Genetics Unit 4

Noncoding RNAS

Molecules transcribed from genes that do not encode polypeptides. About 80% of human cell production involves this, more than mRNAS.

Noncoding RNAS

can bind to DNA, ncRNA, proteins, and small molecules.

scaffold

• when ncRNA binds to a group of proteins

• mechanism of action of noncoding RNAS

guide

• mechanism of action of noncoding RNAS

• ncRNA binds to a protein and directs it to a specific site in the cell

alteration of protein function/stability

• mechanism of action of noncoding RNAS

• binding can change a protein’s structure, affecting its ability to act as a catalyst, bind to another molecule and overall stability.

Ribozyme

• mechanism of action of noncoding RNAS

• RNA molecules that have their own catalytic function

Blocker

• mechanism of action of noncoding RNAS

• ncRNA physically prevents and stops cellular process from happening

Decoy

• mechanism of action of noncoding RNAS

• a ncRNA recognizes and sequesters another ncRNA to prevent it from working.

RNA interference

A mechanism found in most eukaryotes that regulate gene expression or defend against viruses

microRNAS

• noncoding RNA mediator

• transcribed from endogenous genes (cells own DNA), one miRNA inhibits different mRNAS.

siRNAS

• - noncoding RNA mediator

• - come from exogenous sources (not made up by cells), like viruses.

• Mechanism: RISC uses a single strand of 20-25 bp RNA to bind to target to mRNA. Leading to either inhibited translation or mRNA degradation.

CRISPR-Cas

this system provides bacteria with a defense against bacteriophages.

CRIPS locus

site in prokaryotic chromosomes that contains repeated sequences and its segments are derived from bacteriophage DNA.

Adaptation

• - the first phase of defense mechanism in CRISPR

• -  Cas 1/CAS 2 complex cleaves invading bacteriophage DNA and inserts a piece (spacer into the CRISPR gene) between 20-25 bp. AKA spacer acquisition.

Expression

• the second phase of defense mechanism in CRISPR

• the CRISPR gene that is transcribed into pre-crRNA, which is processed into cRNAS. These bind with tracRNAS

interference

• the third phase of defense mechanism in CRISPR

• the cRNA acts as guide to bring the complex to the matching bacteriophage DNA where cas9 makes double-strand breaks to inhibit the phage.

gene inactivation/mutation

  cas9 creates a double strand break. If repaired by nonhomologous end joining (NHEJ), a small deletion typically inactivates the gene.

gene/genome editing

if homologous recombination pair is used (HRR), researchers provide donor DNA with a specific mutation to accurately edit the sequence.

Mutations

a heritable change in the genetic material (DNA sequence). While often harmful and can cause disease, they are also the foundation for evolutionary change and adaptation

change in chromosomes

• type of mutation.

• mutation can involve large scale changes like deletions, duplications, inversions or translocations.

• Can result in abnormal number of chromosomes. Changes in DNA of a single gene.

point mutations

• type of mutation

• Includes:

• 1. Transition: change of pyrimidine (C,T) to another pyrimidine or purine (A,G). MORE COMMON THAN TRANSVERISONS.

• 2. Transversion: change of pyrimidine to a purine or vice versa.

• 3. Insertion/Deletion: the addition or removal of short sequences of DNA.

silent mutation

• a type of mutation. only in protein coding genes

• does not alter the amino acid sequence of the polypeptide due to the degeneracy (encoded by more than one codon) of the genetic code.

missense mutation

• a type of mutation. only in protein coding genes

• changes in a single amino acid

nonsense mutation

• a type of mutation. only in protein coding genes

• Normal codon —> stop codon, resulting in a shorter truncated polypeptide.

frameshift mutation

• a type of mutation. only in protein coding genes

• the addition or deletion of nucleotides not divisible by 3. this shifts the reading frame and changes the downstream amino acid sequence.

promoter

• a mutation that occurs outside of gene coding regions

• May increase or decrease the rate of transcription

regulatory element/operator site

• a mutation that occurs outside of gene coding regions

• may disrupt proper regulation of gene expression.

photolyase

• repairs thymine dimers (caused by UV light) by splitting the dimer bonds. Enzyme uses energy from visible light.

Alkyl transferase

repairs DNA damaged by alkylating agents. it removes methyl or ethyl groups from base by transferring the group to its own cysteine residue. This permanently inactivates the protein

biotechnology

defined as the use of living organisms or their products to benefit humans.

GMO’s

are organisms that have received genetic material via recombinant DNA technology.

insulin

• a hormone required for glucose uptake. Purified from cows and pigs which can cause allergic reactions.

transgenic animals

an organism that has integrated recombinant DNA from a different species into its genome.

Reproductive cloning

methods used to produce two or more genetically identical individuals

cloning of plants

cloning is easy and has been a standard agricultural practice for centuries. Many cells are totipotent, meaning a single somatic cell can regenerate an entire new plant.

cloning of animals

early cloning was restricted using embryonic cells until mid 1990s. They were believed to not be totipotent.

dolly

• proved that differentiated adult cells can be reprogrammed into a totipotent state.

• the first mammal to be cloned into an adult somatic cell

• resulted in premature aging because the donated nucleus already had short telomeres

stem cells

have the capacity to divide (self-renewal) and differentiate into one or more specialized cell types.

Totipotent

• a type of stem cell that can give rise to ALL cell types, including the placenta.

Pluripotent

a type of stem cell that can differentiate into almost every cell type in the body but cannot produce an entire intact individual.

multipotent

a type of stem cell that can differentiate into several cell types, but are more restricted

unipotent

a type of stem cell that can only differentiate into one specific cell type

Induced pluripotent stem cells

Discovered by Shinya Yamanaka in 2006. By injecting four specific transcription factor genes into adult fibroblasts, researchers can repogram adult cells back into a pluripotent state. This avoids the ethical dilemmas associated with using embryos (ES cells)

medical use of stem cells

stems cells are used to repair or replaced damaged tissues. Applications include bone marrow transplants, skin grafts for burns, and potential repairs for heart or cartilage damage.

genome

the total genetic composition of organisms

genomics

the molecular analysis of the entire genome species

mapping

determining the locations of sties, such as genes, along a chromsome.

DNA sequencing

determining the entire base sequence of a genome, which often spans millions to billions of base pairs

associated technologies

genomic is characterized by ‘high throughput” methods, robotics, automation, informatic, and miniaturization.

the “Omics” hierarchy

Genomics (NDA) provides the foundation for transcriptomics (RNAs) and proteomics (protein), following the central dogma of biology.

clone by clone

• an approach for sequencing genomes

• This was the initial approach by the U.S. Human Genome Project starting in 1990. Begins by developing a physical map of the genome before proceeding to sequence the individual cloned fragments.

whole genome shotgun sequencing

• an approach for sequencing genomes

• An innovative approach introduced in 1998 by the Celera Corporation. This method bypassed the initial mapping phase, which significantly accelerated the sequencing work and allowed the project to be completed in four years ahead of schedule.

Human Genome project

Cost: 3 billion

Size of the challenge: sequencing a 3 billion base pair genome with early technology (capable of only 500 bases per run.) would have required 42 million sequencing runs.

Justification: the investment was for potential sceintific discoveries and tech

Human genome project goals

• to obtain genetic linkage and physical maps of the human genome

• obtain the DNA sequence of the entire human genome

• Develop technology for managing genomic information and advancing genetic methodologies.

• Analyze the genomes of model organisms

• Adress ethical/legal and social implications of genomic research.

Public effort

U.S. Human Genome project, began in 1990 and internationally coordinated undertaking led by the NIH.

private effort

-          led by Craig Venter and the Celera corporation.

Next generation sequencing

Technology that can process thousands or millions of sequence reads in parallel, making sequencing much faster and cheaper.

third generation sequencing

• -          Methods that sequence single DNA molecules, used for long-read sequencing.

1000 genomes project (2008)

An international effort to understand human genetic variation by sequencing the DNA of at least 1,000 anonymous participants

prioritizing genomes to sequence

Researchers choose organisms based on their value to basic research, medicine, agriculture, and evolution.

family clustering

when an individual has the disease, their relatives are more likely to have it than the general population

twin concordance

the disease is more likely to occur in both monozygotic (identical) twins than in both dizygotic (fraternal twins)

Specific age of onset

many genetic diseases follow a characteristic timeline

environmental independence

the disease develops even when the individual is not exposed to known environmental triggers (like toxins or pathogens)

population correlation

the disease resembles a known genetic disorder in an animal model

Autosomal recessive

Pattern: An affected individual must inherit two copies of the mutant allele.

Pedigree clues: Affected individuals have unaffected parents. When two unaffected heterozygotes have children, there is a 25% chance of an affected child. Two affected parents à 100% affected children

Traits frequently skip generations. EX: TAY SACHS

Tay Sachs Disease

Affected individuals appear healthy at birth but then develop neurodegenerative symptoms at 4 to 6 months. This disease causes cerebral degeneration, blindness and loss of motor function. They topically die at 3-4 years of age. This disease is 100 times more frequent in Ashkenazi Jewish populations.

Autosomal dominant

Pattern: An individual only needs one copy of the mutant allele to be affected.

Pedigree clues: Needs at least one affected parent. An affected individuals with one affected parent has a 50% chance of passing it to their offspring. Trait does NOT skip generations. Two unaffected parents cannot have an affected child. ex: Huntington’s Disease

Huntington’s Disease

The major symptom of the disease is the degeneration of certain types of neurons in the brain. Leading to personality changes, dementia, and early death. It is the result of a mutation in a gene that encodes a protein named huntingtin

X linked recessive

Pattern: genes are located on the X chromosome. Since males are hemizygous, they are frequently affected than females.

Pedigree clues: mothers of affected males are unaffected carriers.

Daughters of affected males are always carriers (when mother is unaffected)

Criss-cross inheritance: an affected male passes the trait to his grandsons through his carrier daughters.

Affected male never passes the trait to his sons.

X linked dominant

Pattern: rare pattern where a single dominant allele on the X chromosome causes the disease.

Pedigree clues: Affected males pass the trait to 100% of their daughters but 0 of their sons. Affected females à 50% of their children (both sons and daughters)

Key difference: Autosomal traits affect males and females with equal frequency, while X-linked traits show a significant biased based on biological sex.

cancer

a disease that occurs in multicellular organisms characterized by uncontrolled cell division and growth

cancer origin

derive from a single cell. Tumor starts as benign. But once it acquires additional mutations the tumor becomes malignant.

carcinogens

environmental agents that increase the likelihood of developing cancer.

characteristics of cancer

• they originate from a single cell

• it is a multistep process, precancerous —> cancerous

• Once cellular growth has become cancerous the cells become invasive and metastatic.

metastatic

when a cell migrates to another part of the body and becomes fatal

oncogenes

overactive genes that promote uncontrolled cell growth. Only needs one copy to be mutated.

Proto-oncogenes

normal cellular genes that when mutated can become oncogenes.

how proto oncogenes occur

1. amount of encoded protein is increased.

2. A change occurs in the structure of the encoded protein that causes it to be overly active.

3. the encoded protein is expressed in a cell type where it is not normally expressed.

how cancer-causing mutations occur

1. missense mutation

2. gene amplifications

3. chromosomal translocations

4. viral intergration

tumor suppresor genes

gene that prevents cancer, a loss of function mutations in these genes allow cancerous growth to occur.

tumor suppressor gene functions

• negative regulators of cell division. Rb acts as a “brake” to stop cell cycle.

• Maintainers of genome integrity.

how p53 works

1. activates genes that promote DNA repair

2. activates genes that arrest cell division and repress other genes required for cell division

3. activates genes that promote apoptosis.

how a tumor suppressor can be silenced

1. a mutation in the tumor-suppressor itself

2. Aneuploidy

3. epigenetic changes

cancer being multistep

most cancers, particularly those associated with aging require the accumulation of mutations in multiple genes over time. Ex: colon cancer: This typically involves the loss of the APC tumor suppressor, followed by the activation of the ras oncogene, and eventually the loss of the p53 and DCC tumor suppressors.

predisposition

While most cancers are "sporadic," about 5–10% of cases are due to a germ-line mutation inherited from a parent.

Mech: people inherit one defective copy of a tumor suppressor gene, making them heterozygous.

Loss of heterozygosity (LOH)

Cancer occurs if the second, normal copy of the gene is lost or mutated in a somatic cell. This pattern of inheritance often appears dominant in pedigrees because the likelihood of the second mutation occurring eventually is very high. ex: breast cancer, colon cancer, etc.