Introduction to modern genetics

I. Developmental genetics The drawing of conclusions about the developmental role of a particular wildtype gene product from the pattern of defects (e.g., tissue or organ changes) occurring in a mutant II. What makes a good model system? Requirements: easy to maintain grow in small space short generation time (life span) large number of progeny small genome size low amount of repeated DNA easy to mutagenize ability to self fertilize ability to manipulate- transformation Models: Bacteria: E. coli Yeast: Saccharomyces cerevisiae Nematode: Caenorhabditis elegans Insect: Drosophila melanogaster Vertebrates: mouse (Mus musculus), zebrafish (Dano rerio), (also Homo sapiens) ), ( Plant: Arabidopsis thaliana A closer look at some models: Yeast haploid and diploid stages grow and select on medium can transform easily Arabidopsis- “mouse-eared cress” diploid only dozen per 5-6 cm pot 5-6 weeks generation time 10,000 seeds per plant self fertilization Genome Sizes in Mb E. Coli Yeast Neurospora C. elegans Drosophila Chlamydomonas Arabidopsis 42 4.5 15 42 80 160 100 100 Tomato Rice Tobacco Potato Ceratopteris Corn Wheat 714 970 1,600 1,900 5,000 5,000 5,900

III. Mutation Mutation- change in DNA sequence that can change information (protein or how and where to express protein) it encodes Types: 1. chromosomal mutations 2. smaller insertions/deletions 3. point mutations A->T transition A->G transversion 4. frameshift mutataions Amino acid effects of point mutations: tyrosine TAT, TAC TAT -> CAT tyr -> his TAT -> TAA tyr -> stop TAT -> TTT tyr -> phe TAT -> TAC tyr -> tyr T TTT Phe (F) TTC “ T TTA Leu (L) TTG “ CTT Leu (L) CTC “ C CTA “ CTG “ ATT Ile (I) ATC “ A ATA “ ATG Met (M) GTT Val (V) GTC “ G GTA “ GTG “ C TCT Ser (S) TCC “ TCA “ TCG “ CCT Pro (P) CCC “ CCA “ CCG “ ACT Thr (T) ACC “ ACA “ ACG “ GCT Ala (A) GCC “ GCA “ GCG “

misense nonsense neutral in many cases silent G TGT Cys (C) TGC TGA Ter TGG Trp (W) CGT Arg (R) CGC “ CGA “ CGG “ AGT Ser (S) AGC “ AGA Arg (R) AGG “ GGT Gly (G) GGC “ GGA “ GGG “

A TAT Tyr (Y) TAC TAA Ter TAG Ter CAT His (H) CAC “ CAA Gln (Q) CAG “ AAT Asn (N) AAC “ AAA Lys (K) AAG “ GAT Asp (D) GAC “ GAA Glu (E) GAG “

Silent mutations: Changes in base sequence that do not lead to a phenotype. Base substitution mutations in the third position of many codons do not change the amino-acid coding. Lethal mutation: a gene that leads to the death of an individual; these can be either dominant or recessive in nature Conditional mutations: In some instances, a null mutation is lethal to the organism. Therefore, conditional mutations are quite useful to the geneticist. T Temperature-sensitive (ts): low permissive temperature; high non-permissive (or “restrictive”) temperature. Temperature-dependent mutations are usually protein folding mutants affecting protein function or stability.

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Difference between genotype and phenotype? Phenotype: literally means “the form that is shown”; it is the outward, physical appearance of a particular trait Genotype: the specific allelic combination for a certain gene or set of genes How does genotype lead to phenotype? What does a mutation have to do with this? (Hint: What actually causes a disease — the DNA or the protein?) In classical genetics, phenotype can be observed directly, but genotype can has to be inferred from phenotypes of: the individual, the individual’s parents and/or progeny. Genetic variation is essential to classical genetics - genes are defined by mutations that affect gene function. Genes can only be identified when heritable differences among individuals exist. A gene must have at least two variants (alleles) that cause marked phenotypic differences. The likelihood of identifying a “gene” (allele) depends on its degree of phenotypic influence. “Silent” variation (genetic change with no phenotypic manifestation) is undetectable by classical methods. Mutations that have phenotypic consequences can include: those that alter the amino acid sequence of a protein its activity its abundance (stability) those that change the expression of a gene sequences required for mRNA transcription, splicing, protein translation regulatory elements: sites of action of regulatory proteins or RNAs Important refresher Mendel’s First Law: the law of segregation; during gamete formation each member of the allelic pair separates from the other member to form the genetic constitution of the gamete Mendel’s Second Law: the law of independent assortment; during gamete formation the segregation of the alleles of one allelic pair is independent of the segregation of the alleles of another allelic pair Phenotypic variation Penetrance - the frequency of expression of an allele when it is present in the genotype of the organism (if 9/10 of individuals carrying an allele express the trait, the trait is said to be 90% penetrant). Those individuals that do not show the mutant phenotype are sometimes called “escapers”. Expressivity - variation in allelic expression when the allele is penetrant. Not all phenotypes that are expressed are manifested to the same degree. For example, for polydactyly, an extra digit may occur on one or more appendages, and the digit can be full size or just a stub. Therefore, when the “P allele” is present it expresses variable expressivity. Catagories of genetic mutations a. amorph (also called an null mutation)- complete absence of activity b. hypomorph (also called a partial loss of function) - loss of most of activity c. hypermorph (also called a gain of function)- new gene activity d. antipmorph (also called a dominant negative)- dominant negative activity e. neomorph- novel function or activity 44

Mutations are defined based on effect of wild type and mutant dose. The common notation systems is as follows: Use mutation (m), wild type (+), Df (deletions of gene), Dp(+) (duplication of wild type copy), and Dp(m) (duplications of mutant copy). Amorph (complete loss of function) null allele is equivalent to Df (m/Df= m/m= Df/Df) more + copies -> more wild type phenotype more m copies -> no change in phenotype An example would be the complete loss of the coding region for an enzyme. Hypomorph (partial loss of function) more + copies -> more wild type phenotype (m/m < m/Df) more m copies -> slightly more wild type phenotype An example is a misense mutation that reduces an enzymes activity. Hypermorph (gain of function) Think of this type of mutation as an increase in wild type activity of the protein. more + copies -> more mutant / less wild type phenotype (m/m > m/+ > m/Df) more m copies -> way more mutant / less wild type phenotype An example of this type of mutation would be the constitutive activation of an enzyme. Antimorph (dominant negative) In this type of mutation, the protein acts to inhibit a biological process/ activity. more + copies -> more wild type phenotype more m copies -> more mutant / less wild type phenotype An example would be a poison complex. When the mutant protein binds to other proteins in the cell and soaks them up like a sponge, such that they can’t function. Neomorph (novel function) In this type of mutation the protein takes on a whole new function. more + copies -> usually no change in phenotype more m copies -> usually no change in phenotype An example of this type of mutation would be an enzyme that now has a new substrate What is an allele? A gene has a designated sequence in wild-type animals. When a gene is mutated, all mutations do not necessarily affect the nucleotides in the gene’s sequence. Different mutations in the same gene can have different effects. Each different background, sequence of a gene that varies from wild-type, is called an allele of a gene. Nomenclature A mutant name is italic, lower case ( leafy), abbreviated as a 3-letter symbol ( lfy). If different genes were identified by the same mutant phenotype, in each case the name of the mutation is immediately followed by a number ( emb1, emb2). Different alleles of the same gene are given -numbers (emb11,emb1-2) The gene identified by the mutation is italic, upper case ( LEAFY, LFY). The protein encoded by the gene is upper case (LEAFY, LFY). How do you study a biological process using genetics? Forward genetics: disrupt system at multiple random sites, look for interesting change, characterize 45

it and ID affected component (phenotype -> gene). Reverse genetics: disrupt specific component of system, characterize change (gene -> phenotype) IV. Mutagenesis What genes are important for process x you want to study? Decide what we want to study and what phenotype are you going to look for. Mutagenesis: isolation and characterization of functional mutations based on phenotypic effect(s). This usually involves treating the organisms with radiation or chemicals, which induces changes in the DNA. What to mutagenize? Seeds or pollen Types of mutations: Spontaneous, Induced, Insertional chemical, e.g. Ethyl methane sulfonate (EMS, about 100 hits per genome) physical, e.g. X-rays, fast neutrons biological, e.g. transposons, T-DNA (1 hit per genome) (insertional mutations) Targets of mutagenesis somatic mutation are not inheritable gamete mutation are inheritable Problems in mutagenesis formation of mosaic plants from seed mutagenesis plant is dipoloid- many mutations are hidden V. Mutant screening Goal: Maximize your chances to find many informative mutants: a. What exactly is the biological question you want to address by mutagenesis? b. What exactly is the mutant phenotype you are looking for? c. How easy is it to find the mutant phenotype in a plant? d. How many plants can you screen realistically? e. What is the extent of phenotypic variability in the unmutagenized parental line? Screening- looking through mutagenized plants for one with an altered phenotype Screens can include: Visual screening Survival in certain conditions (selection) Biochemical stains Selection- making non-mutated die Selection can include antibiotic selection herbicide resistance (e.g. chlorosulfon) toxic intermediate auxotrophies mutation in biosynthetic pathway Housekeeping genes are necessary for the basic sustenance of a cell. Mutations in housekeeping 46

genes can be lethal. Here are some tricks for studying lethal mutation. • Maintain a mutation as a heterozygote. (homo - see dead seeds as homozygotes • Look for a weak loss of function • Look for conditional mutations, such as temperature sensitivity VI. Analysis of mutants What do I do with all these mutants now that I have them? Mutant characterization • Is it a inheritable mutation? • Backcrossing addresses dominance or recessiveness, and if mutation is single or multiple lesions. • Allelism: Complementation/segregation tests address how many genes/loci you have isolated in a process and defines an allelic series. • Pleiotopy Recessive vs. Dominant alleles In diploid organisms, the phenotype is specified by the concerted action of two alleles of one gene. One of the first questions a geneticist will address is the dominance/ recessiveness of a particular mutation. This gives some clues about the nature of a mutation and how it leads to a mutant phenotype. • Dominant - the allele that expresses itself at the expense of an alternate allele; the phenotype that is expressed in the F1 generation from the cross of two pure lines. • Recessive - an allele whose expression is suppressed in the presence of a dominant allele; the phenotype that disappears in the F1 generation from the cross of two pure lines and reappears in the F2 generation. Backcrossing your mutant line does a few things for your study. • Backcrossing allows you to establish if your mutation is dominant or recessive. • Backcrossing gets rid of other background mutations that may not have anything to do with the process you are studying. When you induce mutations, and then screen, any given mutant will have more than one alteration in it’s DNA. By backcrossing a number of times, and selecting for your mutation, you are diluting out the other background mutations; “cleaning up of the genetic environment”. This establishes that the phenotype you have selected for is the result of a single gene mutation and not multiple genes that contribute to a phenotype. m/m x +/+ —> m/+ If all progeny are mutant, than m is dominant. If all are wt, than m is recessive. By examining cells/individuals heterozygous for a particular mutation (+/m) and comparing their phenotype to those with only wild-type information for the gene (+/+) and those with only mutant alleles (m/m). If m/+ = +/+, the mutation is recessive. One copy of the wild-type gene replaces the function of the mutant gene. Null mutations (amorphs) and hypomorphs mutations are recessive. If m/+ = m/m, the mutation is dominant. Even in the context of a wild-type gene, the mutant gene expresses the mutant phenotype. Examples: mutations causing inappropriately high expression of a gene, hypermorphs, 47

gain-of function mutations, like drug resistance mutations. If +/m has a phenotype intermediate between +/+ and m/m, the mutation is said to be semi-dominant. This may occur because the mutant protein competes with the wild-type or “poisons” its function. Examples include proteins that form complexes or those that bind substrate but fail to function at a later step. When a genetist says a population “breeds true” what is meant is it is a Pure Line - a population that breeds true for a particular trait. The population is homozygous. A test cross is designed to reveal the genotype of an organism that displays a dominant characteristic. Such an organism could be homozygous for a recessive allele, or heterozygous with a dominant allele. (test organism) “M/M or M/m” x m/m (homozygous recessive) If the progeny all show the dominant phenotype the test organism was homozygous dominant. If the progeny show a 1:1 ratio of dominant to recessive then the test organism was heterozygous. Allelism When similar phenotypes occur in mutants does this represent mutations in the same gene or multiple genes. When we study genetics, we often randomly mutagenize organism and look for mutants that are deficient in the process that we’re studying. If we generate many mutants in a process, how do we tell how many of them have mutations in the same gene, and how many different genes there are that are involved in this process? Analyze allelism by crossing two plants with independent mutations • Recessive mutation: complementation analysis • Dominant mutation: segregation analysis Complementation test Geneticists use complementation analysis to determine whether two recessive mutations lie in the same or in different genes. In complementation analysis: m x m* parental cross of two populations that have same mutant phenotype. Parental population is homozygous and recessive. F1 progeny examined m/m* in this case all progeny will have parental mutant phenotype. This is call non complementation, the genes are allelic. A geneticist would conclude that this is the result of mutations in the same gene. Non complementation is allele-specific, which suggest that the two mutant proteins directly interact. The assembly of a complex structure is marginally distorted by one mutant protein but more severely disrupted when two components are both mutant. or m/M, m*/M* in this case all progeny will be wild-type (i.e. no mutant phenotype). This is called complementation (the backgrounds are providing wild-type activity to complement mutant function), the genes are non-allelic. A geneticist would conclude that this is the result of a parental cross with 48

mutations in two different genes. Isolation of a large set of mutants giving a particular phenotype and complementation analysis can quickly reveal the number of genes that contribute to a particular genetic pathway. The number of complementation groups approximates the number of genes in a pathway, if enough mutants were isolated to saturate the appropriate genes. All mutations within a complementation group will fail to complement other members of the same group but will complement mutations in all other complementation groups. e.g. complementation Maize kernel mutants screen for loss of red color c= colorless, recessive c1c1 Mutant male c2c2 c3c3 wt wt Mutant Mutant Mutant c4c4 Mutant wt wt Mutant +=wt red c6c6 wt wt wt wt Mutant Mutant female c1c1 c2c2 c3c3 c4c4 c5c5 c6c6

c5c5 wt wt wt wt Mutant

Result: Complementation groups: c1, c4 c2, c3 c5, c6 A group of alleles in the SAME GENE, where each allele is a slightly different mutation with a slightly different phenotype, is called an allelic series. Another way of saying this is that an allelic series consists of all the known alleles within a single gene. Segregation test Cross homozygous mutants. In first generation, you will always see mutant phenotype because you are studying a dominant mutation. However, in the second generation (F2) if see wt (expect in 1/16), then mutations are non-allelic. If again all mutant then the mutations are allelic. Pleiotropy Genes sometimes have two or more phenotypes. This indicates that the gene product is used in a variety of different processes. Different alleles of a gene may have different phenotypes. The pleiotropy of a gene is determined by studying the effects of various alleles at different times in development and under different conditions. It is important to realize you will only see effects of things you look for. Conditional mutations are very useful for determining pleiotropic effects. For example, if a gene product is required early in development for survival, you may not see that the gene product also effects post-embryonic development like light response. If you had a conditional temperature sensitive mutation, you could grow it at the permissive temperature, and get past the embryonic effects, and then when moved to the restrictive temperature you might see the effects on light response.

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VIII. Gene identification Mapping Where in the genome? Genes are grouped physically in linear arrays, i.e. chromosomes. Each species has a specific number of chromosomes per haploid genome. The diploid genome has two copies of each chromosome. Genes can be inherited independently from each other, but if they lie close together on the same chromosome their physical association is revealed as non-random inheritance in genetic crosses To map a gene, one must analyse the linkage between the mutant phenotype and markers whose physical position in the Arabidopsis genome. Genes can be mapped by recombination only if they exist in at least two distinguishable allelic versions. This is true for both visible markers and molecular markers. Linked genes are preferentially inherited together, but meiotic crossing over will break the linkage. In the following mapping cross, A and a are alleles of a gene that may be linked to gene B. AB/ab x ab/ab -> parental types: ab/ab, AB/ab, recombinants: Ab/ab and aB/ab The percentage of recombinants out of the total progeny is termed the recombination rate. 50% recombination (the maximum) equals random segregation and corresponds to absence of linkage. The unit for recombination rates is the centiMorgan (cM). A positional cloning project can be briefly summarized as follows: The genetic map position of the gene X to be cloned is first determined with respect to two other genes, m1 and m2, which serve as markers. M1 and m2 must flank gene X on either side on the chromosome. M1 and m2 should also have been cloned earlier, a prerequisite that is almost always met, because m1 and m2 are typically molecular markers (e.g. RFLPs). Now the next step is to isolate clones for all the genomic DNA that lies between m1 and m2. This is achieved by `chromosome walking’, the successive selection of overlapping clones from a genomic library by hybridization, starting with clone m1. The resulting set of overlapping clones is termed a `contig’. At this point, one of our clones from the contig must contain gene X, but we don’t know yet which one it is. To determine which of the clones contains gene X, the contig is scanned for specific loci that show complete genetic linkage to gene X. Phenotypic rescue In a technique known as phenotype rescue normal copies of the gene are “put back” into the organism that carries the defective gene. Some of these normal genes succeed in taking over the function of the defective genes, resulting in offspring that have normal function or otherwise alleviating the mutant phenotype. As a result, the mutants are “rescued” from the effects of mutant gene product. This allows the scientist to confirm that the gene that they think is responsible for the mutant phenotype is indeed the one. It plays a checking role in the process. Phenotypic rescue experiments are by no means foolproof. Sometimes the inserted gene doesn’t make any of the product it’s supposed to--or it may make too much, too little, at the wrong time, or in the wrong place. Determining the molecular lesion Once a scientist has determined the genetic lesion then the gene product in the mutant animal maybe sequenced to determine the DNA change that has occurred to produce the mutant phenotype. The sequence of a gene is the precise order of the nucleotides in a specific region (usually the coding-region of the gene of interest). 50

IX. Then what? • When and where is the wild-type gene activity required? • Temperature shift experiments can reveal time of gene action • Genetic mosaics can reveal site of gene action • Suppression analysis A genetic mosaic is a creature whose body is built of a mixture of cells of two or more different genotypes. Suppression analysis Another genetic trick is the isolation of suppressors that reverse or at least alleviate a particular mutant phenotype. Such mutations often provide important clues about other genes that have similar function, act in the same pathway or complex, or regulate a known gene.

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