Daily Newsletter: November 12, 2013 - Lac Operon

Daily Newsletter

November 12, 2013 - Lac Operon

---

One of the most well studied gene regulation systems is the Lac Operon found in Escherichia coli. To understand this system, it is important to first understand that bacteria do not experience Transcription and Translation in exactly the same way as eukarotes (the mechanics are very similar, but there are some distinct differences).
The core difference between prokaryotes and eukaryotes is the nucleus. Since bacteria lack a nucleus, transcription takes place in the cytosol. There is no need for a tag to get out of the nucleus, so there is no need for capping. Also, there are no introns, so no need for splicing. Basically, there is no processing of RNA to make mRNA. There is no pre-mRNA. When a gene is transcribed, the transcript is mRNA.















Since there is no membrane separating transcription from translation, you can couple these to processes. As mRNA is made, it can be translated (polyribosome).

Bacteria have a single circular molecule of DNA (a genophore, not a chromosome). They have to conserve their genetic space, so bacteria combine genes for a metabolic process in a single mRNA. IMPORTANT: bacteria can combine genes for a metabolic pathway into a sequential sequence with a single promoter. Thus, when you transcribe, you get all the genes for a given metabolic pathway.
The word OPERON describes this unique arrangement of prokaryotic genes: One promoter and one operator for a given series of metabolically linked genes.
The lac Operon holds three genes that give the cell the ability to take in and use the sugar LACTOSE. [Image Note: Bacteria have two promoter regions, -10 & -35, for a given gene or operon]


For E. coli, glucose is the preferred sugar. When glucose is present, there is no need to use lactose: these genes are not transcribed. When there is no glucose, E. coli has to use other sugars. IF lactose is present, the genes for lactose utilization will be made. Conversely, if there is no lactose, they genes remain locked down.

• Glucose Present: No transcription
• Glucose Absent: Minimal transcription
• Glucose Absent, Lactose Present: Transcription of the lac operon.

NOTE: In this example, there are two ways to control the expression of a gene or operon:

1. You can block the operator of the gene. This prevents RNA polymerase from making RNA.
2. You can alter the promoter (or the interaction between transcription factors and DNA) to prevent binding of the Transcription Complex (RNA polymerase).

Negative Transcription Regulation (Repression) in the lac operon: There is a repressor for the lac operon. This is a protein that can bind to the operator of the lac operon (that region immediately downstream from the promoter). This creates a physical block that prevents RNA polymerase from transcribing.

The lac operon repressor (LACI) is a protein that is constitutively (always) expressed. This indicates that the lac operon is normally turned OFF. Notice that the gene for the regulatory protein is upstream from the lac operon.  The lac repressor (LacI) binds to the major groove of the DNA at the operator. As you can see in the annotated image of the repressor, you have a DNA-binding region (active site), and a regulatory domain. The regulatory domain has the ability to bind to Lactose (the inducer).

You must have a way to unlock the operon, or to put it another way, to inactivate the repressor. An inducer is a ligand that can bind to a regulatory domain, changing the shape of the regulatory domain, and thus inactivating the DNA-binding domain. In the case of the the lac repressor, the ligand is allolactose (a derivative of lactose). When allolactose binds to the lac repressor, the repressor is inactivated, and the operon cleared. This is seen in the image below.

Therefore, the lac operon is partially regulated by the presence of lactose in the environment. If there is no lactose in the environment, then there is no need to transcribe the three genes needed to use lactose.

Remember, we don't want to expend energy for things we don't need. Below is the size of the three gene products:

• β-Galactosidase 1,024 Amino Acids
• β-Galactoside Permease 418 Amino Acids
• β-galactoside transacetylase 203 Amino Acids

Total Amino Acids, 1645. Using the assumption of 4 ATP per amino acid added to a protein, that makes 6580 ATPs needed just for protein synthesis. With this many amino acids, you are also looking at 4935 nucleotides (remember 3 nucleotides = codon = 1 amino acid), plus at least 3 stop codons. Each nucleotide added to a transcript takes the equivalent of 1 ATP, so you are looking at 4944 ATP minimum to make the transcript. Just to make one example of each protein (gene product), you are looking at 11,524 ATP. Do you just make one example of each protein? NO! Do you have though the idea that this is energy consumptive? Would you make it if there was no lactose around?

Tomorrow we will look at the positive regulation of the lac operon. Remember, you still will not transcribe this operon if glucose is present. You only transcribe when glucose is absent. So how does the operon know when glucose is absent? That will be our discussion tomorrow.

---

Daily Challenge

In your own words, describe how lactose (allolactose) is used to regulate the transcription of the lac operon. In your discussion, make sure that you explain the concept of an operon, and discuss the differences between eukaryote and prokaryote gene transcription.

Daily Newsletter: November 11, 2013 - Introduction to Gene Regulation

Daily Newsletter

November 11, 2013

Introduction to Gene Regulation

---

Suggested Reading

These brief articles are a supplement to the readings from your textbook on Gene Regulation. You do not have to finish these articles today, but they will help you understand gene regulation at a deeper level. They also make great references for your next milestone paper.

• Regulation of Transcription and Gene Expression in Eukaryotes
• Operons and Prokaryotic Gene Regulation
• Positive Transcription Control: The Glucose Effect
• Negative Transcription Regulation in Prokaryotes
• Obesity, Epigenetics, and Gene Regulation
• Environmental Influences on Gene Expression

---

• Do we express all of our genes at the same time? Why?
• Do we need all of our genes expressed all the time? Why?
• Why do we have so many genes?

These are just a few of the questions you need to start asking yourself. Humans have hundreds of thousands of genes. Many are needed all the time (constitutive), but others are only needed when the cell get's certain signals. So how do we control the expression of all this genetic knowledge?

During mitosis, for example, did you see the production of DNA polymerase and the replication complex during the start of G1, or did you only see it after you passed the first restriction point? Do we keep DNA polymerase around just in case we are going to do some nuclear division? or do we unlock its expression only when needed?

Consider: The first restriction point determines if you are going to prep for division. When you have enough cyclin-dependent kinase available, you pass the restriction point. CDK signals the cell to get ready for division. How does this signal work? It changes gene expression (i.e., we activate regulated genes).

Think about the human body and homeostasis. Think about hormones. Are you always producing everything, or do you need to trigger some events? Could that trigger then be a regulated gene?

Remember that you need at minimum the equivalent of 4ATP per amino acid incorporated into a protein. Add to this 1 ATP equivalent for each nucleotide during transcription. You should quickly realize that gene expression is energy expensive.
Your goal today is to start reading about gene regulation, and more specifically, come to an understanding of the necessity of gene regulation.

---

Daily Challenge

Why do we need gene regulation? Today, reflect on the need and use of gene regulation. Why would an organism need to have some genes that it could turn on or off? Why would you need to control gene expression? Can the environment affect gene regulation? Can gene regulation affect evolution?

Case Study
In BOLO, you will also find a case study dealing with genetics.

Daily Newsletter: November 7, 2013 - Beyond Mendel

Daily Newsletter

October 18, 2012 Beyond Mendel

---

As discussed in class yesterday, after the rediscover of Mendel's work, people began investigating inheritance in other systems. As research continued, scientists began seeing that the Mendelian ratios did not always work. There were variations.

• Sex linked traits: Many species have what is known as a sex chromosome. Normally, every chromosome of a given set is the same size and shape, and most importantly, they carry the same genetic information. Not so with sex chromosomes. One variation of the chromosome is shorter, and does not carry the same information. When the standard and shorter chromosome (X and Y) are in the same cell, you have a Hemizygotic state. The suffix hemi come from ancient Greek, and means half. This refers to the idea that some of the genes in an XY pairing are haploid, not diploid. If the X has a gene that the Y does not possess, then it is always expressed (as would be the case in haploid organisms).

• Epistasis: Epistasis involves a two gene system. While the genotype follows Mendelian Laws, the phenotype does not. The reason is that one gene completely masks the effect of the second gene. Below is an epistatic example. Can you describe how this is different from a standard dihybrid cross?

• Pleiotrophy: This is when an allele has widespread impact in an organism, and so is not limited to one trait. For example, would a change in microtubles affect only one aspect of a cell? Would it affect only one cell type? Pleiotrophy may also cause problems at different stages of development, such as varying affects at different ages. Here is an example of pleiotrophy.
• Heterosis: Hybrid Vigor. Inbreeding can depress the adaptive strength of a species by allowing recessive traits a greater chance to express. When you begin crossing inbreed strains, you suddenly see an increase in adaptive vigor. This is useful with coordinated breeding of animals and plants, as the hybrid produced is stronger than the parental strains. It also informs our understanding of endangered species, and helps researchers work on ways to increase not only the adaptive vigor an an endangered species, but also the population size (without increasing inbreeding depression).

• Gene expression from environmental cues: Some genes are only triggered during certain environmental conditions. Remember the concept of signal pathways and signal transduction. The body picks up an environmental signal, and then tells the cells to change in some way. There are two terms you need to understand when dealing with Environmentally Cued Gene Expression:
  • Expressivity: This describes how much gene expression you get in an individual when exposed to the proper cues.
  • Penetrance: This is a population concept. When exposed to a given condition, how many individuals of the population express the gene?

Some additional reading for those interested in genetics:

• Gregor Mendel
• Mendel's 7 genes and their locations on pea chromosomes
• Gregor Mendel and the Principles of Inheritance
• Pedigree Analysis (Howard Hughes Medical Institute)

---

Blood Typing 

 The ABO blood type system is an example of codominance, meaning that in a heterozygous individual, both alleles are expressed, giving the individual two phenotypes (not a blending).  In other words, the alleles are considered dominant.  The ABO blood type is also multi-allelic, meaning there are more than two alleles for this one gene.  IMPORTANT:  The ABO blood type is derived from a single gene that has multiple alleles, of which two are considered dominant (A type and B type).

The gene (FUT1) for the ABO blood type system is a glycosyltransferase.  This enzyme adds carbohydrates onto proteins during post-translational modification.  The gene is found on chromosome 19. The antigen carrying proteins (the transmembranal protein that is glycosylated) currently has an unknown function.  The following shows the glycosylation patterns of the H antigen (the correct designation for the ABO antigen).

The fucose-galactose-N-acetyl-glucosamine glycosylation seen in O is common throughout the system.  It is the precursor to the other forms.  The FUT1 gene has allelic variation based on several SNPs.  The result is a difference in glycosylation patterns.  Specifically, we see the an additional N-acetyl-glalactosamine on A and an additional Galactose on B.

• If you have the allele for A, you produce the A glycosylation pattern. 
• If you have the allele for B, you produce the B gylcosylation pattern.
• If you have the allele for A and B, you produce both A and B glycosylation patterns.
• If you have neither the A or B allele, then you produce the precursor O configuration only.
• If you are Heterozygous A or B, meaning you have the (Ai^o) or (Bi^o) genotype, then you will produce the O glycosalation pattern.  Remember though, A and B are dominant to O.

An important discovery for the ABO system was the discovery of Antigen H.  This discovery began in 1952 by Y.M. Bhende.  Dr. Bhende, working in what is now known as Mumbai, India, discovered a patient who reacted to all ABO blood types.  They built antibodies against all ABO blood.   This led to the realization that O blood was antigenic to this patient.  What is now known is that the specific glycosylation of O is an antigen, and is the precursor to the A and B phenotypes.  Antigen H is the antigen found on O blood.  Antigen H is the precursor to antigen A and antigen B, as such, antigen H is found in A, B, AB and O blood.

Individuals who do not produce antigen H, described genotypically as (h,h) (heterozygoun recessive for the H antigen), are intolerant to all ABO blood (they build a reaction against it).  This blood group (h,h) is known as the Bombay Blood Group.  It represents the an additional allelic variation to the ABO blood type system.

---

Daily Challenge

Even with multiple alleles in a population (consider the ABO, Bombay blood groups) and multiple genes (epistasis), Mendel's laws still hold at the level of the genotype.  Explain how the laws of inheritance still hold true, and how variations such as multiple alleles, co-dominance, incomplete dominance, and epistasis all serve to increase population diversity.

Daily Newsletter: November 6, 2013 - Mendel's Second Law of Inheritance

Daily Newsletter

November 6, 2013 -

Mendel's Second Law of Inheritance

---

Yesterday we talked about inheritance of one genetic trait (gene). But humans (and other organisms) are made up of more than one genetic trait. Every protein made by a cell is encoded in at least one gene (quaternary proteins would be encoded by multiple genes). While most genes are "house keeping" genes, i.e., needed for the organisms survival that don't have many variations among individuals(ribosomes, the genes for glycolysis, respiratory chains, etc...), some genes have greater variance. This is how we get the diversity of life, and more importantly, the uniqueness of individuals (this uniqueness is needed for evolution).

So, what happens if you are interested in more than one trait? This is where Mendel's second law comes into play. He was curious as to whether he could follow the inheritance probabilities of two traits, so he looked simultaneously at two traits in the pea.

NOTE: Going above two traits becomes mathematically more difficult, and we generally don't look at those problems at this level. When you take genetics you may see some of these higher order problems.

Mendel's experiments helped him propose what is now known as the Law of Independent Assortment. We now know that the traits Mendel looked at were found on different chromosomes (DNA molecules). The math would have been horrible if they had been on the same chromosome! As research into genetics progressed, and we realized that genes could be on the same chromosome, Mendel's second law (and the expected probabilities) became the model by which variations were assessed. Gene Mapping utilizes Mendel's probabilities for a dihybrid cross as the starting point.


With the Law of Segregation (first law), Mendel showed that for each individual trait, a pea plant (and by extension a human) has two possible allele that they can carry for a single trait (gene). When ovum and sperm (pollen) are produced, the parent donates only one allele to the ovum or sperm; the parent donates only one allele to the next generation. Therefore a new individual is composed of a set of genes (and alleles) from the mother, and another set from the father.


So, what happens when you look at two different traits (genes)? Ultimately, what Mendel discovered is that the two different traits do no interfere with each other. An allele from the first gene is donated independently, and is uninfluenced by, the allele from the second gene. So, you have a 50/50 chance of donating a given allele from the first gene, and a 50/50 chance of donating an allele from the second plant. [When looking at two distinct traits, we refer to the mating as a dihybrid, i.e., two trait, cross]

The math gets a little harder, but the idea is the same. The easiest way of showing what happens is to look at the Punnet Square for a visual interpretation of the probabilities. Below is a great picture of a Punnet square:

As you can see, on the top we put the Male Genetic Donation, and on the left side we put the Female Genetic Donation. There is a 50/50 chance the male will donate a given allele, same with the female. Look at how this is represented. Male donation is either B or b. Each has its own column. For the female, each possible donation has its own row. You then just cross-reference column and row to find out the possible offspring. The Punnet square also provides a rapid visual. 4 possible offspring, 3 of which are purple.

The Punnet square can be expanded to look at a dihybrid cross (two traits). Below is a good image of a dihybrid Punnet square. Note that the PHENOTYPIC ratio is 9:3:3:1. This is a critical ratio that will be seen repeatedly in biology.

---

Daily Challenge

Explain the concept of Independent Assortment. I made a point that this does not always occur when genes are on the same chromosome. So, what happens to Independent Assortment when genes (traits) are on the same chromosome? Why is it important?

Daily Newsletter: November 5, 2013 - Mendel's First Law of Inheritence

Daily Newsletter

November 5, 2013 -

Mendel's First Law of Inheritence

---

To understand Mendel, we must first understand some terms and put them into context.

• Gene - A segment of DNA that holds a sequence of nucleotides that provide the instructions on how to create either RNA or a Protein. These are known as gene products.
• Chromosome - This is a molecule of DNA and associated proteins. The chromosome is only visible during specific phases of nuclear division. The word chromosome is used loosely (weak sense) as a synonym of DNA.
• Haploid - A cell that has only one copy of each DNA molecule (chromosome).
• NOTE: each organism has a known number of DNA molecules (chromosomes).
• Humans have 23 different DNA molecules (23 different chromosomes).
• Diploid - A cell that has two copies of each DNA molecule.
• Humans have 46 chromosomes total: 2 copies of each of the 23 different DNA molecules.
• Mutation - A change in the nucleotide sequence of DNA.
• Genetic Variation - Different versions of the same gene generated by mutation. These could be subtle or pronounced alterations.
• Phenotype - The specific physical form an individual's genetics produces. Generally we start by looking at one well defined physical trait, such as flower or seed color.
• Allele - Genetic Variation in a single gene that results in different phenotypic expression.
• There can be many different genetic variations in a population.
• An individual can only possess a number of allele equal to the # of chromosomal copies.
• So, a human is diploid, possessing 2 copies of each gene.
• Humans can therefore have at most 2 alleles for each gene.
• There can be more than 2 alleles though for the entire human population; each individual can only have a maximum of 2.
• A haploid individual has only 1 allele for each gene.
• Homozygous - A diploid organism that has the same allele for a given gene.
• Heterozygous - A diploid organism that has different alleles for a given gene.
• Genotype - The specific alleles an individual possesses for a given trait.

The following picture will help you visualize the concept of Alleles, Homozygous, and Heterozygous.
Mendel's work with the garden pea, Pisum sativum, resulted in two laws of inheritance. The focus of his work was to determine the inheritance pattern of specific traits. For example, if you have a pure breeding strain that produces white flowers, and a pure breeding strain that produces purple flower, what is the percentage of offspring which will possess purple flowers? His work was based on probability mathematics, and as we have discussed previously, mathematical certainty is needed in the establishment of laws.
Mendel's First Law is known as the Law of Segregation. Remember, Mendel did not know about genes or even DNA. He was working solely with gross physical characteristics that could be observed with the naked eye.
Going back to flower color, Mendel first wanted to see what would happen if you took pure-breeding white flowered peas and crossed (mated) them with pure-breeding purple flowered peas (F₀ generation with homozygous purple and homozygous white individuals). Many of Mendel's contemporaries held the view that the offspring were produced by a blending of characteristic. What Mendel saw directly contradicted this view. He saw only purple flowers, this contradicted the idea of blending.
Mendel decided to self-cross (self-pollinate) this generation of purple flowers (F₁ heterozygous individuals). The next generation held both purple and white flowered individuals, but in a very specific ratio - 3:1. He repeated his experiment, and even used different characteristics. The same thing happened: pure-breeding parents produced offspring with a specific trait, and when self-crossed, these produced offspring in which the original parental traits appeared in a 3:1 ratio.
Mendel inferred the following from his mathematical calculations:

• Each individual possesses two "factors" which determined the specific trait, e.g., Flower Color. (Mendel's Factors = Genes).
• When an ovum or pollen is produced, it holds only one Factor (Gene).
• When an ovum and pollen join, the new individual will carry one factor (gene) from the ovum (mother) and one factor (gene) from the pollen (father).
• There is a 50% chance (probability) that the mother will donante one factor over another, and a 50% chance (probability) that the father will donate one factor over another.
• You thus have four possible outcomes.

An easy way to view these possible outcomes is to use a Punnett square, a simple visual probability tool. The square shown to the right demonstrates the inheritence (genotype and phenotype) probability of pea flower color. On the left hand side of the square you will see the female (pistil) allelic contribution, and on top the male (pollen) allelic contribuition. The mother can donate either B or b; the father can donate either B or b.
If the offspring came from the joining of B from the mother and B from the father, then it will be Homozygous B (dominant) and Purple. An offspring with Bb will be heterozygous and Purple, while an offspring with bb will be homozygous (recessive) and white. There is a 1/4 probability of BB (25%), a 1/2 probability of Bb (50%), and a 1/4 probability of bb (25%). The most common is the heterozygous condition (remember this).
We now understand more regarding the mechanism which Mendel inferred, and have molecular data to support his initial findings. Mendel's factors are genes, and alleles describe the differences between factors. Cells undergo meiosis to produce gametes (sperm/ovum, pollen/ovum, etc...), and it is this process that causes the seperation of genes. We will talk more about meiosis next week.
But why did the offspring of the pure-breeding plants produce only purple flowers?
The traits he picked had variations based on a mutation of a single gene. Today we would call this type of mutation a knock-out mutation, because a function was knocked out. The purple color is produced by a fully functional gene. It produces a functional pigment. The white color is produced because the gene that codes for the pigment is flawed; it can't produce the pigment. What you have is one functional gene product (dominant) that masks a non-functional gene product (recessive). Mendelian genetics become much more murky when you have multiple genes coding for a trait or when you don't have a complete knockout.

---

Daily Challenge

How does the first law of inheritance affect our understanding of how genes are passed from generation to generation, and how does it affect our understanding of evolution.  Consider the consequences of a single mutation, such as the one seen in Sickle Cell Anemia.  How does that move from generation to generation, and how does natural selection play a role once we understand inheritance?