Genetics and Stem Cells

Concept Check

• Review where (and how) gene expression can be controlled.

• Explain how transcription factors are involved in gene regulation.

• Compare cell potency (totipotent, pluripotent, multipotent), including their capabilities and examples.

• Explain the function of stem cells in the body, including their role in tissue maintenance and repair.

• Describe reprogramming of cells, including the methods and applications.

Review - Gene Expression

• Chromatin Remodeling: The region of the chromosome must be opened up in order for enzymes and transcription factors to access the gene. This involves changes in the structure of chromatin, making DNA more accessible.

  • Histone modification (acetylation, methylation, phosphorylation)

  • DNA methylation

• Transcription Control: The most common type of genetic regulation, involving the turning on and off of mRNA formation.

  • Promoter and enhancer elements

  • Role of transcription factors (activators and repressors)

• Post-Transcriptional Control: Regulation of the processing of a pre-mRNA into a mature mRNA.

  • Splicing (alternative splicing)

  • mRNA editing

  • Regulation of mRNA stability

• Translational Control: Regulation of the rate of initiation of translation.

  • Initiation factors

  • Ribosome binding

  • miRNA regulation

• Post-Translational Control (protein activity control): Regulation of the modification of an immature or inactive protein to form an active protein.

  • Protein folding

  • Glycosylation, phosphorylation, and other modifications

  • Protein degradation through the ubiquitin-proteasome pathway

• Steps in Gene Expression:

  1. Chromatin remodeling: "Open" DNA (some DNA not closely bound to proteins).

  2. Transcription: Primary transcript (pre-mRNA).

  3. RNA processing: Mature mRNA with Cap-0-0-Tail.

  4. mRNA stability: Degraded mRNA (mRNA life span varies).

  5. Translation: mRNA to Polypeptide.

  6. Post-translational modification: (folding, glycosylation, transport, activation, degradation of protein) to Active protein.

Epigenetic

• Histone proteins and DNA nucleotides can be modified chemically. Modifications affect nucleosome spacing and gene expression. This type of gene regulation is called epigenetic regulation. Epigenetic means “around genetics.”

  • DNA methylation: Typically represses gene transcription.

  • Histone acetylation: Typically enhances gene transcription.

• The changes that occur to the histone proteins and DNA do not alter the nucleotide sequence and are not permanent but can be heritable across cell divisions.

Genes

• A gene can be turned on or off depending upon the location and modifications to the histone proteins and DNA.

Promoter

• Genes are organized to make the control of gene expression easier. The promoter region is immediately upstream of the coding sequence. This region can be short (only a few nucleotides in length) or quite long (hundreds of nucleotides long).

  • Core promoter: Includes the transcription start site and binding sites for general transcription factors.

  • Regulatory promoter: Contains specific regulatory sequences that bind transcription factors.

• In addition to the general transcription factors, other transcription factors can bind to the promoter to regulate gene transcription. These transcription factors bind to the promoters of a specific set of genes. They are not general transcription factors that bind to every promoter complex but are recruited to a specific sequence on the promoter of a specific gene.

Transcription Factors

• Pre-initiation complex (PIC): Includes RNA pol II, AGRA, TF II F, TF II E, TBP, TF II D, TF II, TF II B, TF II H, and DNA

  • General Transcription Factors Bind Promoter

  • DNA + RNA Poly-II + TBP + Transcription Factors (TF) leads to RNA polymerase binding and transcription beginning.

• Transcription factors are proteins involved in the process of converting, or transcribing DNA into RNA. Transcription factors include a wide number of proteins, excluding RNA polymerase, that initiate and regulate the transcription of genes.

  • Activators: Enhance transcription

  • Repressors: Inhibit transcription

• Prokaryotic organisms regulate gene expression in response to their environment. Eukaryotic cells regulate gene expression to maintain homeostasis in the organism.

Eukaryotic Transcription Factors

• Several types of factors affect transcription:

  • Enhancer: A DNA sequence that enhances transcription and can be located far from the promoter.

  • RNA polymerase and basal factors bind at promoter

  • Multiple transcription factors interact with eukaryotic promoters and enhancers

  • High transcription rates are attained by binding of transcription factors to specific genes

  • Transcription factors are often expressed in a tissue-specific manner

  • Eukaryotic TFs function by recruiting other proteins to build large complexes that interact with the transcriptional machinery to activate or repress transcription

  • Activators bind at promoter

  • Activators bind to distal sites in promoter or to enhancers

  • Coactivators connect activators to basal factors

  • Coactivators/regulators act on local structure of gene

  • Basal factors (TFS) and RNA polymerase bind to promoter and TATAA box.

  • Activators are proteins that recognize specific short DNA sequences inducing the efficiency of the promoters.

  • Co-activators are proteins required for a more efficient transcription. They do not bind DNA,

  • Regulators of chromatin structure

Transcription Factors - Binding Site

• Specific sequence on DNA - transcription factors bind

• Eukaryotic RNA Pols cannot bind to promoters on their own (unlike prokaryotic RNA Pols).

• Transcription factors are required for eukaryotic RNA Pol binding.

• General transcription factors allow binding of RNA Pol binding to promoters and a basal level of transcription.

• Gene-specific factors stimulate transcription further (or repress it) and allow fine regulatory control.

• A transcription factor molecule binds to the DNA at its binding site, and thereby regulates the production of a protein from a gene.

Gene Mutations

• Germ-Line: cells that will become eggs/sperm, passed on to offspring

• Somatic: occur in cells of individual

• Causes: chemicals, radiation, DNA mistakes, spontaneous mutations.

Types of Mutations

• Nucleotide: changes in DNA base(s). These can be:

  • Point mutations (substitution of a single base)

  • Insertions (addition of one or more bases)

  • Deletions (removal of one or more bases)

• Chromosome: changes in chromosomes, number or arrangement

  • Duplications

  • Inversions

  • Translocations

Mutations: Sickle Cell Anemia

• Sickle cell anemia is the result of a missense mutation. The mutated protein leads to sickle-shaped red blood cells and sickle cell anemia. The mutation occurs at codon number six of the beta globin gene, resulting in the substitution of glutamine with valine.

Diseases caused by chromosomal mutations:

• Down syndrome: trisomy of chromosome 21. Symptoms: characteristic facial features, usually a delay in mental development, often visual impairment, hearing impairment, skeletal structure defects, cardiac malformation, defects of the gastrointestinal tract

• Edwards syndrome: trisomy of chromosome 18. affected fetuses are usually miscarried; if the child is born, shows a number of deformities and intellectual disability

• Klinefelter syndrome: an additional X chromosome in a male karyotype. slightly above average height, less prominent tertiary sexual characteristics and infertility in people with one additional chromosome X

• Patau syndrome: trisomy of chromosome 13. most fetuses are miscarried, live-born children die in early childhood; numerous defects of internal organs and anomalies in external structure are observed

• Turner syndrome: monosomy of the X chromosome in a female- most fetuses are miscarried; in other cases, shortness, abnormal body proportions, delayed puberty, and infertility are observed

• XXX syndrome: trisomy of the X chromosome in a female- more prominent tertiary sexual characteristics, usually increased height, possibly marginally reduced IQ and fertility

• XYY syndrome: an additional Y chromosome in a male's karyotype- more prominent tertiary sexual characteristics, usually increased height, marginally reduced IQ

DISEASES ASSOCIATED WITH FRAMESHIFT MUTATION

• Crohn's Disease- Crohn's Disease has an association with the $NOD2$ gene. The mutation is an insertion of a Cytosine at position 3020. This leads to a premature stop codon, shortening the protein that is supposed to be transcribed. When the protein is able to form normally, it responds to bacterial liposaccharides, where the 3020insC mutation prevents the protein from being responsive.

• Cystic Fibrosis- (CF) is a disease based on mutations in the CF transmembrane conductance regulator (CFTR) gene. There are over 1500 mutations identified, but not all cause the disease. Most cases of cystic fibrosis are a result of the _\DeltaF508 mutation, which deletes the entire amino acid. Two frameshift mutations are of interest in diagnosing CF, CF1213delT and CF1154-insTC.

  • They both lead to a small decrease in the function of the lungs and occur in about 1% of patients tested.

Stem Cells

• Totipotent: early stage embryo, fertilized egg (In vivo). Can give rise to all cell types including extraembryonic tissues.

• Pluripotent: 8 cell embryo, Blastocyst embryonic stem cells. Can give rise to all cell types of the body but not extraembryonic tissues

• Multipotent: bone marrow, Adult blood stem cells, Different types of blood cells. Can differentiate into a limited range of cell types

  • Specialized: Blood cells Neural cells Cardiac muscle

• Stem cell potency:

  • Totipotent: Capable of giving rise to all cell types of body and extra-embryonic tissues

  • Pluripotent: Capable of giving rise to all cell types of body; ESCs or iPSCs

  • Multipotent: Capable of giving rise to all cell types of a particular tissue or organ

  • Nullipotent: Not capable of giving rise to other cell types

Stem Cell Research

• Zygote → Totipotent cell

• Blastocyst- Trophoblast

  • Primitive Endoderm

  • Epiblast → Pluripotent cell

• Multipotent cell

• Nullipotent cell

• Reprogramming

Therapy

• Gene therapy: fix gene, add correct gene

• Ethics: germ-line cells, passed on to children

Somatic cell gene therapy

• The functioning allele of the gene is introduced into target cells - therefore techniques to get the gene to the target location are needed, or specific cells must be removed, treated and then replaced (this is called ex vivo therapy)

• Introduction into somatic cells means that any treatment is short-lived and has to be replaced regularly. The specialised cells containing the gene will not divide to pass on the allele.

• There are difficulties in getting the allele into the genome in a functioning state. Genetically modified viruses have been tried but the host becomes immune to them so cells will not accept the virus vector on second and subsequent treatments. Liposomes are used but these may be inefficient

• Genetic manipulations are restricted to the actual patient

Germline cell gene therapy

• The functioning allele of the gene is introduced into germline cells - delivery techniques are more straightforward

• Introduction into germline cells means that all cells derived from these germline cells will contain a copy of the functioning allele. The offspring may also contain the allele

• Although more straightforward, it is considered unethical to engineer human embryos. It is not possible to know whether the allele has been successfully introduced without any unintentional changes to it, which may damage the embryo.

• Genetic manipulations could be passed on to the patient's children.

Cell Fate

• Cell Determination - What type of cell

• Cells divide and become specialized

• Transcription factors

• All cells are offspring of the original zygote - how do cells "know" what kind of cells they should become?

• During development regulatory mechanisms turn specific genes on and off, leading to differences in gene expression in different types of cells, even though all cells of an organism have the same genome

Importance of Cell Migration

• Embryonic development

• Tissue turnover

• Wound healing

• Immunity

  • Cells and tissue layers of a Gastrula: Ectoderm (External Layer), Mesoderm (Middle Layer), Endoderm (Internal Layer)

• Germ cells - Become Gametes; Oocyte or Sperm

Development

• Cells Differentiate - Regulated

• Stem Cells - Embryonic - Totipotent

  • Germ Cells - Become Gametes

• Oocyte → Sperm →ZYgote → Morula → Blastocyst → Embryo- Inner cell mass: PGCs → ES cells → Embryonic germ cells

Stem Cells

• Stem cells - can replicate themselves; can create new cell types

• Stem Cell - Can produce new cells

Research

• Embryonic: Totipotent - can become all cells in body (also placenta)

• Blastocyst stage (inner cell mass) - pluripotent - can become all cells in body

• Adult: multipotent - can become specific type of cells

• Induced: adult cell - factors added - can become specific types of cells

Reprogramming

• Reversal of a differentiated cell type to an undifferentiated state then, redifferentiation into the cell type of choice in vitro is known as reprogramming

• The process can be divided into two stages:

  • Dedifferentiation - Conversion of adult somatic cells into the pluripotent state

  • Redifferentiation - Conversion of the pluripotent cells into differentiated cells of choice

• Start with differentiated/specialized cell

Concept Check

• Review where (and how) gene expression can be controlled.

• Explain how transcription factors are involved in gene regulation.

• Compare cell potency (totipotent, pluripotent, multipotent).

• Explain the function of stem cells in the body.

• Describe reprogramming of cells.

Stem Cells & Cancer - Lecture 33 - Spring 2024

Concept Check

• Describe how gene therapy works, including delivery methods and ethical considerations.

• Explain cell differentiation in the process of development, including the signaling pathways involved.

• Explain cell reprogramming, including the factors and mechanisms involved.

• Describe SCNT and how Dolly was created, including the challenges and implications.

• Compare apoptosis vs necrosis, including the morphological and biochemical differences.

• Explain the genes involved in cancer (oncogenes, tumor suppressor genes), including their roles and mechanisms of action.

Genetic Code

• Genetic code refers to the instructions contained in a gene that tell a cell how to make a specific protein. Each gene’s code uses the four nucleotide bases of DNA (adenine (A), cytosine (C), guanine (G) and thymine (T)) in various ways to spell out three-letter “codons” that specify which amino acid is needed at each position within a protein.

• The genetic code is redundant in that more than one codon may code for the same amino acid.

  • For example: CUU, CUC, CUA, CUG all code for leucine.

Therapy Stem Cells

• Harvested

• New Tissues

• ICM = cells removed & grown in culture - differentiate into different cells

Clones

• Genetically identical copy of an organism

• Naturally occurring

  • Twins - fertilization of a single egg with a single sperm. As those cells divide and multiply, at some point very early in embryonic growth they split into two individuals.

• Created in the lab- There are three different types of artificial cloning:

  • Gene cloning produces copies of genes or segments of DNA.

  • Reproductive cloning produces copies of whole animals.

  • Therapeutic cloning produces embryonic stem cells for experiments aimed at creating tissues to replace injured or diseased tissues.

Difference

• Reproductive: Clonal embryo to implant for producing a child

• Therapeutic: Clonal embryo to generate ES cells for treatment

Dolly

• Dolly the sheep was born on 5 July 1996.

• 1996, Scientists in Scotland have announced the birth of the world's first successfully cloned mammal, Dolly the sheep.

• was a female domestic sheep.

• first mammal to be cloned from an adult somatic cell, using the process of nuclear transfer.

• The sad part of Dolly the Sheep was she died on February 14th, 2003, nearly six years after her birth progressive lung disease was caused because her cells were already old she also had premature arthritis.

• It turns out that her telomeres are only 80% as long as those in a normal one-year-old sheep.

• The examination of DNA from Dolly's cells revealed that her telomeres were abnormally short.

• Cloning process:

  • Remove a donor Egg Cell

  • Enucleation: Remove the nucleus from the egg

  • Mammary Cell: Remove cells from a sheep udder

  • Select a single cell

  • Combine Cells using an electric shock. The combined cell now has a single nucleus from the nuclear donor.

  • The combined cell begins to divide normally to become a blastocyst.

  • The blastocyst is placed in the uterus of a Surrogate Ewe

  • The blastocyst develops into a fetus and in about five months a lamb is born - Dolly

Medical Advances by Cloning

• Cloned animals can be genetically engineered to be more nutritious or to carry human traits that could lead to development of new medicines.

• How Dolly the sheep was cloned:

  • Mammary cells removed from a 6-year-old ewe in its last trimester of pregnancy.

  • Cells placed in a culture dish and starved of nutrients to the verge of death.

  • Rescued cell inserted into unfertilized sheep egg (containing no DNA), Egg thinks it is fertilized and embryo production begins.

  • Egg implanted into a ewe ready for pregnancy.

  • After 148 days, Dolly, a healthy lamb, was born. It took scientists 277 tries before producing Dolly.

Dolly the sheep

• Dolly the sheep may have been the world's most famous clone, but she was not the first. Cloning creates a genetically identical copy of an animal or plant. Many animals - including frogs, mice, sheep, and cows - had been cloned before Dolly. Plants are often cloned - when you take a cutting, you are producing a clone. Human identical twins are also clones.

• So, Dolly was not the first clone, and she looked like any other sheep, so why did she cause so much excitement and concern?

• Because she was the first mammal to be cloned from an adult cell, rather than an embryo.

• This was a major scientific achievement but also raised ethical concerns.

SCNT

• Somatic Cell (body cell) Nuclear Transfer

• DNA from one cell put into egg cell w/out nucleus

• Activated - grows Somatic Cell (Body Cell) Nucleus Egg Cell Nucleus Removed Egg Grows Into Embryo - Somatic Cell DNA

Telomeres

• In a normal cell every time a cell divides, telomeres (ends of DNA) become shorter

• Telomerase (enzyme) restores telomere length

• Aging occurs (due to telomere shortening), and cells die - Dolly

• In cancer cells, telomeres remain unchanged (cell does not die)

Therapeutic Cloned Cell

• Induced

• Forms Embryo

• Stem Cells Harvested

• Nuclear Reprogramming

• Genes Inserted

• Induce Reprogramming

• Resemble ES Cells

Cell Death

• Damaged/Old/Incorrect Cells -- Cell Death

  • Apoptosis: Programmed - Neat

  • Necrosis: Unprogrammed Death

  • Process of Apoptosis: Cell begin apoptosis → Formation of blebbing → Nucleus condensing → Apoptotic body → Partition of cytoplasm and nucleus into apoptotic bodies → Phagocytosis

• Apoptosis:

  • Programmed cell death.

  • Death cycle is programmed by the cell itself

• Autophagy:

  • 'Self-eating'

  • Catabolic process involving lysosomes.

• Necrosis:

  • 'Death' caused by external factors like trauma or toxins.

  • Not programmed.

Types of Cell Death

Apoptosis

• Programmed Cell Death

• Elimination of Unwanted cells

• Cell Shrinks

• Nuclear Fragmentation

• Phagocytosis of Apoptotic Bodies (No Inflammation)

• Nuclear Condensing and Degeneration

Necrosis

• Always Pathological

• Cell Enlargement and Swelling

• Loss of Membrane Integrity

• Leakage of Content and Disintegration

• Inflammation

Differences Between Necrosis and Apoptosis

Necrosis:

• increase in cell volume

• loss of plasma membrane integrity

• leakage of cellular contents

Apoptosis:

• cell shrinkage

• plasma membrane blebbing

• formation of apoptotic bodies

Cancer

• Cancer begins when cells acquire the ability to grow uncontrollably and ultimately invade and damage the body’s normal tissues.

• Genes are segments of DNA located on chromosomes and can mutate over time to become cancerous.

• These mutations can result from a variety of causes, including diet and lifestyle choices as well as exposure to certain environmental factors.

Cancer

• Uncontrolled Cell Division

• Tumor Suppressor Genes: Removed or Inactivated

• How cancer starts:

  • Normal cells

  • Abnormal cells multiply

  • Cancer in-situ

  • Angiogenesis - Malignant or invasive cancer

• Normal Cells vs Cancer Cells

  • Normal Cells: Small, uniformly shaped nuclei; Relatively large cytoplasmic volume; Conformity in cell size and shape; Cells arranged into discrete tissues; May possess differentiated cell structures; Normal presentation of cell surface markers; Lower levels of dividing cells; Cell tissues clearly demarcated

  • Cancer Cells: Large, variable shaped nuclei; Relatively small cytoplasmic volume; Variation in cell size and shape; Disorganised arrangement of cells; Loss of normal specialised features; Elevated expression of certain cell markers; Large number of dividing cells; Poorly defined tumor boundaries

Risk Factors for Cancer

• Cigarette smoke

• Ultraviolet rays

• Preserved meats

• Excess body weight

• Pollution

• High Quantities

• Artificial sweeteners

• Alcohol

• Pesticides

Causes of Cancer

• Inherited mutations in genes that affect cell cycle, DNA repair, or apoptosis: these mutations give a genetic predisposition for cancer.

• Somatic mutations to these same genes caused by:

  • Exposure to risk factors- environmental mutagens (carcinogenic chemicals, radiation)

  • hormones

• weakening of immune system (as in AIDS).

• Oncogenic (tumor) Virus infections

  • Epstein Barr virus (causes Burkitt lymphoma)

  • Human Papilloma Virus (causes cervical cancer).

• Tumor viruses transform human cells into cancer cells by:

  • Introducing viral cancer-causing oncogenes into host cell DNA

  • Causing Translocation and overexpression of host protooncogenes.

How Being Overweight Can Cause Cancer

1. Fat cells make extra hormones and growth factors

2. Hormones and growth factors tell cells in our body to divide more often

3. This increases the chance of cancer cells being produced…

4. …which can then continue to divide and cause a tumor

Mutations

• INITIATION → PROMOTION/PROGRESSION → METASTASIS

• Normal cell

• Mutation → Chromosomal damage

• Immortalized Cell

• Transformation

• Mutation

  • Tumor Suppressor Gene = Inactivated

  • DNA Repair Gene = Inactivated

  • Proto-Oncogene = Stimulated

• Mutation inactivates tumor suppressor gene CELLS PROLIFERATE

• Mutation inactivates DNA repair gene Transformed cells

• Mutation inactivates several more tumor suppressor genes

• Mutation of proto-oncogene creates an oncogene CANCER

• Invasion of blood cells → Establishment of tumors Multiple tumor sites in the body

Genes & Cancer

• Genetic changes that contribute to cancer affect three main types of genes:

  • Proto-oncogenes- Proto-oncogenes are involved in normal cell growth and division. However, when these genes are altered in certain ways or are more active than normal, they may become cancer-causing genes (or oncogenes), allowing cells to grow and survive when they should not.

  • Tumor Suppressor Genes- Tumor suppressor genes are also involved in controlling cell growth and division. Cells with certain alterations in tumor suppressor genes may divide in an uncontrolled manner.

  • DNA Repair Genes- DNA repair genes are involved in fixing damaged DNA. Cells with mutations in these genes tend to develop additional mutations in other genes. Together, these mutations may cause the cells to become cancerous.

• Mutated genes do not behave normally

Cancer Genes

• Oncogenes and Tumor Suppressors

• There are two broad categories of genes to think about when considering cancer-forming mutations:

  1. Oncogene - An oncogene is a gene whose normal activity promotes cellular proliferation or division. Oncogene = gas pedal

  2. Tumor Suppressor - a gene that inhibits events leading towards cancer. Tumor Suppressor gene = brakes

p53

• The p53 gene is a tumor suppressor gene, i.e., its activity stops the formation of tumors.

• Mutations in p53 are found in most tumor types and so contribute to the complex network of molecular events leading to tumor formation.

Concept Check

• Describe how gene therapy works.

• Explain cell differentiation in the process of development.

• Explain cell reprogramming.

• Describe SCNT and how Dolly was created.

• Compare apoptosis