2 UNIT GENETICS How cells Reproduce
I. Reproduction A. Asexual reproduction One parent 2 identical offspring 1. Who uses it? a. prokaryotes b. single celled eukaryotes (protists, yeast) c. some multicellular organisms (plants, starfish) d. some somatic cells (body cells) in multicellular organisms 2. ConsequenceAll offspring are genetically identical (bacteria in lungs multiply) B. Sexual reproduction 2 parents offspring that are NOT identical to either parent
II. Eukaryotic Cell Cycle-(2 phases) A. Interphase 3 subphases: (90% of cells time in interphase) 1. G1 (Gap 1) before DNA synthesis a. cell increases/duplicates the organelles b. growing in size 2. S (Synthesis): DNA replication (copy necessary) a. chromosomes replicate and remain joined at their centromeres (middle of the chromosome) = sister chromatids (identical) alone = chromosome, duplicated = chromatid b. continued growth of the cell 3. G2 (Gap 2) after DNA synthesis a. protein synthesis (getting ready for division) 1
b. general metabolic activity (ATP is available) B. Mitosis (M)- 4 subphases: 1. Prophase a. DNA condensing (starting to become visible under light microscope), coiling more b. Nuclear envelope and nucleolus- begin to break up and disappear c. Spindle begins to form (made up of microtubules) One end of spindle joined to: one sister chromatid at its kinetochore at the centromoric region
Other end of spindle joined to: pole at microtubule organizing center (MTOC) or centrioles = animal cells d. Sister chromatids (replicated chromosomes) move towards the center of the cell (equator) 2. Metaphase "middle" a. Sister chromatids at the metaphase plate (equator) b. Spindle fully formed Some spindle fibers at the equator are not attached to chromatids, what are they attached to? Attached to other spindle fibers from the opposite pole c. Nuclear envelope disappeared 3. Anaphase a. Sister chromatids separate (called chromosomes again) b.Spindles shorten those joined to chromosomes c. Spindles lengthen those joined to opposing spindle fibers (egg shape)
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4. Telophase/Cytokinesis begins once all chromosomes at spindle poles a. DNA uncoils (becomes less visible) b. Nuclear envelopes and nucleoli- reappear (2 each) c.Spindle disappear d. Cytokinesis "splitting of the cytoplasm" 1. animals: cell at equator form two identical cells (called cleavage)
2. plants cell plate formation. Vesicles of cellulose align and fuse at the equator (2 cells) can't pinch because of cell wall
PRACTICE: To the left is a picture of the chromosomes of a cell in G1 of the cell cycle. Draw what this cell will look like at: 1) G2 2) Metaphase 3) After cytokinesis
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Meiosis and Sexual Reproduction
Definitions/Terms to get started: Gamete a sex cell (sperm, egg) Somatic cell- any cell that is not a gamete (skin cell, brain cell, etc) Homologous chromosomes two chromosomes that are nearly identical (one inherited from mom, one from dad); they have the same genes in the same location but may have different "versions" of those genes sometimes they can be identical Different species have different amounts of chromosomes Autosomes chromosomes 1-22. Chromosomes found in both males and females. Everyone has two copies of them Sex chromosomes determine the sex of the individual (xx = female, xy = male). 2 chromosomes Diploid cells two sets of chromosomes (2n; n=set). Somatic cells are diploid. Goes through mitosis [Humans=46] Haploid cells have a single set of chromosomes (n). Formed by meiosis from a diploid cell [N=23]. Located in Gametes I. Meiosis overview A. Chromosomes number reduced by: one half (a diploid cell in either the testes or overies 4 haploid gametes B. What cells use it? Specialized reproductive cells C. Purpose: 1. To keep the genetic material from doubling with each generation 2. Shuffle the genes to create immense variation
II. Meiosis Stages A. Interphase same as mitosis (G1 S G2) B. Prophase I-similar to prophase of mitosis BUT: a. chromatids are aligned as tetrads (4 chromatids lined up together) b. synapsis ("crossing over" DNA breaks and rejoins between non-sister chromatids. Form hybrid chromosomes.
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C. Metaphase I -similar to metaphase of mitosis BUT... a. tetrads that align at metaphase plate b. Tetrads attached to spindle D. Anaphase I homologs separate "sisters chromatids remain joined"
E. Telophase I and Cytokinesis 1. Spindle disappears 2. Nuclear envelopes and nucleoli may reappear (depends on the species) 3. DNA- may uncoil 4. Cytokinesis results in- 2 daughter cells Have not formed 4 haploid cells yet F. Prophase II Telophase II- same as mitosis
G. Summary: MI- homologs separate MII sister chromatids separate 4 haploid gametes are formed (not identical to parent cell)
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PRACTICE: To the left is the same picture that you saw in our previous lecture of a cell and its six chromosomes in G1. Draw what the cell would look like at: 1) G2 2) Metaphase I 3) Metaphase II 4) After Telophase/Cytokinesis II What is n = ? in this diploid cell? (after you have completed this, revisit your picture of mitosis with the same cell, see the similarities and differences?)
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III. Meiosis leads to enormous genetic variability A. Independent Orientation Orientation of homologs at metaphase I plate. (see slide!) Total number of combinations in a diploid? 2n Ex: If n = 2 there would be 22___ combinations (unique gametes) What about humans? 223 = 8 million B. Random fertilization 1. (2n)(2n) = 223 x 223 2. human = about 64_ trillion combinations C. Crossing over during synapsis of prophase I- completely random, unpredictable infinite number of variations gametes
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Inheritance and Human Genetics
I. Gregor Mendel Father of Genetics A. Four advantages to using pea plants: 1. easy to grow 2. come in many varieties (7 traits) 3. can control fertilization (self or cross-fertilization) 4. can use pure-breeding lines B. Principle of Segregation- "Diploid cells have pairs of genes on pairs of ___homologous chromosomes_______. The two genes of each pair are segregated (separated) from each other during _meiosis___, so they end up in different gametes. II. Terms/Definitions A. Genes a unit of information about specific traits (ex. Pea Color) B. Alleles alternate forms of a gene (ex. Yellow or Green) C. Pure-breeding or homozygote individuals have two identical alleles (AA or aa) A: dominant (yellow) D. Hybrid or heterozygote individuals have different alleles for one trait (Aa) E. Dominant allele allele expressed in a heterozygote A/a = yellow F. Recessive allele allele that is masked in a heterozygote A/a = green masked G. Genotype allelic composition of a gene A/a a/a 3 different types
A/A
H. Phenotype what does the expressed trait look like/act like Ex. Yellow or green
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Tester homozygous recessive for all genes being examined Ex. a/a III. Single gene crosses: (Using Punnett Squares) I. A. Homozygote x homozygote- (A/A x a/a) 1. genotypes- 1 (all the same)
2. phenotypes- 1 (all the same) B. Monohybrid x monohybrid- (A/a x A/a) 1. genotypic ratio = 1:2:1 [Aa/Aa]
2. phenotypic ratio = 3:1
C. Monohybrid x tester- (A/a x a/a) 1. genotypic ratio =
2. phenotypic ratio =
IV. Dihybrids A. Mendel's Principle of Independent Assortment as meiosis ends, genes on pairs of homologous chromosomes have been sorted out for distribution into one gamete or another, independently of gene pairs of other chromosomes. B. What gametes form by Independent assortment? 1. AABB individual? A;B 2. AAbb? A;b 3. AABb? AB; Ab 4. AaBb? (1/4)A;B or (1/4)A;b or (1/4)a; B or (1/4)a;b - known as a dihybrid C. Dihybrid x Dihybrid = _____________ phenotypic ratio (You can do 16-square punnett--or take my word for this!) A/a ; B/b x A/a ; B/b (* 1 A and B gives you the dominant) 9 = AaBb (both dominant traits) 3 = AAbb (dominant for one; recessive for the other) 3 = aaBB (recessive for one, dominant for the other) 1 = aabb (recessive for both)
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^PRACTICE. KNOW RATIOS^
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D. Dihybrid x tester = _1: 1: 1 :1 _ phenotypic ratio 1 = both dominant 1 = one dominant, one recessive 1 = one recessive, one dominant 1 = both recessive V. A few things Mendel did not observe A. Multiple alleles 3 or more alleles (version of genes) for one gene rather than just two [A or a] A1 (red) A2 (brown) A3 (blonde) Example with red blood cells: Three alleles: IA = A antigen IB = B antigen i = no Antigen (on surface) Different possible genotypes: IA/ IA (type A) ; IA//I = type A blood IB /IB (type B) ; IB/I = type B blood i/ i: (type O) IA/ IB = type AB
B. Codominance = when 2 dominant alleles are expressed in a heterozygote
C. Incomplete dominance- one allele of a pair is not fully dominant over the other in a heterozygote, so an "_intermediate__" phenotype is observed. Ex: A = red, a = white A/A x a/a = all heretozygotes; look pink A/a x A/a = 1:2:1 (red AA:pink Aa :white aa)
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To the left is a diploid cell, (2n= 4) that will begin the process of meiosis. Below this, draw what the cell would look like after these chromosomes have replicated and aligned in metaphase I. metaphase I:
** in powerpoints**
Now, go back and reconsider what you just sketched...don't we need to consider independent orientation? Draw the second possible orientation that would arise the other half of the time beside your first sketch. Okay, now draw the cells that would arise from these two orientations at the end of meiosis I (assume there is no crossing over that occurs in prophase I). You should have four cells:
Lastly, draw the eight gametes that could form from the four cells above. You should now have all the possible gamete combinations that can form from this one diploid cell.
How many different unique combinations are there? ______ Lets assume that A-tall, a- short, and B- brown eyes; b-blue eyes. Now, take this male who is A/a; B/b and mate him to a female who is short with blue eyes. What proportion of their children would have the same phenotype as the mother? _______
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Inheritance and Human Genetics (cont.)
I. Probability & Pedigrees for single gene traits A. Rule of Multiplication (Product rule)- the probability of a compound event is the product of __the separate probabilities__. For example, what is the probability that a woman might have four sons? 1/16 x x x = 1/16 B. RARE Autosomal Dominant DISORDERS 1. Need __1 dominant allele___ to have the disease ex: A/a has disease. (mostly) a/a doesn't. A/A has disease 2. Affected individuals usually have one _affected___ parent Doesn't skip generations 3. Examples: a. Huntingtons Disease (affects brain. Effects after age 30-40) chances for diagnosis is (50%) if you have the genotype you WILL develop the disorder b. Achondroplasia C. RARE Autosomal Recessive DISORDERS 1. Need __both diseased alleles (Recessive)___ to have the disease 2. Affected individuals usually have _unaffected___ parents 3. __inbreeding___ increases frequency in a population 4. Examples: Aa
D. Examples of some COMMON autosomal single gene TRAITS 1. hitchhiker's thumb 2. tongue curling
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3. How does the recessive allele differ here from one above such as Taysach's? E. RARE Sex-linked recessive DISORDERS 1. Males inherit only one X, so _only one recessive gene_ is needed in males to have disease (females would need _2__ copies) 2. Males affected usually have _unaffected_ parents 3. _inbreeding__ increases frequency in population 4. Examples a. color blindness b. c. II. Abnormal number of chromosomes (_47- can only have trisomal on the 21st chromosome which gives you down syndrome _) in humans A. Cause nondisjunction if homologous pairs don't separate the way they are supposed to one cell gets both chromosomes, the other gets none 1. Non-disjunction in meiosis I- the tetrads don't split apart (four in one, one ends up with none)
2. Non-disjunction in meiosis II- the homologous pairs don't split apart (two in one, none in the other)
B. Aneuploidy in autosomes 1. Monosomy only have one copy of the allele
2. Trisomy have three copies of the allele (two from one parent, one from other)
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3. Risk increases: in sex cells as people get older (why it's dangerous for older people to have kids: down syndrome or miscarriage)
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C. Aneuploidy in sex chromosomes 1. XXY kleinfelters syndrome (males) Sterile, tall and thin, feminization 2. XYY male, normal "criminal genotype" 3. XO monosomy females Turners syndrome Sterile, wide necks, short stature 4. XXX normal, fertile females
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DNA Structure, Packaging, & Replication
I. What is the genetic material?? A. Fred Griffith- 1928: showed that the genetic material could be transferred (geneteic material from heat killed S strain to the live R) **SEE THE SLIDE** B. Consensus at this time: "eukary. chromosomes carry genetic material and consist of _DNA_____ and __proteins______ C. Genetic material was either _DNA____ or __protein____
I. DNA is the genetic material: historical highlights A. Oswald Avery, Colin MacLeod, Maclyn McCarty- 1944: Built on Griffiths experiment. Proteases did not block the transfer DNAses did block the transfer Therefore DNA, not protein = genetic material B. Alfred Hershey and Martha Chase 1952: 1. Studied T2 bacteriophage = viruses that infect bacteria Consist of DNA and protein 2. Labelled two batches of phages with radioactivity a. only protein b. only DNA 3. Infected bacteria with the two batches, agitated, spun to separate phage proteins (light) and bacteria cells (heavy). a. Results when proteins labeled: Pellet (bacterial cells) were not radioactive b. Results when DNA labeled: pellet was readioactive 4. Conclusion- phage do not transfer protein into bacteria. Phage transfer DNA, which is the genetic material
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II. Structure of DNA (historical highlights) A. Edwin Chargaff 1950 performed biochemical experiments 1. Ratio C:G = 1:1 2. Ratio of A:T = 1: 1 B. Rosalind Franklin and Maurice Wilkins-1951 structural biologists 1. Used _x-ray diffraction_ as their main tool A "shadow of DNA" 2. Their data suggested: Suggested a helix of some sort C. James Watson and Francis Crick- 1953 1. Helix diameter- was uniform 2. Determined that there were two _helices "double helix" 3. Determined that the bases paired: ... based on Chargaffs data
A-T
G-C
III. Review of Structure of DNA, what we know today... A. DNA is a _double_ helix of two long _polynucleotide_ strands B. The two strands of the double helix are _antiparallel_____. 5' --------------------------------------- 3' 3'---------------------------------------- 5' based on structure of the sugar C. Polynucleotides = strinsg of nucleotides
D. Nucleotides = phosphate group, sugar (dioxiribose) and a nitrogenous base (A, T, C or G)
E. Bases bond with each other with _hydrogen_ bonds, how? A = T (two hydrogen bonds) C = G (three hydrogen bonds)
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IV. DNA Replication S subphase of interphase_ A. First stage of __ mitosis or meiosis B. Watson-Crick: "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material" A-T A-T T-A G-C G-C C-G A-T A-T T-A Each strand of a double helix becomes a template for a new strand, how? Because the parent strand is conserved during the process, half of every doublestranded DNA is "old" and half is "new". This is termed _semi conservative___ replication. C. _helicase__ enzyme that breaks hydrogen bonds between bases to unwind DNA into single strands D. DNA polymerase enzyme that adds nucleotides to growing DNA polymer
DNA Polymerase only adds nucleotides to the _3'_ end of a polynucleotide. Because of the _antiparallel__ nature of DNA and because the replication fork (polymerase) only moves in one direction, this means that replication is __continuous____ on one strand and __discontinuous____ on the other.
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Transcription/Translation
I. Overview Transcription translation A. DNA _mRNA___ __protein___ central dogma of life B. DNA bases somehow code for 20 amino acids? 4 nucleotides each code for 1 AA? No. would only give us 4 amino acids 2 nucleotide sequence (AA or AT...) No. would only get 16 amino acids (24) 3 nt sequence (AAG or AAT...) yes. Would get 64 combos (34) = 64 [have leftovers] C. Transcription One of the two DNA strands is the template and is rewritten into the "language" of RNA 1. Template single strand of DNA 2. Complementary strand of: DNA G A C T _mRNA C U G A enzyme performs reaction
D. Translation translate the mRNA into the language of proteins (with the help of transfer RNA)
1. Every triplet of bases (three bases) in mRNA = codon 2. Each codon codes for: 1 amino acid (AA) 3. More than one codon codes for one _amino acid_ Ex: UUU and UUC both code for Phe
II. Transcription in eukaryotes A. Location: occurs in the nucleus B. 3 Players 1. RNA polymerase adds RNA nucleotides to the 3' end of a growing mRNA 2. Single stranded DNA template
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3. RNA nucleotides (ribose + phosphate group + base) floating in nucleus (from metabolism) C. Importance sequences in DNA 1. Promoter signal for "start" of transcription 2. Coding region Sequences that will be expressed in the protein = exons Sequences that will not be expressed in the protein = introns (trash) 3. terminator sequence that signals "stop" of transcription [not transcribed]
D. Post-transcription processing of mRNA 1. 5' cap Purpose: protection from degradation 2. 3' poly A tail 3. introns are removed and exons are spliced together
E. Transcript shuttled from _nucleus___ to _cytoplasm____ = mature mRNA III. Translation A. Location cytoplasm B. 5 Players 1. mRNA (instructions for process) 2. ribosome (forms peptide bonds) 3. transfer RNAS (tRNA) that carry amino acids 4. free amino acids 5. enzymes that attach AA to tRNAs
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C. tRNA: 1. Function: a. carry the appropriate AA b. recognize the appropriate codon in mRNA 2. Structure a.. anticodon- triplet sequence complementary to a specific mRNA codon
Ex: UUU in mRNA, so tRNA: anticodon would be ____ b. AA attachment site (holds a specific AA) D. Ribosomes "protein factories" 1. Function a. bring mRNA and tRNA together b. form peptide bonds between AA's
2. Structure a. Large and small subunits made of- rRNA (ribosomal RNA) and proteins b. P site: binding site for tRNA (hold the tRNA with the growing amino acid chain)
c. A site: binding site for a tRNA (bringing the next amino acid)
E. Important sequences on mRNA a. AUG- codon, "start": check chart and see is coding for Met b. UAA, UAG, UGA "stop" (don't code for amino acids)
F. 3 Phases of translation
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1. Initiationa) small subunt of ribosome binds mRNA b) large and small subunit clamp together c) initiator tRNA (carrying Met) sits in the P site
2. Elongation a. Codon recognition- a tRNA carrying the next AA sits in the A site b. Peptide bond formation links the 2 AA's in the P and A site
c. Translocation: tRNA carrying the growing AA chain moves from the A site ot the P site, the "empty" tRNA leaves the P site 3. Termination recognition of the stop codons polypeptide is freed Overview Example: DNA: TAC GAA TCG ATT mRNA codon: AUG CUU AGC UAA tRNA anticodon: UAC GAA UCG AUU Amino Acid: UAC- met GAA- leu UCG- ser AUU- not a sequence IV. Mutations What is an "allele"? a specific DNA sequence that codes for one version of a gene What is a "mutation"? a change in DNA (spontaneous) during DNA replication A. Base substitutions single base pair change Ex) AAG ACG -> AGG ACG B. Base insertions/deletions- causes "frameshifts" every codon downstream of the mutation changes, very different proteins C. Effect on proteins? Might affect protein structure function affected?
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D. How do mutations arise? 1. Spontaneously during what process? DNA replication 2. Ionizing radiation x-rays, cause double-stranded breaks in DNA (chromosome literally breaks in half, may join again correctly or may not), low level of xray is relatively safe but one get too many is dangerous) 3. Non-ionizing radiation (UV) causes framed mutation Occurs in a cell before replication, cells dividing out of control (passed to daughter cells) 4. Chemicals cause base substitutions E. Consequence of mutations arising in different types of cells: 1. Somatic cell (body cell) lead to cancer 2. Germ cell (pre-gamete) germ line mutation (inherited) pedigrees
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Controls over Genes
What do we mean when we say a gene is turned "on or off" or "controlled"? We might mean transcription of that gene is actively occurring. Some genes are never or rarely transcribed, and some are always (continually) transcribed. Alternatively, a gene could be transcribed but then the mRNA is never translated. This would be another level of control. I. One example of gene control in prokaryotes: negative control over the Lac operon
Controls expression of 3 enzymes involved in: break down of lactose A. Lac operon = DNA, 3 components: 1, 2, and 3 (code for enzymes 1, 2, and 3) 1. Three genes coding for- 1, 2, and 3 (code for enzymes 1, 2, and 3) DNA
2. Promoter- RNA P can bind and begin to transcrip of genes 1, 2 and 3 SEQUENCE 3. Operator repressor protein can bind
B. Other players: 1. Regulatory gene codes for repressor protein 2. Repressor protein that binds operator (DNA) 3. Lactose (modified to allolactose) can bind repressor protein C. If lactose not present, transcription is on or off? Genes 1, 2, 3 1. Repressor- bound to operator, blocks RNA P from binding to the promoter 2. RNAP- is blocked, therefore.. no transcription D. If lactose present, transcription is on or off? 1. Lactose- binding to repressor 2. Repressor- changes shape and can't bind operator 3. RNAP- can bind promoter transcription goes!
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II. Purpose of regulation in multi-cellular eukaryotes... so what makes a red blood cell different from a muscle cell? A. Differentiation cells are specialized in structure and function by having a unique set of genes expressed
B. All cells in our body contain the same: DNA C. Differentiated cells "pick and choose" what? Genes will be transcribed and translated into proteins_
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