Week 9 & 10 Reflections

We focused on natural selection as an evolutionary-genetic force during the Tuesday lectures of Weeks 9 and 10. Natural selection is a force that can be quantified by considering relative fitness (w), and the selection coefficient (s), which is more widely used by geneticists. Relative fitness values are generally obtained by comparing some measure of fitness (for example, virus doubling time, or fecundity in an animal species) in a reference genotype (often ‘wild-type’) to that observed in a genetic variant (often ‘mutant’). The selection coefficient can be calculated by simply subtracting the value for w from 1 (s = 1-w), and can be thought of as the ‘percent advantage (or disadvantage)’ of the mutant relative to wild-type. We spent time discussing one molecular test for the effects of natural selection on protein-coding sequences: the Ka/Ks test. Ka is the frequency (or rate) of replacement substitutions per replacement site; Ks is the frequency (or rate) of silent substitution per silent site. This is at its core a very simple test that is also very broadly applicable – all you need is two homologous protein-coding sequences to compare to each other. This test is being widely used today, applied to genome-scale data, to look at the average effects of different selective forces across many gene sequences. The genome-wide distributions of Ka/Ks values for Drosophila orthologs were all very small (most <0.3), suggesting pervasive purifying selection. A similar trend was observed in the gene-duplicate data for six different model organisms, though in this case some of the younger duplicates (low Ks values) showed evidence for positive selection.

We also revisited recombination, from an evolutionary perspective, this week. Ultimately, recombination can be described as a ‘natural selection facilitator’ because it makes natural selection more efficient by selection many different types of genotype combinations to act upon. We next progressed to study relationships between the effective population size (Ne) and genome size and content. This work (like the gene duplicate work) was done by Mike Lynch and John Conery in the early 2000s. Lynch and Conery looked for correlations between Ne and genomic traits such as genome size, numbers and sizes of introns, transposon abundances, etc. They found strong and significant correlations – as Ne decreases as you move from prokaryotes to unicellular eukaryotes to multicellular eukaryotes, genome size increases. Further, intron abundances and lengths increase, and transposon abundances increase. The big (and still somewhat controversial) conclusion from this study is that for species evolving in relatively small Ne (for example, trees and humans), genetic drift is too strong and natural selection is too weak to keep deleterious genomic features (such as transposons and other forms of ‘extra genome space’) out of the genome. In other words, the underlying cause of our remarkably complex and transposon-laden genomes is an inability of natural selection to maintain a ‘tidy and streamlined’ genome similar to those observed in prokaryotes.

On Thursday, we discussed the exciting topic of next-generation DNA sequencing technologies. We focused mostly on the Illumina approach. This method starts with the random fragmentation of your starting target genome (what you want sequenced) into smaller pieces (usually 200-1,000 bp). ‘Adapter’ molecules are then ligated onto the ends of the fragmented genome pieces, a different specific adapter goes on each end of the molecule. These adapters have DNA sequences that are known to the experimenter – these known sequences are essential for the downstream methods, for example by providing known priming sites. Next, adapter-ligated genome pieces are washed over a ‘flow cell’ (= fancy microscope slide) which contains a lawn of oligonucleotides (‘oligos’) bound to the surface that are complimentary to the adapter sequences. The adapter sequences hybridize with the lawn oligos, thereby initiation a ‘bridge amplification’ process involving nearby oligos as primers which results in an amplified ‘cluster’ of DNA molecules (all identical to the molecule that started the amplification process) at a particular spot on the slide. Many, many millions of these clusters are created across the flow cell simultaneously. After cluster generation, sequencing occurs with reversible dye-labeled terminators, similar to the Sanger technology (see textbook section 6.8). However, with Illumina ALL of the nucleotides in the reactions are dye-labeled terminators. In the first ‘cycle’, all four dye-labeled terminator nucleotides (each with a different particular dye attached) are washed over the slide and the appropriate nucleotide incorporates in the first position. Then, a fancy fluorescence microscope scans along the slide and ‘reads’ what dye (= base) is present for each cluster. After the first cycle, an enzyme comes along that cleaves off the dye, making the incorporated nucleotide now free to accept another base in position 2 in the next cycle. This process continues on for up to ~250 cycles, yielding 250-bp reads for many hundreds of millions of different DNA sequences.

We also discussed Pacific Biosciences single-molecule real-time (SMRT) DNA sequencing technology. With this newer approach, longer reads are achieved (~5,000-bp) and sequencing is done without terminators! A DNA polymerase is anchored to the bottom of a sample well and then a single molecule is replicated in the well by the polymerase. The nucleotides used have fluorescent dyes attached to the terminal phosphate; when the nucleotide is incorporated by the polymerase, a fluorescence pulse is emitted which is detected below the plate.

Week 8 Reflections

We started the week discussing gene regulation – how cellular systems regulate the amounts of protein products (RNA, protein). The famous lac operon system was introduced; this set of genes in E. coli provided the foundation for much of our understanding of regulation at the transcriptional level. We then turned to discuss the similar gal system in yeast as a model for eukaryotes before considering some post-transcriptional forms of regulation such as alternative splicing and RNAi. On Thursday we turned to population and evolutionary genetics. Much of the discussion centered on the Hardy-Weinberg Principle of population genetics which provides important avenues for calculating genotype frequencies and allele frequencies. The class discussed some of the key underlying assumptions of the H-W Principle as well as two major implications of that principle for population genetic processes. Lecture material transitioned to a bit about evolutionary genetics with special emphasis on the forces of evolution. Mutation, although the most fundamental of the forces that provides the key ‘variation substrate’ for other evolutionary forces to act upon, is also a very weak evolutionary force because it is not able to cause rapid change in allele frequencies (unless population sizes are very, very small). Genetic drift was also discussed – the role of ‘sampling error’ of gametes from one generation to the next. When population sizes are small, there is a high probability that one particular allele might be completely lost (or completely fixed) in the population simply because that allele happened not to be ‘sampled’ purely due to chance from one generation to the next. An analogy used to help make drift more understandable is flipping a coin – with one million coin flips, you are very likely to end up with something very close to 50% heads and 50% tails (and almost certainly heads and tails getting sampled at least once) whereas with a much smaller number of coin flips (six, for example) there is a much greater chance of not getting a ‘50/50’ result and not either getting heads or tails at all in the series of coin flips.

We also discussed Muller’s Ratchet – a theoretical evolutionary concept not covered in the book. The idea behind the ratchet is that if you have a small isolated population that reproduces asexually, you might expect to lose the most-fit class of individual in that population (individual with fewest deleterious alleles) by drift. On top of this, because the population is small, mutation is a stronger force – individuals are accumulating new deleterious mutations on top of this. With no input of genetic variation from migration and no recombination, such populations would be expected to over time become less and less fit (individuals harboring increasing numbers of deleterious alleles) until it reaches extinction. How optimistic! This might seem rather unrealistic, but the theory has served as a model for helping evolutionary geneticists understand why other evolutionary forces (such as recombination) came about. Also, there are some asexual genetic components of endangered populations (e.g., mitochondrial DNA) that might be subject to the ratchet. Why ‘ratchet’? The idea here is that every time the population gets worse (loses most-fit individual) due to drift, this is a turn of the ratchet toward extinction.


Week 9 Sneak Peek: We will finish up Thursday’s lecture material, and then continue our population/evolutionary-genetic unit by discussing natural selection – Darwin’s key premises and deduction leading to his theory, and the many different sub-types of selection that are possible. We will focus on one test for the effects of natural selection on protein-coding sequences. I will also be posting some supplemental reading on this topic – it is a primary research article that I will be covering. Reading this paper is not absolutely required, but might help you understand things a bit better. Remember: no class on Thursday, and no recitation all week!

Week 6 Reflections

This week we wrapped up a little bit of recombination, then turned our focus toward mutation and then cancer genetics.  We turned attention to the molecular mechanisms of DNA recombination, overviewing a few early (but ultimately incorrect) models before learning about the double-stranded (ds)DNA break pathway, which is currently held as the correct model.  dsDNA break-mediated recombination can give rise to both recombinant as well as non-recombination chromosomes depending on how chi structures are resolved, and can also result in gene conversion events.  The example of yeast mating-type switching provided an example of dsDNA break-mediated recombination occurring between repetitive DNA sequences on the same molecule (between hidden and silenced ‘mating cassettes’ and the expressed MAT gene itself).

Next, we first learned about different pathways by which DNA can mutate, ranging from base substitution changes resulting from the natural fluctuations in electron distributions in nucleotides (for example, the keto versus enol form example in thymine) to the significant genome alterations that can result from mobile genetic elements such as retrotransposons. We also learned about how mutations can be classified in a variety of ways.  Our discussion of mutation concluded with the topic of mutation rates, covering two different methods for how such rates can be experimentally estimated (reporter gene versus mutation-accumulation line approach). Reporter gene approaches rely on the phenotypes associated known to occur when the gene is mutated. The phenotypic change (for example, from blue to white colony of E. coli cells) reports the occurrence of a mutation. With mutation-accumulation lines, on the other hand, organisms are propagated across many (usually hundreds) of generations in the lab, and then their DNA is directly sequenced afterwards to count mutations at the DNA-level. This strategy offers a much more direct avenue (not relying on assumptions about reporter gene phenotypes) to understanding the mutation process. On Thursday, our attention turned toward cancer genetics. This lecture also highlighted the utility of temperature-sensitive mutants in genetic analysis with the yeast model organism. We learned about how cyclin-CDK complexes regulate cell cycle progression.

Week 7 Sneak Peek: We will wrap up cancer genetics on Tuesday, and transition to some basic discussion about gene expression (for example, transcription basics). Thursday will be midterm #2… Study hard! Bring your calculators! The basic format will be the same as the first midterm – about 45% of the points coming from multiple choice and about 55% coming from short answer. This exam will cover all material up through (and including) next Tuesday’s lecture content. Material covered in-between Midterm #1 and #2 will be the main emphasis of the questions, though you will still need the knowledge base of material presented early in the class to succeed. Also, reminder that next week on Friday is Veteran’s Day, and OSU will not be in session.

Week 5 Reflections

It was all about recombination! We learned about the inheritance patterns of linked genes nearby one another on a chromosome, most importantly how to quantitatively express the incidence of co-inheritance of linked markers in terms of recombination frequencies (or, genetic distance as measured in cM). In addition, we gained an understanding of how patterns of genetic distance between marker genes with clear associated phenotypes led to the creation of genetic maps: maps revealing the relative ordering of genes along chromosome physical space. These genetic maps have been enormously helpful to both scientists learning about genetic and other biological processes in their study systems, and also to plant and animal breeders interested in producing high-value crops and livestock in the world of agriculture. The three-point testcross – appearing in lecture, homeworks, and recitation – illustrates the basic logic that underlies the construction of genetic maps.

We also learned about recombination rates (not covered in the book).  This is an expressed as the genetic distance (in cM) divided by the physical distance (in Mb).  Looking at recombination this way, it becomes very apparent that different regions of DNA can vary extensively in term of how ‘recombinogenic’ they are.  A human chromosome example was provided, showing lots of recombination hotspots, and coldspots, across the length of the chromosome space.

We started to talk about the molecular mechanism of homologous recombination on Thursday and I discussed things a bit differently than the treatment in the book.  Understanding the molecular DNA-level actions and movements of DNA during recombination has been a central area of genetics research for decades. This was an area of great controversy for a very long time, until the development of the double-stranded DNA break and repair (DSBR) model was established, which has been well-accepted and experimentally supported every since. The first model was the Holliday Model which posited that the whole process began with a pair of single-stranded DNA nicks at homologous positions at the two participating DNA molecules. After nicking, reciprocal strand invasion occurred (forming Holliday or chi structure) followed by branch migration followed by endonucleolytic resolution. Though many of the details were right, the Holliday model was ultimately discounted because there was no known enzymatic activity that could cause ssDNA nicks at the same exact (homologous) sites in two DNA molecules – still nothing known to this day that can do that. The Meselson-Radding (M-R) Model modified things by suggesting that a single-stranded nick could start the process – the free end of the nicked strand (“donor strand”, gray in diagram) could then carry out strand invasion of the “acceptor” DNA molecule (pink in diagram) which would then cause a displacement loop (or, D-loop) strand from the acceptor that could undergo base-pairing with homologous sites in the invading molecule. This model was widely regarded as essentially correct, but there was still one puzzle related to recombination – gene conversion – that could not be explained by the M-R model. Gene conversion, originally only observed in certain species of fungi, is a process whereby there is allelic “conversion” during meiosis that is in opposition to predictions of basic Mendelian genetics (e.g. one ‘a’ allele is “converted” into a ‘A’ allele in the example shown). Since this phenomenon was only observed in gametes, it was presumed to be closely associated with a meiosis-specific process such as recombination. This led researchers to develop the double-strand break & repair (DSBR) model. The DSBR model has been strongly supported over the last few decades in all lab experiments and is also able to effectively account for gene conversion. At the end of the DSBR mechanism there are tracts of DNA at the end of the process where there is base pairing between nucleotides from the “donor” strand and the “acceptor” strand. There is the possibility of nucleotide mispairing between these two strands (because they originally came from different dsDNA molecules) – these mispairings are recognized by the mismatch DNA repair enzymatic machinery that ultimately removes one of the mismatching nucleotides and replaces it with a correct complementary base. Thus, there is the possibility in this DSBR path for one allele to be converted into the other allele! Since this was all worked out, we’ve come to discover that gene conversion outcomes occur much more frequently than recombinant chromosome outcomes of the molecular recombination process in virtually all eukaryotic life forms, from fungus to human.

Week 6 Sneak Peek: Next week we will quickly recap recombination, and then transition to talk about the mutation process (Ch. 12) on Tuesday. On Thursday we will talk about the molecular genetics of cancer (Ch. 13), and how yeast (a single-celled organism…) provided key insights into the genetic underpinnings of cancer in humans.

Week 4 Reflections

Tuesday was the first midterm – we are about halfway done with grading and aim to return them to students next week during lecture.

On Thursday, we moved into the world of mammalian and human chromosome biology, discussing some basic terminology and conventions used in karyotype studies. We discussed some interesting chromosome evolution stories in the human lineage, demonstrating the utility of nonrecombining chromosomes (e.g., Y) for certain genetic applications. We learned about methods for studying chromosome biology, such as chromosome painting and G-banding. After the not-thinking break, we started to move into the topic of recombination…

Week 5 preview: The upcoming week will be dedicated to the topic of recombination, linkage, and gene mapping. We will cover chapter 4 – make sure you look at the material ahead-of-time.  It is important to wear your ‘thinking caps’ for Chapter 4 material, and to be engaged in lectures and recitations.

Week 2 Reflections

At the beginning of the week, we went through mutation screening methods with Neurospora and associated complementation testing. These studies provide great examples of the type of logic that geneticists use – make sure you understand the logic that determines whether different particular genetic mutants go into the same versus different complementation groups. Next, we entered into classical Mendelian (aka transmission) genetics – how the results of thousands of crosses between pea plants of varying phenotypic traits and combinations of traits led to our current understanding of how different versions (alleles) of genes segregate, and how different alleles, and allele combinations, lead to different phenotypic outcomes. After revisiting the basic concepts and Punnett square approaches to understanding how alleles are transmitted, probability concept approaches were introduced that offer much faster and practical ways to solve these problems (especially when it comes to many genes). Mendel was EXTREMELY lucky to have worked with seven allele pairs (each pair for one of seven different genes) that each affect different “easy to score” phenotypic traits where there are very simple “full dominance” relationships between each of the allele pairs. We also entered into variations on dominance concepts such as incomplete dominance and codominance. Most phenotypic traits of interest to the health sciences (e.g. susceptibilities to genetic diseases and cancers, ability to defend against infectious microbes and parasites, aging) and agricultural sciences (e.g. plant drought tolerance, resistance to pests, livestock growth rates) are influenced by many genes. We will study these more complicated scenarios later in the term.

Week 3 Sneak Peek: Next week we will finish up Ch. 2, discussing more about variations on dominance concepts and epistasis. Ch. 3 will follow where discussion will turn toward the biology of chromosomes and the genetics of sex chromosomes. We will also cover the chi-square test.

Reflections: Weeks 0 and 1

Greetings, and welcome to my weekly BI311 blog summaries for students! I will be posting these on Friday afternoons for the rest of the term. These summaries will provide some basic reflections on the material covered over the previous week, as well as a sneak peek at information that will be presented the following week. I encourage everyone to take the few minutes to read through these blog posts to help learn and understand material presented in lecture. There will NEVER be material found only in the blog that will appear on the midterms and final. Rather, the blog summaries serve to supplement and provide a different ‘voice’ for the material covered during lectures, recitations, and in the homework. Enjoy!

Week 0 and 1 Reflections: In the first few lectures we reviewed DNA basics, with special emphasis on its features that make it an effective molecule for heredity (replication) and coding biological functions (transcription and translation, for example). There was also a strong emphasis on some of the basic laboratory techniques (restriction analysis, agarose gels, PCR, terminator-based DNA sequencing) used by scientists to study genetic processes – it will be important to have a strong understanding of these methods: what each one achieves, and how each one works. These methods will repeatedly come up throughout the course as we discuss more and more about genetic processes. We also started down the path of understanding the logic of genetic analysis in understanding biochemical pathways, including complementation testing. This sort of analysis underpins many genetic studies and the conceptual logic is very important to understand. In recitation, you got to know Sulochana and brushed up on some important concepts and terminology. These early lectures were meant to mostly be review – covering key concepts and topics that I will presume students know and understand as we move forward in the class.

Week 2 Sneak Peek: We will quickly review material from Chapter 1.3 on Monday, and then switch focus to the fundamental processes of transmission genetics that were revealed by Gregor Mendel’s famous pea plant experiments. Chapter 2 is very foundational – important to make sure you understand the concepts presented here to succeed in this class.