus sequence

A sequence for which there is agreement (or consensus) that it is an authentic representation of the sequence for each DNA base pair in the genome

genome assembly

After we sequence, we must put the reads back together again, in the correct order.

gene knockout

Both copies of a gene have been altered to an inactive form

key concept of genome sequencing projects

Characterizing whole genomes is fundamental to understanding the entire body of genetic information underlying the physiology and development of living organisms, and to the discovery of new genes such as those having roles in genetic disease

what does CRISPR stand for?

Clustered Regularly Interspaced Short Palindromic Repeats

what were the 5 model organisms for genome projects before the human genome project?

E. coli, S. cerevisiae, C. elegans, D. melanogaster, M. musculus

who was GH Hardy?

English mathematician

revive and restore mission

Enhance biodiversity through the genetic rescue of endangered and extinct species.

why is CRISPR preferred to other genome editing methods?

Faster, cheaper, accurate and efficient compared to any other genome editing methods Allows scientists to quickly target, delete and repair any mutated sequence of DNA

how can knockout genes teach us about what genes do?

Gene knockouts may reveal the function of the gene • Sometimes there is no obvious phenotype - Single gene may only make small contribution to overall phenotype - Another gene with similar function may compensate

who was Wilhelm Weinberg?

German half-Jewish physician and obstetrician-gynecologist, practicing in Stuttgart

Dolly

Ian Wilmut and his colleagues at the Roslin Institute cloned Dolly

When is a population in disequilibrium/not evolving?

If the equation fails to predict genotype frequencies (i.e., there is a statistically significant difference between what was observed and what was predicted by HWE)

When is a population in equilibrium/not evolving?

If the genotype frequencies predicted by HWE are close to what is actually observed in the population

narcolepsy in humans vs dogs

In Humans can take different forms. A few of those are: -Excessive Daytime Sleepiness (EDS) -Loss of muscle tone due to strong emotional stimulus -Sleep paralysis (can last up to 15 mins) - Complex mode of inheritance because there is a complicated interaction between environment and genetics. This makes it hard to work with genetically. • Not just in dogs, also in miniature ponies, and Brahman bulls Dogs breeds identified with narcolepsy -Dachshunds -Poodles -Labrador retrievers -Dobermans Original theory about narcolepsy: Automosal recessive Autoimmune disease -85% of narcoleptic patients share an association with a gene located in the MHC But then a Narcolepsy mutation was found in dogs -Located in hypocretin receptor 2 gene (Hcrtr2) -Usually turned on in posterior hypothalamus - Narcoleptics' brains are missing the cells that produce, Hcrtr2, an important wakefulness-promoting hormone So then they genotyped a bunch of humans for this mutation and found that narcolepsy is really a neurodegenerative disease -Massive reduction in neurons.

dogs and cancer

It is estimated that about 10 percent of dogs in the United States develop cancer. Most of the cancers found in humans also affect dogs. Another reason dogs are so appealing to cancer researchers as research subjects is that dogs are exposed to the same elements as people on a daily basis.

canine genome

NIH funded project -Cost $30 Million -Sequencing performed by the Broad Institute at MIT Why was the dog genome sequenced? -Dogs are an animal model for Cancer and other diseases -More Mendelian disorders have been identified in dogs than any other vertebrate species Tasha was the boxer that was sequenced -7.5X sequencing coverage -Chosen because she is highly inbred -1X skims of 9 other breeds, 4 wolves and a coyote were performed so that SNPs could be identified (found about 2.5M SNPs)

high throughput sequencing

Rapidly sequencing large amounts of DNA

how do we make a knockout mouse?

Researchers inactivate a normal gene using a method such as CRISPR-Cas technology • Results in a heterozygous animal • When these heterozygous animals are crossed to each other, one-fourth of the offspring will be homozygous for the inactivated gene • An animal that is homozygous for the inactivated gene is a knockout

what accounts for 90% of genetic variation between people?

SNPs

Why do we need to sequence a genome?

Sequencing a genome gives us a comprehensive view of all of the base pairs that make up an organism

phenotypic accommodation theory

The ability to developmentally alter structures in the body (e.g. ear bones) to accommodate selective changes in another trait (e.g. the ears)

CRISPR example

Used to generated polled Holstein calves - Dehorning is unpleasant - Horns can cause damage to humans and cattle - Precision Breeding and CRISPR

Hardy-Weinberg equilibrium definition

Uses observed allele frequencies to predict genotype frequencies that would occur under "equilibrium" conditions (no evolution).

How does CRISPR work?

Works like a find and replace Cas 9 protein - cuts DNA Guide RNA - recognizes the sequence of DNA to be edited Spacer - short DNA sequence that follows the DNA region targeted for cleavage by CRISPR system

Is Phenotypic Accommodation found in nature?

YES -What was tested? Bushy tailed woodrats -Why? Woodrats have large ears and rely on hearing to detect predators and mates -Where: Northern California (costal forests to arid deserts -With longer ears comes greater soft tissue mass and the altered shape of the ear bone may help support the load

polymorphic

a gene that commonly exists as 2 or more alleles in a population

contigs

a set of overlapping DNA segments that together represent a consensus sequence for a particular region of the genome

gene knock in

a type of gene addition in which a gene of interest has been added to a noncritical site in the genome

gene pool

all of the alleles of every gene in a population

Gene modification

alters the sequence of a gene

missense mutation

change of a single base pair causes an amino acid change in the resulting protein

comparative genomics

considers the genomes of closely related and distantly related species for evolutionary insight

What are the four selection scenarios?

directional balancing disruptive stabilizing

why are organisms different in different places?

genetic differentiation local adaptation phenotypic plasticity

what is a population?

group of individuals of the same species that are capable of interbreeding with each other

why would you use CRISPR-Cas technology to introduce a missense mutation into a gene?

missense mutations in mice can mimic disease-causing mutations in humans This allows us to study the effects of disease in mice

how are local populations usually separated from one another?

moderate geographic barriers

genomics

molecular analysis of the entire genome of a species

what are the four main evolutionary processes studied in population genetics?

natural selection, genetic drift, mutation, and gene flow

who contributes to the gene pool of the next generation?

only the individuals who reproduce

transgenic

organism that has integrated recombinant DNA into its genome

Hardy Weinberg equation

p^2 + 2pq + q^2 = 1

molecular pharming

production of medically important proteins in the mammary glands of livestock

Genetically modified organisms (GMOs)

received genetic material viarecombinant DNA technology

polymorphism

refers to the observation that many traits display variation within a population

genome sequencing projects

research endeavors aimed at determining the DNA sequence of the entire genome of a species

population genetics

studies the extent of genetic variation within a population

Biotechnology

technologies that involve theuse of living organisms, or their products, to benefit humans

gene addition

the addition of a cloned gene into a site in a chromosome of a living cell

bioinformatics

the analysis of the information of content of entire genomes

gene redundancy

the phenomenon in which an inactive gene is compensated for by another gene with a similar function

sequence assembly

the process of building up all of the individual sequence reads into a consensus sequence

genome

total genetic composition of an organism

functional genomics

understanding gene and protein function in biological processes

single nucleotide polymorphism

variation in a DNA sequence occurring when a single nucleotide in a genome is altered

why would you use CRISPR-Cas technology to inactivate a gene?

we can figure out how the loss of gene function affects phenotypes

genome wide screening for dog cancer

§In order to study cancer scientists, prefer to use purebred dogs registered at the AKC. § §WHY? §AKC has access to pedigree information. The family history can then be used to determine if a genetic association to cancer exist. §Inbreeding also reduces the amount of genetic diversity, making it easier to find the cancer alleles. § §In order to do a genetic association study, we need phenotypes, §Vets are responsible for the phenotypes in this case. They look at tumor growths in multiple breeds of dogs and compare them. §This allows vets to characterize a bunch of different tumors § §Then we genotype the dogs. §We then use the phenotypes, the genotypes and the pedigrees to help us identify regions of the genome associated with cancer §When the genetic defects that cause cancer are found in dogs, then therapies will be created to help treat cancer in humans.

uses of bioinformatics

• Genome Assembly • Figure out what the genome is composed of • Understand Gene Function • Identify Genomic Variation • Facilitate Generation of New Technologies ( examples: SNPChips

NIH vs Craig Venter

•1995-Craig Venter announced that he and his team of about forty had succeeded in sequencing the entire genome of: - H. influenzae -Mycoplasma genitalium.- smallest known genome of any living organism - •By sequencing these small genomes Venter realized: -He would need a very powerful computer infrastructure. -That "shotgun" sequencing was more effective. •Sequencing at that time: -One sanger sequencing machine can sequence 384 DNA samples in three hours, reading about 500 bases -Allowing for poor quality templates, this means 300,000 bases per day, or 300 Million bp in three years.

PRIVATE human genome project - Craig Venter

•1998 Celera Genomics •Aim to sequence the human genome in 3 years •'Shotgun' approach - no use of maps for assembly •Heavy technology use: automation and computers •Had access to public project's data

earth biogenome project

•Aims to sequence, catalog and characterize the genomes of all of Earth's eukaryotic biodiversity over a period of 10 years

Darwin's lop eared rabbits

•Among the domesticated species that Darwin studied is rabbits. Specifically lop-eared rabbits -Lop-eared - floppy eared rabbits that fall vertically from their heads rather than ears that point up. •Why was Darwin excited about Lop-eared rabbits? -Because he observed that their lengthy ears were linked to an extreme deformity in the side of their skull, misshaped ear bone •In the 1800's rabbits were bred for overly large ears. -Long ears > Lop eared > bone morphology

International Human Genome Sequencing Consortium

•Approach was conservative and methodical •Had to wait for technology •First produced a physical map of the genome that would serve as a scaffold for later assembly of the sequence data

problem with using contigs

•Contigs eventually run into repetitive DNA sequence regions that prevent the contigs from continuing. •This leads to multiple contigs. •Then we have to figure out how to join the contigs together in the correct order and orientation.

What's in a genome?

•Genes (i.e., protein coding) •But. . . only <2% of the human genome encodes proteins •Other than protein coding genes, what is there? • genes for noncoding RNAs (rRNA, tRNA, miRNAs, etc.) • structural sequences •regulatory sequences • non-functional "junk" ?

PUBLIC human genome project - Watson/Collins

•Human Genome Project •Proposed in 1985, endorsed in 1988, officially launched in 1990 •Worldwide effort - both academic and government institutions •Assemble the genome using maps •Public project

Who proposed the theory of natural selection and when?

•In the 1850s, Charles Darwin and Alfred Russel Wallace independently proposed the theory of natural selection

what drives microevolution?

•Mutation •Random genetic drift •Migration •Natural Selection •Nonrandom mating

Microevolution

•describes changes in a population's gene pool from generation to generation

genome annotation

•the process of assigning biological function to sequences within a genome. Annotation allows us to understand of the genome sequence