T3 Genetics

Genetics basics

genes

prokaryotes V eukaryotes

definitions:

  • gene: a sequence of DNA that codes for a specific trait
  • DNA: genetic matieral
  • locus: the position of the gene on the chromosome
  • alleles: alternative forms of a gene

genome: the totality of all genetic information of a cell/ organism including non-coding DNA

  • the genome of a specifies is not a good indicator of complexity
  • found by identifying common sequences (expressed sequence tags, presence of transposons and pseudogenes(non-functioning DNA) make this hard)
  • for example, humans have 46 chromosomes, around 3 billion b.p and 21,000 genes and only 1.5% of those are coding sequences

human genome project: an innitiative to sequence the human genome, completed in 2003, led to 4 main things:

  • Mapping - know where all the chromosomes and its gene loci are
  • Ancestry: figure out evolution
  • Medicine - find new proteins for medicinal uses
  • Screening - detecting diseases

size of genomes can usually be represented as this:
eukaryotes> prokaryotes> virus and bacteria
plants just kinda are all over the place due to polyploidy

mutation: a change in the base sequence of a gene/ chromosome, and lead to the production of new alleles

types of mutations:

  • point mutations: change in a single nucletide in the base sequence
    -- insertion
    -- deletion
    -- substitution
    -- inversion
    can lead to silent (no change)/ missense (change 1 amino acid)/ or nonsense (premature stop codon)/ frameshift (changes reading frame)


  • block mutations: change made to a segments of a chromosome:
    -- duplications
    -- deletions
    -- inversions
    -- translocations
    image

can lead to disorder such as:

  • sickle cell anemia
    caused by a mutation on the non-trasnscribed strand GAG to GTG, which causes the 6th codon on the mRNA strand to become GAG to GUG, changing it from glutamic acid to valine

this causes the proteins in RBC to be insoluble and fibrous, causing it to have a sickle shape, be weak, inefficient in carrying oxygen and easily clot up.


however those who are heterozygous for sickle cell anemia enjoy heterozygous advantage against malaria, as the virus breeds in RBC
image image

applications

Karyotyping: arresting a genes during mitosis and taking a screenshot of a person's chromosomes, used to determine sex and genetic conditions pre-birth

autoradiography: used by John Cairne to determine the length of chromosomes
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showed that DNA replication creates a replication bubble and that it is bidirectional
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their differences:

diploid v haploid:
2n v n

  • diploid has two alleles of each trait, somatic cells are diploid and they are present in most animals and many plants
  • haploid cells have 1 allele of each trait, are present in humans as gametes, present in bacteria and fungi

autosomes v heterosomes:

  • autosomes are body cells
  • heterosomes are sex cells, boys XY girls XX, therefore it is the fathes gamete that determines the sex of the child

eukaryotes

chromosomes:

  • have a pinching point called a centromere, the short arm is p and the long arm is q.
  • each chromosome has unique banding patterns
  • as we inherit both of our parent's chromosome, we have two sets of each allele, these two chromosomes are called homologous chromosomes (same structure and loci of the gene, diff alleles)
  • genes can be located using 3 categories, number of the chromosome. arm, g-band location
  • the position of the centromere of a chromosome can be split in 4 categories:
  • meta-centric - equal p and q
  • sub meta centric - basic chrom shape, p a bit shorter than q
  • acrocentric - p much shorter than q
  • telocentric - no p
    image

chromosome number: diploid number of chrom present in an species

  • does not determine genetic complexity (i.e. tomatoes have 24 chrom, 950 million b.p and 32000 genes)
  • different number of diploids means they cannot create offspring, if offspring is created, then they wlll be infertile

down syndrome:

  • caused by a trisomy on chrom 21, which is caused by non-disjunction event in one of the parental gametes
  • causes mental and physicaly delays

process: cells are taken from feotus then chemicallly induced to undertake cell division so wecan arrest in mitosis, then stained and photographed.
sample can be take in 1 of 2 ways:

  1. Chronic Villi sampling: removal of placental tisssue via tube inserted trhough cervix, done at 11 weeks, 1% miscarraige
  2. amniocentesis: extraction of amniotic fluid with needle, at 16 weeks, 0.5% misarrange
    image

DNA of eukaryotes is linear and bound with histone proteins for storage, they bind with an octamer of histones to form a nucleosome:
image

meiosis: the process by which sex cells are made
happens via 2 divisions:

  1. to split the homologous chromosomes (dip->hap)
  2. to split the sister chromatids (created during replication in interphase, are identical copies of one another)
  • important because most sexually reproducing organisms are diploid, meaning in order for the sex cells to fuse they must be halved, to form a diploid, if this does not occur the number of chrom will double (polyploidy)
    image

genetic variation

stages:
meiosis is the same as mitosis except that it happens twice:
both are precceded by interphase and have the substages of prophase, metaphase, anaphase, telophase, within each division.
Meiosis 1 (splitting of homologous chrom):
P: chrom condenses, nuclear membrane dissolve, homologous chromf form bivalents crossing over occurs
M: spindle fibers from centromere attacthes to bivalent chrom at centromere and moves them to align in the center of the cell
A: spindles pull at the chrom, splitting the homologous chrom
T: , chrom decondense, nuclear membrane may reform, cytokinesis occurs


Meiosis 2:
P: chrom condenses, nuclear membrane dissolves, centromeres move perpendicular to last position
M: spindles connect to chrom at centromere and align in the middle
A: spindles pull the sister chromatids apart
T: chrom decondense, membrane refrom cytokinesis occurs
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mitosis v meiosis:

  • both preceded by interphase, go through same pathway (PMAT) and cytokinesis
  • difference
    image

abnormal situations

crossing over:

  • a source of genetic variation that occurs only in prophase 1
  • homologous chromosomes will form bivalents through a process called synapsis , with points of connection being called chiasmata or chiasma
  • crossing over occurs when the nonsister chromatids exchange genetic information, creating recombinants, creating 4 genetically unique haploid daughter cells (not present in either parent)
    image

random assortment:

  • refers to how the placement of homologous chromosomes can contribute to genetic variation during metaphase 1, i.e. maternal on left/right and paternal on left/right
    • the orientation of homologous pairs is random, therefore increase variation, also the orientation will affect which haploid gets which genes
  • can figure out the number of possible combos of any species with the equation, 2^n
    image

fusion of random sex cells:

  • allows for increased variability, as each zygote will be different
  • other than identical twins who spilt via fission of ONE fertilized zygote

images:
image
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non-disjunction: when the chrom fails to seperate correctly. can happen in either:

  • anaphase 1 - forming 4 affected daughter cells

  • anaphase 2 - forming 2 affected daughter cells


    image


  • there are a few conditions related to having extra or missing chrom:

  • down syndrome (trisomy 21) - (occurs as one of the parental gametes experienced non-disjunction and has 2 21 chrom, therefore resulting in 3 in the offspring)
  • turners syndrome (monosomy X)

  • Klinefelter's syndrome (XXY)


  • research shows that chance of non-disjunciton occuring increases with age, especially for mothers ("to developing oocytes being arrested in prophase I until ovulation as part of the process of oogenesis")


    -- risk increase signifigantly after age of 30 for women


    -- risk higher in meiosis 1


    -- as avg maternal rate increases, so does avg rate of down syndrome


click to edit

drawing meiosis:

  • making sure to include interphase, M1, M2, and cytokinesis
  • Int -> chrom not replicated yet so (I-> X)
  • M1 splitting of homologous chrom (XX -> X,X)
  • M2 splitting of sister chromatids (X -> I, I)
  • cyto splitting of cytoplasm and forming individual cells
    image

somatic (meaning body) mutations:

  • cannot be inherited
  • occurs in one body cell
  • only tissues derived from it are

germline (meaning being able to develop into a new thing + successive) mutations:

  • can be inherited
  • occurs in gametes
  • every cell affected

polyploidy: when an organism contains more than two sets of the same chromosome in all cells (i.e. more than diploid)

  • occurs if meiosis does not happen before fertilizations
  • common in plants due to lack of separate sexes and self-pollination
  • not common in animals due to consequences of having more/less chrom

PCR + gel electrphoresis + DNA profiling：
PCR: aka polymerase chain reaction is a lab technique used to replicate large amounts of DNA, each reaction cycle doubles the amount therefore 30 cycles creates around 1 billion copies

  • uses heat:
  1. denature at 95C for 1 min, to split the strands in two
  2. annealing, at 55C for 1 min, primers attach to the strand
  3. extending, 72 C for 2 min, heat tolerant DNA polymerase (Taq)attaches to primers and begins replicating
    image

gel electrophoresis: seperating the dna sample via electrocurrents

  1. DNA sample is put on gel and an electric current is applied which causes the sample to move to the positive terminal
  2. small samples move farther, creating a map or the different dna
    can be applied to DNA and protein:
  • for dna: DNA can be cut into different lengths using restriction endonuclease, generating different samples, the samples are attracted to the positive terminal due to the negative phosphate group on the DNA
  • when placed in agarose gel the fragments will move cuz of the current and can be further idetified through radioography
  • for protein:their size (affect speed) depends on how they fold, and their charge as well(usually non linear), therefore to make it linear, proteins are treated with anionic detergent, and create a uniform negative charge
  • sample are then placed in polyacrylamide gel and it seperates, the samples can then be identified via staining it with a cerain antibodies

DNA profiling: a technique used for paternity testing and crime investigations, allows scientists to identify people based on their DNA, as in DNA there are satalite DNAwhich contains STRs,(repeating sequences) the number of repeats are unique to each satalite locus on a chrom, therefore can be used to identify a person

  • procedure:
  1. sample is collected and amplified using PCR
  2. restriction endonuclease cuts out the STRS to generate fragmetns
  3. fragments seperated with gel electrophroesis generating unique profiles


number of loci used to make profile is dependent on the compared population

  • the profiles must be a complete match
  • all fragments in offspring should also appear in parents

gene transfer + stem cuttings
gene transfer: the process of taking a desired gene and placing it in a host for production (also called gene modificaiton), modified organisms are called transgenic
process:

  1. isolating gene and vector:
  • dna separated via centrifuging, nucleus is heavy and will sink to bottom, then amplify using PCR, can also use reverse transcription to get dna
  • for vector(used to carry gene into foreign cell), commonly use bacterial plasmids as they are capable of autonomous self replication but can also use modified viruses and artificial chrom
  1. digestion with restriction enzymes:
  • both vector and gene must be cut with restriction enzymes at specific recognition sites, they enzymes wil generate sticky ends (complmentary overhangs)
  1. ligation of vector and insert
  • gene of interest is inserted in plamid vector that has been cut with same restriction enzymes, and they stick due to complementary overhangs
    then spliced together by DNA ligase (forming a covalent phosphodiester bond)to form recombinant construct
  1. gene of interest intorudce to host or organism through transinfections (euk) or transormaiton (pro), antibiotics can be used to identifiy if the incorportion was succesful or not by monitoring growth , then the successful ones are isolated and purfied and made to produce
    image

Inheritance

basics:

  • zygosity:
    -- homozygous means the maternal and paternal alleles in offspring are identical
    -- heterozygous means they are not the same
    -- hemizygous means only 1 allele, i.e. sex chrom
    image
  • modes of inheritance:
    -- genotype refers to type of gene combos possibile from breeding
    -- phenotype refers to the observable characteristics presented as a result from the genes present and can be influecned by environment factors
    -- complete dominance: dominant mask recessive, unless recessive is homozygous
    -- co-dominance: pairs of alleles are expressed equally, heterzygotes have joint effect, when represetning use superscripts (big letter still show in capital or lower case)
    image
    example of co-dominance:
  • blood type: blood type is catagorised by the structure of its surface glycoprotein, one gene has multiple alleles that control for Bloodtype, (A, B, O)
  • A and B are condominant (alter strucutre) with O being recessive(doesnt change anything)
    this means that these combos are possible for blood type:
    image
  • however because humans produce antibodies in response to foriegn bodies, it means that:
    image
    image

mendel and his discoveries:

  • he crossed purebred plants and took their seeds and crossed them again and came to a few conclusions to which we derived 3 laws from:
  1. law of segregation: when gametes form, alleles are separated so that each gamete carries only 1 allele for that gene
  2. law of independent assortment: segregation of alleles for one gene occurs independently to that of any other gene, except for genes located on the same chrom
  3. principle of dominance: the dominant allele will mask the recessive alleles
    image

punnet squares:

genetic diseases:

  • autosomal recessive - only occur if both are present, heterzygous inviduals are carriers, i.e. cystic fibrosis
  • autosomal dominant - only need 1 copy of allele, i.e. huntingtons disease
  • codominant alleles only need 1 faulty allele, but heterzygous ppl will have less severe sideffects, i.e. sickle cell anemia
  • sex-linked - where the mutated gene is located on any sex chrom, as y is much shorter than X, X tends to be the linked gene
    image
    they are different from autosomal traits as they are hemizygous, and are "bias" toward genders, females more x linked dom traits, males more x linked recssive
    (only females can be carriers, male offspring must recieve X from mom)(written like codominant alleles)

cyctisc fibrosis:

  • autosomal recessive disorder, mutation on CFTR gene on chrom 7
  • those with this disorder produce lots of thick mucus in their lungs, clogging their airways and secertory ducts of the digestive system, casuing resiraratory issues and pancreatic cysts

Huntington's disease:

  • autosomal dominant disorder, mutation on HT gene on chrom 4
    -has a repeating nucleotide sequence (CAG), that in small amounts 10-25 is okay, over 28 it gets unstable ad continually repeats, causing the huntingtin protein to misfold and degenerate
  • usually occurs in late adulthood, causing uncontrollable spasmodic movements and dementia

over 4000 identified genetic disorders, but since such disorders drasticlly affect chances of living, they are rare.
recessive condition tend to be more common due to the presence of carriers, whereas dominant conditions may often be lateset, as does not affect reproduction

red-green color blindness + haemophillia:

  • X-linked recessive conditions, more common in males than females
  • haemophillia, cant stop bleeding due to defective gene on cogaulation on X chrom, therefore cant form fibrin, meaning they bleed a lot
  • red-green color blindness, mutation in red/green photoreceptors, located on X chrom, can be diagnosed using the ishihara color test

mutations can be induced or spontaneous:

  • they can be induced through mutagens:
    -- raditation - gamma UV xray
  • chemicals - reactive o2 species
    -- biological agents - bactieria / virus
    (carinogens are an exmaple of a mutagen)

link to chernobyl and hiroshima, caused increase in cancer develpment, reduced t cell counts, organ specific issyes, thyroid disease, chernobyl still is unsafe for human living

reading pedigree charts

lethal alleles: alleles that cause an organism to die when present in homozygous condition, must be an essential gene that is dominant or recessive
example: achondroplasia, genetic condition causing dwarfism, autosomal dominant
however is homozygous dominant, offspirng will die, heterzygous dom lead to dwarfism and recessive leads to normal height.
usually punnet grids lead to 1:3, however due to certain death of homozygous dominant, the ratio is 2:1

extra:

  • epistasis: where one gene controls the expression of another gene
    example, in mice the gene for hair color is determined by the gene for pigment production, if no pigment, no hair color, and mice is albino (same for baldness and hair color)
    image image
  • pleiotropy: 1 gene affects multiple traits, therefore lead to severe consequences
    example, sickle cell anemia, lead to increased lethary and low O2 levels, can clot easily, break easily, lead to organ failure or heart attack
  • x-inativation, one of the two X crhom in females is inactivated, so that they do not produce twice as many x-lined genes as males. the process is random in mammals, meaning it can be different in diff cells. X chrom is inactivated permanetly by packaging it as heterochromatin (forming a structure called a Barr body)
    image
  • mosaicism: tge presence of two populations of cells with disticnt genotypes within a single organism, can be caused by either mutation or divsion errors. more pronounced the earlier it occurs, as it affects a greater amount of cells
    image

cloning: clones are groups f genetically identialy organisms or cells derived from a single parental cell, organisms that reproduce asexually will produce clones, and it can also happen in sexually reprouducing organisms (just more rare)
process:

  • somatic cell transfer: making a clone from an adult
    , can be used to clone, or can be used for theraputic cloning (i.e. making an organ)1. somatic cells (body cells) are taken from an adult donor and cultured
  1. an egg from a female is taken and the nucleus is removed (enucleated egg)
  2. somatic cell nucleus is injected in egg
  3. an electric current is applied to the egg to force it to divide and develop into embryo
  4. embryo is implanted into uterus of surrogate
    image

GMO debate:
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example: Bt corn:
bt corn is a type of genetically modified maize that incoorporates an inseticide producing gene that is deadly to certain types of larvae, (corn borer), however people worry that it is killing the monarch butterfly, as its pollen can get dusted on milk weeds (butterfly main food source).
studies found that in lab conditions, when feeding butterflies bt corn dusted milk weed leaves, the mortality rate was higher, however another study found that in real life, there was a minimal correlation

natural cloning: most plants, all bactiera, most fungi and come animals all have antural ways to clone
animals:

  • binary fission: dividing the self equally by two, producing identical daughter organisms
  • budding: cells split off the parent organisms, generating a smaller daughter org that will eventually seperate
  • fragmentation: new org grows from seperated fragement of parent (starfish)
  • parthogenesis: embroys formed from unfertislized ova, female makes diploid eggs
    image

plants:

  • are able to do vegatative propagation whereby small pieces can grow indepenetly, almost all root and shoot plants can do this (e.g. onion ad garlic bulbs are all geneticlaly identcal), underground stems can form new plants such as potatoes, and runners (horizontal stems) can grow roots and shoots out of them, also some do it via spores
    image

humans:

  • monozygotic : 1 fertilisation occurs from on zygote(100% dna shared), split into two genetically identical embryos
  • dizygotic are not identical twins but are t image wins, two seperate fetrilidations (50% of dna shared)
    image

artificial cloning: can be done in two ways:

  • by splitting a normally fertilised embryo in the lab, as embryos are pluripotent therefore they should be able to develop into a whole organism, the downside of this is that it needs to have early in the developmental cycle (cycle 8) and you do not know what the offspring will look like
  • another way is somatic nuclear transfer

types of vector delivery:

  • non- viral:
    most common method is using a plasmid due to its abilities, there are a few ways that you can get the plasmid in:
    -/electroporation - making temporary holes in membrane(mm)
  • heat shoccking - destabilising mm
  • particle bombarment - dna coated particles shot through a gene gun
  • microinjection - a glass micropipette is used to inject the vector directly into the cell
  • lipoinfection- vectro transferred within a lipsome
  • viral: use virus to inject vector in, advatage: protein synthesis will be driven by endogenous expression patterns, bad: injection can damage exisitng host genes
    viruses either have DNA(adenovirus) or RNA(retrovirus) genome,
    "Retroviruses use an enzyme called reverse transcriptase to convert the RNA sequence into a DNA copy prior to integration"

gene therapy: involves removing the defective genes and replacing them with working genes/ DNA, usually viral vectors are used due to their ability to integrate new DNA into the host
example: ADA deficiency - autosomal recessive
caused combined immunodeficiency disorder, those who do gene therapy have shown a steady increase in level of ADA in cells


gene silencing: by inserting the Cre gene into one parent and the LoxP gene into another, when they breed to have offspirng, the offspring will have the Cre-LoxP system in their body, which will remove the gene located between the Lox sites to create a conditional knockout (gene slienced)


links to siRNA - a double stranded rna that is 20-25 bp in length, it inerferes with gene expression by causing mRNA transcripts to break prioro translation.
process:

  • when unwound, one strand degrades(passegner strand) and the other will hybridise(guide strand) to its complementary rna sequence and recruit a RISC protein, the RISC protein will then destroy the target mrna, preventing translaiton of that gen image e