Biology 2010 Lecture Notes

Unit 5. Genetics, Inheritance, and Evolution

Some key terms and phrases

• DNA replication:  origin of replication, replication fork; leading strand, lagging strand, Okazaki fragments; DNA polymerase, primase, ligase, nuclease; PCR
• viral genetics:  lytic vs lysogenic cycles, prophages; retroviruses, reverse transcriptase, cDNA
• bacterial genetics:  transduction, tranformation, conjugation; plasmids; restriction enzymes, RFLP analysis
• eukaryotic chromosomes: DNA, histones, nucleosomes; chromatin, chromosome, chromatid; centromere, kinetochore; telomere, telomerase
• mitotic cell cycle: interphase (G1, S, G2, G0), mitosis (prophase, metaphase, anaphase, telophase), cytokinesis; cyclins and cyclin-dependent kinases, growth factors
• meiosis and sexual life cycles: haploid vs diploid cells, homologous chromosomes; gametes vs zygotes; alternation of generations; stages of meiosis, synapsis and crossing over
• Mendelian genetics: alleles, traits, genes; homozygous vs heterozygous, dominant vs recessive; Mendel's first and second laws of genetics; multiple allele systems, polygenic traits; gene linkage, sex-linked traits
• Population genetics: population, gene pool, Hardy-Weinberg equilibrium; genetic drift, genetic bottlenecks, founder effects, gene flow, mate selection, natural selection; stabilizing selection, directional selection, diversifying selection; speciation

1. Introduction
1. two basic problems in biology
1. started with no life on Earth, now have at least 2 million species--Why?
2. the species show varying levels of relatedness--Why?
2. the most commonly accepted answer originally given by Darwin and Wallace--evolution by means of natural selection
1. there is natural variation in a population
2. some of the variation is inheritable
3. some of the inheritable variation leads to a greater chance of successful reproduction
4. this leads to differential reproductive success and changes in the genetic make-up of the population
3. this unit we want to focus on the sources of variation required by Darwin's theory
1. last unit dicussed the centrality of proteins and protein regulation to metabolism (and cell definition); we also learned that DNA was the key to protein synthesis
2. this unit we will look at how DNA is passed on to the next generation and at the implications for evolution
2. General process of DNA replication
1. review structure of DNA
1. chains of nucleotides (bases) held together by sugar-phosphate linkages (phosphodiester bonds)
   
2. double-stranded with the two strands held together by hydrogen bonds between complementary bases (the triple-stranded DNA of science fiction makes no sense)
1. application: DNA-DNA hybridization studies
2. application: RFLP analysis and genetic probes
3. strands are ant-parallel
   
2. semiconservative process, each new molecule is half old, half new
3. starts at origins of replication where proteins attach and pull the stands apart; replication works both ways from origin
4. DNA polymerase attaches energized nucleotides in the correct sequence from the replication fork
1. only adds nucleotides to 3' (without phosphate) end, so new strand only grows 5' to 3' direction
1. this creates a leading strand and a lagging strand with the lagging strand in pieces called Okazaki fragments which much be joined by ligase
2. only adds to an existing strand, so must have a primer ready; primer is a stretch of RNA created by primase
5. DNA polymerase aided by helicase, which unwinds and separates the double helix, and single-strand binding proteins, which hold the template straight
6. errors occur in the process, usually about 1 nucleotide wrong every 10,000; DNA polymerase also proofreads its work, reducing the number of mistakes to 1 in a billion
1. NOTE: a number of other enzymes (some of which are products of tumor suppressor genes) check DNA constantly, looking for errors; these are cut out and repaired as found
7. Application -- the polymerase chain reaction (PCR)
1. purpose is to increase the amount of DNA from a variety of sources
2. makes use of a minimal set of chemicals:  template DNA (what you want to be amplified), nucleotides (to build your new molecules of DNA with), primers (specific to the genes or random, depending on what you are trying to do--you cannot amplifiy the entire genome), DNA polymerase (special, heat-insensitive version)
3. uses heat to separate the strands of DNA; a typical cycle includes a melting phase, an annealing phase, and an elongation phase; the cycles are repeated 30 or 40 times, increasing the amount of DNA tremendously
8. Application -- DNA sequencing
1. the process is similar to that used in PCR, the main difference is that four separate sets of reactions are run simultaneously; in each set small amount of a single nucleotide analogue (dideoxyribonucleotides-these are missing the -OH group at the 3' carbon) is added
2. during the elongation cycle, the analogues are added at random to the growing strand; this blocks elongation of the strand
3. when the PCR system is complete, you have a mixture of DNAs in each tube; if these are separated by size in gel electrophoresis the sequence of the DNA can be read by comparing the samples
3. Viral replication, genetics, and evolution
1. used by Hersey and Chase to demonstrate the importance of nucleic acids
1. labeled T2 virus with radioactive sulfur (proteins) or radioactive phosphorus (DNA) and checked to see which entered the cell
2. found DNA entered the cell causing the destruction of infected cell
1. a quick review of viral structure
1. non-cellular
2. genetic material can be dsDNA, ssDNA, dsRNA, ssRNA (includes viruses whose genetic material acts as mRNA, viruses whose genetic material serves as template for mRNA--must have a special protein in the capsid toserve as the enzyme for this process--and retroviruses whose genetic material is first transcribed into DNA by reverse transcriptase--a capsid protein--and then transcribed back into mRNA by the host enzymes)
1. reverse transcriptase is an important tool in modern biology; it allows us to transcribe mRNA for a eukaryote protein into a DNA sequence without introns; this DNA sequence can be inserted into a bacterium and the bacterium made to produce a functioning version of the eukaryotic protein
2. life cycles
1. lytic life-cycle
2. lysogenic life-cycle
1. viral genome incorporated into host genome as a provirus and replicates with host DNA
2. at some point, viral genome exits host genome and lytic cycle continues
3. sources of variation
1. mutation
2. inclusion of host genetic material, especially in lysogenic forms
4. Bacterial replication, genetics, and evolution
1. a quick review of bacterial structure
1. bacterial cells
2. genetic material usually single molecule of double-stranded DNA, but there may be other, smaller circular molecules of DNA called plasmids
2. bacterial reproduction (binary fission)
1. can be as fast as every 20 minutes
2. follows basic pattern of DNA replication described previously: DNA polymerase attaches at a single origin and proceeds in both directions, using primers, etc. as needed
3. as DNA replicated, the cell grows; when large enough the cell splits into two pieces, each with half of the DNA
4. an interesting point: DNA replication and cell division not closely tied; another copy of the DNA could be started before the cell divides
3. sources of bacterial variation
1. mutations during the copying process
1. chance of mutation in any one gene is about 1/10,000,000, but since 20,000,000,000 cells of Escherichia coli are produced in the human gut each day as many as 2000 mutants for each gene are produced daily; of course, some of these are the same
2. E. coli has about 4,600,000  nucleotide pairs and about 4500 genes coded for in its DNA; since about 2000 mutants are produced for each gene, it is conceivable that 9,000,000 variants of E. coli are produced in each human each day
3. repair mechanisms usually involve cutting out wrong nucleotides (excision repair) using a nuclease and replacing them using DNA polymerase and ligase
   
4. application: the Ames method of testing for carcinogens using his^- mutants
2. genetic recombination and gene transfer
1. transformation - uptake of naked foreign DNA from the environment
1. process was involved in the discovery of the importance of DNA: Griffith, Avery, and Streptococcus pneumoniae
2. uses specialized surface proteins which attach and protect the DNA; calcium ions help in the process
2. transduction - incorporation of foreign DNA carried by virions
1. also involved in deciding DNA important: Hershey, Chase, and bacteriophage T2
2. generalized version of transduction: bacterial DNA pieces (result from lytic phage breaking up the host DNA) are packaged into the capsid instead of the viral genetic material by mistake; the virion injects this material into another host, where it takes up residence in the new host chromosome
3. specialized version of transduction: uses a temperate (lysogenic) phage; when genome leaves chromosome it takes some DNA with
3. conjugation - incorporation of DNA from other bacteria by a direct cell-to-cell transfer
1. involves plasmids - small, circular DNA molecules separate from the main DNA molecule that contain their own origin and replicate independently of the main DNA molecule (may become part of the main DNA molecule, in which case it is called an episome); usually only carries a few genes
1. example: R plasmids carry resistance to certain antibiotics; can be transferred by conjugation
1. application: the spread of drug resistance
2. example: transposons are groups of genes that can change their position in the DNA molecule; may move onto plasmid and be transferred
4. application: plasmid-based recombinant DNA technology
1. the idea is to create special plasmids containing genes of interest
1. plasmid should have origin, restriction sites, new genes, and an easy way to tell whether bacteria contain plasmid
1. double resistance systems
2. the Blu-Gene system--the plasmid contains a gene coding for ampicillin resistance and a gene coding for the protein beta-galactosidase; the gene for beta- galactosidase contains the only restriction sites for certain restriction enzymes
3. the pGLO system--uses the gene for the green fluorescent protein of jellyfish as one of the markers, antibiotic resistance as the other
   
2. process (example based on the Blu-Gene system)
1. cut the plasmid with restriction enzyme, mix with desired DNA (trimmed with same restriction enzyme), then treat with ligase--end up with a mixture of plasmids, some with DNA inserted at the restiriction site, some without
2. mix the plasmids with bacteria--end up with some bacteria without plasmids, some bacteria with the original, unmodified plasmid, and some bacteria with the new, modified plasmid
3. plate the bacteria on a selective, differential medium
1. in the Blu-Gene system the medium contains ampicillin and X-Gal, a blue compound that is a competitor of galactose in beta- galactosidase (the enzyme turns X-Gal from white to blue); bacteria without any plasmids are killed by the ampicillin; bacteria with unmodified plasmids form blue colonies (because of the build up of modified X-Gal), bacteria with the desired gene form white colonies (the gen is inserted in the middle of the beta-galactosidase gene, making it non-functional)
2. How could this be done using the gene for green fluorescent protein on a plasmid?
   
4. select the colonies with the desired characteristics and grow in the appropriated nutrient solution, with antibiotics added to prevent bacteria without the plasmid from taking over
5. Eukaryote replication (asexual)
1. basic ideas
1. not all eukaryotes use exactly the same methods
2. more complicated than bacteria because of the number of chromosomes involved
1. karyotypes and ploidy level
2. chromosome structures
1. chromatids
2. centromeres and kinetochores
3. many eukaryotes use sexual reproduction, causing additional problems
2. generalized cell cycle
1. interphase
1. G1 phase - normal metabolism and growth
2. S phase - grow and replicate DNA; form sister chromatids
3. G2 phase - grow and prepare for cell division
2. cell division
1. mitosis (nuclear division)
1. movement of chromosomes toward the centrosome depends on microtubules associated with the kinetochores; the chromosomes walk up the microtubules using motor molecules while the microtubule breaks down behind them
2. movement of the centrosomes away from each other depends on nonkinetochore microtubules; these interact with each other through motor molecules, at the same time the microtubules grow longer at the ends
2. cytokinesis
1. actin-mediated in animals and many protists, microtubule-mediated in plants
2. timing variable among different species and at different stages of the life-cycle; in most text-book examples cytokinesis starts during late anaphase or telophase
3. results
1. amount of DNA at each stage and phase
2. number of chromosomes and chromatids at each stage and phase
4. controlling the cell cycle
1. more important than in bacteria because so many chromosomes to deal with
2. three basic control points
1. initial control takes effect late in G1 phase; if conditions right proceeds into S phase, if wrong moves to G0 phase (actually most human cells are in G0)
2. second control point at the end of interphase seems to be related to the concentration of a protein called cyclin (G1 control point may also involve cyclins)
1. cyclin is degraded during mitosis, reformed during interphase
2. cyclin binds to cyclin-dependent kinases creating active versions of the kinases called MPF; these activate the enzymes leading to mitosis by adding a phosphate group
3. third control occurs in mitosis; kinetochores (near centromeres) block mitosis until all chromosomes are attached to microtubules; when all are attached, anaphase-promoting complex is formed which leads to both the completion of mitosis and the destruction of cyclin
3. numerous growth factors, density of cells (density-dependent inhibition and contact inhibition) also play a role
3. sources of variation are essentially the same as in bacteria, but the chance of mutation is higher because of the increased amount of DNA, lower because of increased generation time
6. Sexual reproduction and sexual life cycles
1. most eukaryotes also include a sexual phase in their life-cyle, leading to greater variation
1. always involves the fusion of two cells; resultant cell will then, of course, have twice as much DNA and twice as many chromosomes as the original cell; the chromosomes will be in homologous pairs
1. the cells that fuse are called haploid gametes
2. resultant cell is a diploid zygote
2. must be some way to reduce the number chromosomes or this process will break down; the solution called meiosis (or reduction division)
1. process of meiosis
1. interphase: similar to interphase of mitosis
2. prophase I: similar to prophase of mitosis except that homologous chromosomes pair up (synapse) and exchange pieces (crossing over); move to metaphase plate as a pair (tetrad)
3. metaphase I: tetrads line up at metaphase plate
4. anaphase I: pairs are pulled apart by the spindle; only half of the original chromosomes in each end of the cell now
5. telophase I and cytokinesis I: similar to mitosis but sister chromatids remain together
6. prophase II, metaphase II, anaphase II, telophase II and cytokinesis II are all similar to mitosis
3. timing of sex and meiosis highly variable
1. most common life-cycle is primarily haploid: the haploid cells reproduce asexually by mitosis; at some point they form special gametes (by mitosis); the gametes fuse to form diploid zygote; the zygote undergoes meiosis to produce haploid cells and the cycle continues
2. a common variation (among plants) involves alternate multicellular generations: haploid cells reproduce mitotically to form the thallus of a haploid multicellular gametophyte; certain cells in the gametophyte form gametes by mitosis; gametes fuse to form zygote; zygote grows by mitosis to form a diploid sporophyte; sporophyte makes special reproductive cells (spores) by meiosis; the haploid spores reproduce by mitosis to form a haploid gametophyte
3. animals (almost unique) have a diploid body with special cells that form haploid gametes by meiosis; gametes fuse to form diploid zygotes which reproduce by mitosis to form a diploid body
4. implications for variation
1. each diploid cell contains two copies of each gene - some mutations may be hidden from natural selection, leading to different versions of the genes in the population; these versions are called alleles
2. at meiosis I chromosomes are independently separated; this means that the haploid cells probably do not have the same combination of chromosomes and, therefore, alleles as the previous haploid generation; the diploids they make will not be the same as the previous diploid generation
3. ex. genetic relatedness in humans
7. Implications of sexual life cycles - Mendelian genetics
1. variation comes about in a predictable way from sexual reproduction
2. Mendel's rules
1. each "diploid" organism has two copies of each gene
2. genes come in different versions called alleles
3. usually one of the versions is dominant to the other
4. Mendel's First Law: the copies separate during meiosis (so that haploid cells only have one copy of each gene)
5. Mendel's Second Law: Copies of different genes separate independently of each other (they move into gametes independently)
3. examples, modifications, and exceptions
1. sickle-cell anemia as dominant-recessive pair
2. cystic fibrosis as a pleiotropic trait
3. Huntington's chorea as a late developing trait
4. blood types and multiple allele calculations
5. epistasis and polygenic traits
6. height, weight, skin color as polygenic (multifactorial) systems influenced by environment
7. sex-linked genes and chromosome maps
1. Morgan's fruit flies
1. white-eyed males
2. graybody normal wings (hybrid) with blackbody vestigial wings
1. expected 575 of 4 phenotypes
2. observed 965 gray/normal, 944 black/vestigial, 206 gray/vestigial, 185 black normal
2. chromosome maps based on degree of linkage
3. modern mapping techniques
1. microsatellites as markers for linkage mapping
2. chromosome walking using sequenced fragments of DNA
1. start with a known sequence
2. cut DNA with two restriction enzymes to create two gene libraries
3. probe one of the libraries with DNA matching the 3' end of the known sequence; clone and sequence the appropriate fragment
4. probe the other library with DNA to match the 3' end of the new sequence; clone and sequence the appropriate fragment
5. probe the first library with a probe matching the 3' end of the new sequence, etc.
6. process continues, in theory, until the entire DNA molecule is sequenced
8. cytoplasmic inheritance
1.  example: Kearns-Sayre syndrome:  progressive heart and muscle weakness, degeneration of the retina
4. applications
1. family histories, pedigrees, and genetic counselling
1. technology-based carrier recognition
2. amniocentiesis and fetal testing
3. newborn testing (PKU)
4. chromosomal aberrations and karyotyping
8. Implications: population genetics and evolution
1. Darwinian natural selection is straight-forward in asexual organisms: variation appears during the replication process, selective processes occur on the offspring
2. sexual organisms have a more complicated set of rules
1.  Hardy-Weinberg calculations in sexual populations
1. calculating the gene pool and the structure of the next generation
2. application:  determining the number of carriers in a dominant/recessive system
1. 1/10,000 born with PKU
2. 1/2,500 born with cystic fibrosis
2. the basic assumptions underlying the previous calculations:   no natural or mate selection, a large population with no migration in or out, and no mutation; suppose the assumptions are violated
1. effects strong natural selection
2. effects of small populations
3. In summary, each generation of diploids gets a new mix of genes which are acted upon by a number of forces and processes, including:  natural selection, mate selection; genetic drift; genetic bottlenecks and founder effects; non-random migration.  Selective and random processes can result in stable genotypes, directional selection, or disruptive selection; the latter two lead to the development of new species.
9. Beyond Darwin: human evolution in the future
1. Are humans still evolving through natural selection?
2. prospects for the Lamarckian evolution of humans
1. eugenics programs
1. What is a defective gene?
2. recombinant DNA methods--current and future status:
1. recombinant plants and animals
1. retarded ripening (using an anti-sense gene); plants producing their own pesticide or  new drugs
2. growth hormones in fish, pigs, rats; etc.; drugs in sheep milk; mice with sickle-cell anemia
2. recombinant humans
1. genetic treatment of defective tissues--use of stem cells from adult or embryonic tissues
2. genetic treatment of gametes
3. genetic screening and treatment of zygotes and early stage embryos
3. ethical and safety questions
1. release of modified organisms into the environment
2. genetic modification of humans