Experiments – AP Biology

the blueprint of life [11]: DNA replication 1

DNA replication

Definition: the process of copying a parental DNA molecule to form two daughter DNA molecules.

Introduction to DNA replication

DNA replication is essential for cell proliferation, i.e. mitosis, meiosis.
DNA replication is a complex endeavor involving a series of enzyme activities.→see “DNA polymerases”
DNA replication is performed in a semiconservative and semidiscontinuous mode.→see “DNA replication is semi-conservative”
DNA replication has 3 stages: initiation, elongation and termination.→next section
DNA replication is tightly regulated, involving various protein-protein, protein-DNA interactions.
DNA replication of prokaryote and eukaryote shares similar features, but is distinctive in details.

Chemical Reaction of DNA replication

Essentials

1. Substrate: deoxynucleoside triphosphates(dNTPs)
2. Template: a primer-template junction

DNA is synthesized by extending the 3’ end of the primer (free 3’-OH is required)

– RNA primer or priming from a nick in DNA

3. Enzymes: DNA polymerases etc

4. Energy supply: Hydrolysis of pyrophosphate (PPi) is the driving force for DNA synthesis

5. Ions involved: Mg++ or Zn++

DNA polymerases

DNA polymerase I
- Pol I was the first enzyme discovered with polymerase activity, and it is also the best characterized one.
- Although abundant in cells (400/cell), Pol I is NOT the primary enzyme involved with bacterial DNA replication.
- Main functions of Pol I: (1) Fill any gaps in the new DNA that result from the removal of the RNA primer by its 5’ -3’ polymerase activity; (2) Remove a new mispaired base by proofreading (校读)3’-5’ exonuclease (外切酶) activity. (3)Remove the RNA primer by its 5’-3’ exonuclease activity

The 3′–>5′ exonuclease activity intrinsic to several DNA polymerases plays a primary role in genetic stability; it acts as a first line of defense in correcting DNA polymerase errors. A mismatched basepair at the primer terminus is the preferred substrate for the exonuclease activity over a correct basepair. (source)

DNA polymerase III
- The primary polymerase in DNA replication, although lower in abundance (15/cell)than pol →referred to as “replicase”
- functions: (1) 5’→3’ polymerase activity; (2) 3’→5’ exonuclease activity – proofreading
- Catalytic efficiency: much higher than pol I→High processivity and polymerization rate
- A multi-unit complex: “holoenzyme” (全酶)

DNA replication is semi-conservative

Bet you all have learned it in high school, and the famous experiment by Meselson and Stahl. We still need to go over the points again as they are essentially important for what we will learn next.

The key to the mechanism of DNA replication is the fact that each strand of the DNA double helix carries the same information-their base sequences are complementary (we talked about this in THE BLUEPRINT OF LIFE [2]: PRIMARY AND SECONDARY STRUCTURE OF DNA).

During replication, the two parental strands separate and each acts as a template (that’s right, the template for DNA replication is DNA itself!)to direct the enzyme-catalyzed synthesis of a new complementary daughter strand with the normal base-pairing rules (A-T, C-G)

This semi-conservative mechanism was demonstrated experimentally in 1958 by Meselson and Stahl.

Hypotheses:

In the experiment:

E. coli cells were grown for several generations in presence of the stable heavy isotope ¹⁵N so that their DNA became fully density labeled (both strands are ¹⁵N labeled: ¹⁵N/¹⁵N)

The cells were then transferred to medium containing only normal ¹⁴N and, after each cell division, DNA was prepared from a sample of the cells and analyzed on a CsCI gradient using the technique of equilibrium (isopycnic) density gradient centrifugation, which separates molecules according to differences in buoyant density.

After the first cell division, when the DNA had replicated once, it was all of hybrid density, in a position on the gradient half way between fully labled (¹⁵N /¹⁵N )and fully light (¹⁴N/¹⁴N). After the second generation in ¹⁴N, half of the DNA was hybrid density and half fully light.

Thus, two of the hypotheses were denied, left us with the semi-conservative mechanism.

After each subsequent generation, the proportion of ¹⁴N/¹⁴N increased, while some DNA of hybrid density persisted. Thus the semi-conservative mode of DNA replication is confirmed: each daughter molecule contains one parental strand and one newly-synthesized strand.

Mendel’s Genetics [6]: Examples of epistasis

In Mendel’s dihybrid cross, each gene locus(the position of a gene along a chromosome, often used to refer to the gene itself.) had an independent effect on a single phenotype. Thus, the R and r alleles affected only the shape of the seed and had no influence on seed color, while the Y and y alleles affected only seed color and had no influence on seed shape. In this case, there were two separate genes that coded for two separate characteristics.

But what happens when two different loci affect the same characteristic? For instance, what if both of the loci in Mendel’s experiment affected seed color?

Let’s begin with a relatively simple example.

example (1) 9:3:3:1→9:7

enzyme A serves to convert white substrate in an unnamed plant to white product; enzyme B synthesizes purple pigment and converts the white product to purple product.

In this case, If BOTH A & B exist, purple product is yielded;

If ONLY A OR B exists, synthesis of purple pigment cannot be completed, and purple product won’t be produced;

If NEITHER ENZYME exist, purple pigment, surely, cannot be synthesized, and purple product won’t be produced.

Based on the info above, the dihybrid F2 generation looks like this:

When the situation gets a little bit more complicated:

example (2) 9:3:3:1→9:3:4

enzyme A serves in the synthesis of red pigment and converts white substrate in an unnamed plant to red product; enzyme B synthesizes purple pigment and converts the red product to purple product.

In this case, If BOTH A & B exist, purple product is yielded;

If ONLY A exists, synthesis of red pigment can be completed but synthesis of purple pigment cannot, and red product will be produced;

If NEITHER ENZYME exist or ONLY B exists, surely, neither red or purple pigment, can be synthesized, and the the yield will be white.

Based on the info above, the dihybrid F2 generation looks like this:

A more complex situation:

example (3): 9:3:3:1→12:3:1

Here two enzymes compete for the same substrate. Enzyme A converts the substrate to a purple product, and enzyme B to a red product. BUT enzyme A has much higher affinity for the substrate than enzyme B. The difference in affinity is so marked that enzyme B can only work effectively without the presence of enzyme A

SO as long as enzyme A is present, the yield is be purple; only when enzyme B exists without enzyme A would the yield be red; and only when neither B or A exists would the yield be white.

Based on the info above, the dihybrid F2 generation looks like this:

[F2] purple: red: white=12: 3: 1

The three examples above lead us to a new concept: Epistasis.

Sometimes genes can mask each other’s presence or combine to produce an entirely new trait. Epistasis describes how gene interactions can affect phenotypes.

In a strict sense, 12: 3: 1 is the only ratio which was originally referred to as epistasis, because the presence of enzyme A can completely make the genotype of the B gene. But the term is now used wherever genes interact to alter the expected ratios.

There are several other variations of the 9:3:3:1 ratio caused by interaction between the gene products, including 9:6:1, 15:1, and 13:3.

In every case the ratios are derived by summing together the four phenotype classes 9, 3, 3, or 1 of the basic ratio.

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For more examples and explanations of the Epistasis, see Epistasis and Its Effects on Phenotype | Learn Science at Scitable

Mendel’s Genetics [5]: The dihybrid cross

Last sections we discussed inheritance where only a single gene was involved. The 3:1 ratio is the basic Mendelian ratio and everything that follows depends upon it.

The obvious next step is to look at a situation where the inheritance of two different inherited characters are studied at the same time, a dihybrid cross.

Again, we learn dihybrid cross by experiments. Let’s see what Mendel did:

He crossed two pure-bred starins of pea plants, one producing only round yellow seeds and the other only wrinkled green seeds.Round seeds are dominant over wrinkled ones; yellow seeds are dominant over green ones. Both phenotypes are determined by one single gene.

(Why did Mendel use the seed characteristics as his focus of study instead of other traits of the pea plants? )

A cross between the two parents produced a F1 generation that consisted only of round yellow seeds. Self-fertilization of F1 yielded F2 generation, the seeds of which showing considerable diversity.

Four different phenotypes could be identified. Of 556 seeds analyzed, Mendel found 315 round yellow seeds, 108 round green seeds, 101 wrinkled yellow seeds and 32 round green seeds. Close to a ratio of 9: 3: 3: 1, which is referred to as the dihybrid ratio.

dihybrid cross — THE 9:3:3:1 IN MENDEL’S EXPERIMENTS, image from Instant Notes in Genetics

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Interestingly, if we only focus on the round/wrinkled phenotype, the ratio of round seeds and wrinkled seeds is 3:1. the monohybrid ratio. Similarly, if we only focus on the yellow/green phenotype, the ratio of yellow seeds and green seeds is the monohybrid ratio as well. Just as we said “The 3:1 ratio is the basic Mendelian ratio and everything that follows depends upon it”

Now I want to introduce a little bit mathematics into this section. Let’s see what happens when we use polynomial multiplication to explain the relationship between 9: 3: 3: 1 and 3:1:

(yellow: green)× (round: wrinkled)

= (yellow·round): (yellow·wrinkled): (green·round): (green·wrinkled)

known: yellow: green=3:1; round: wrinkled=3:1

SO: (yellow: green)×(round: wrinkled)=(3:1)×(3:1)

=(3×3):(3×1):(1×3):(1×1)=9:3:3:1

Therefore, (yellow·round): (yellow·wrinkled): (green·round): (green·wrinkled)=9:3:3:1
Does this reasoning inspire you? What happens when it comes to a tri-hybrid ratio?

(3:1)×(3:1)×(3:1)=

(3×3×3):(3×3×1):(3×1×3):(3×1×1):(1×3×3):(1×3×1):(1×1×3):(1×1×1)

=27 : 9 : 9 : 9 : 3 : 3 : 3 : 1

See? Quite easy. The ratios for the three groups of phenotypes are simply multiplied across, and the 27 : 9 : 9 : 9 : 3 : 3 : 3 : 1 ratio is obtained.

SO what happens when it comes to tetra-hybrid, penta-, hex-? What happens when it comes to n-hybrid?

I’m sure the answer is easy for you now, make the n phenotypes multiplied across, and the expected ratio is obtained. In plain mathematics, (3:1)^n

——————————————————————————-As noted before the 3:1 ratio can be distorted by factors such as incomplete dominance or lethal alleles. These also affect the 9:3:3:1 ratio, but other factors can also modify this ratio.

The two different genes must not act on the same character. For instance, if the proteins encoded by the two genes are involved in the same biochemical pathway then the ratios of phenotypes resulting from the genotypes will be altered.
If the two genes lie close together on the same chromosome, linkage happens and the ratio won’t be 9:3:3:1 either, as we mentioned in Review of Mendel’s Genetics, the very beginning of our Genetics study.

Testcross

As with the monohybrid cross, it is also possible to conduct a testcross with F1 generation of the dihybrid cross.

Mendel’s Genetics[3]: Variations of the 3:1ratio

Variations of the 3:1 ratio

The simple 3-to-1 monohybrid ratio is not always observed in instances where only one gene is responsible for a particular phenotype.

A number of factors:

Partial or incomplete dominance

Complete dominance means the phenotype of first filial generation(heterozygous) is exactly identical to that of one of the parents(both homozygous). Partial or incomplete dominance means the first filial has phenotype somewhere between that of both parents.

For example, When two pure-bred snapdragons, with white and red petals respectively, cross, their first filial generation has pink petal rather than red or white.

In this case, the homozygous phenotypes are red, or white petals while heterozygous one is between white and red: pink petal.

Thus, it is conceivable that when it comes to the second filial generation, which was produced by the self fertilization of the heterozygous F1, F2 should have three different phenotypes, white, pink, and red. And we can also deduce the ratio of them is 1:2:1.

Codominance

Codominance is similar to incomplete dominance, but here the heterozygote displays both alleles(两种等位基因均被表达).

For example, in humans the MN blood group is controlled by a single gene.

In humans the main blood group systems are the ABO system, the Rh system and theMN system.

Only two alleles exist, M and N. Children whose father is an NN homozygote with N blood and whose mother is a MM homozygote with group M blood are MN heterozygotes and have group MN blood.Both phenotypes are identifiable in the hybrid. And the ratio also switches from 3:1 to 1:2:1.

Lethal alleles

Some alleles affect the viability of individuals that carry them.

In most cases the homozygous recessive does not survive but the heterozygotes may have a normal lifespan.

The best-known example of lethal alleles is the inheritance of yellow coat color in mice.

Yellow fur can arise in strain of mice with different colors, or instance, black. Yellow coat color is dominant to black coat. Mice with BB alleles are back, with BB^yare yellow, with B^yB^y alleles are supposed to be yellow as well, but B^yB^y alleles are lethal and any mice with this genotype die in utero(in the uterus : before birth).

SO it is conceivable that when two yellow mices are mated ratio of the different phenotypes of their first generation is 2 : 1, rather than 3 : 1 or 1 : 2 :1.

NOTE: The allele B^yis recessive in its relation to its effect on viability (only homozygous B^yB^ys die, while the heterozygotes survive ), but dominant in relation to coat color(heterozygotes present in yellow fur in stead of black fur.).

Other examples where alleles are lethal when homozygous but have a dominant effect when heterozygous, include :

tailless Manx cats

genetics — a manx cat, image from the Internet

a breed of domestic cat(Felis catus) originating on the Isle of Man, with a naturally occurring mutation that shortens the tail.

The Manx taillessness gene is dominant and highly penetrant; kittens from two Manx parents are generally born without any tail. Being homozygous for the taillessnees gene is lethal in utero.Thus, tailless cats can only be heterzygous. Because of the danger of having homozygous taillessness gene, breeders avoid breeding two entirely tailless Manx cats together.(wikipedia: Manx (cat))

short-legged Creeper chickens.

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(Annie: If none of the homozygous yellow mice can survive before birth, then where did all the heterozygous yellow mice come from in the first place???????)

Answer: Through mutation. The presence of one mutant allele alters development so as to produce characteristic changes to the animal, but when two of the mutant alleles are present, development is so aberrant as to cause death.

This may occur in utero as described above or resulted in shortened life expectancy as found in several examples in humans, such as Tay-Sachs disease, Huntington’s syndrome（亨丁顿舞蹈症） or sickle-cell anemia（镰刀形红血球病）. (from Instant Notes)

Mendel’s Genetics[2]: The monohybrid cross

Keywords:

Phenotype

Any character (trait) which can be shown to be inherited, such as eye color, leaf shape or an inherited disease, such a cystic fibrosis, is referred to as a phenotype.

Description: A fly may be described as having a red-eyed phenotype. A child may be described as displaying the cystic fibrosis phenotype.

Genotype

The pattern of genes that are responsible for a particular phenotype in a individual is referred to as genotype.

Dominance

In hybrids between two individuals displaying different phenotypes, only one phenotype may be observed. This phenotype is referred to as the dominant trait and the un-shown one the recessive.

For instance, if the wife has wide eyes while the husband has small eyes, and their little girl has wide eyes, then the wide eyes are dominant to small eyes.

Pure-breeding lines

Organisms which have been inbred for many generations in which a certain phenotype remain the same.Pedigree breeds of dogs or cats are commonplace examples of pure-breeding lines.

A puppy from two purebred dogs of the same breed, for example, will exhibit the traits of its parents, and not the traits of all breeds in the subject breed’s ancestry.

Homozygous: Individuals with two identical copies of a gene.

“True breeding (pure-breedind) organisms are always homozygous for the traits that are to be held constant.”

Heterozygous: Individuals with two different copies of the gene.
Alleles: The different variants of a gene.

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Mendel made a cross between two pure-breeding lines of pea plants, one of which had violet petals and the other white petals. The hybrids produced in this cross were referred to as the F1 (first filial) generation.

In Mendel’s experiment, the ratio of violet pedals and white ones in the second filial were very close to 3 to 1, which applied to the theoretic reasoning shown above.

He did many other experiments focusing on different types of genotypes of the pea plants and the results were shockingly similar. The hidden phenotype in the first filial reappeared in the second filial and the ratio of the dominant to the recessive phenotype were all close to 3 to 1.

The 3:1 ratio is referred to as the monohybrid ratio and is the basis for all patterns of inheritance in higher organisms.

One simple extension of the 3:1 phenotype ratio is a 1:1 ratio, produced when a heterozygous F1 individual is crossed to the homozygous-recessive parent. The process is known as testcross.

Testcross is useful in any condition when it is necessary to determine whether an individual is heterozygous or homozygous. Conceivable that if F2 all have dominant phenotype, then the tested parent is homozygous-dominant; if F2 have a 1:1 ratio of dominant and recessive phenotype, then the tested parent is heterozygous.

Review of Mendel’s Genetics

Here I found a great page story-telling Mendel’s Genetics.Can’t be more suitable as a revision of what we learned about genetics and inheritance in high school. >>Mendel’s Genetics

I believe by reading the link page you have remembered the principles of Mendel’s Genetics. We’ll summarize these principles again in next posts.

While Mendel’s research was with plants, the basic underlying principles of heredity that he discovered also apply to people and other animals because the mechanisms of heredity are essentially the same for all complex life forms.”

It must be a cliche to summarize the success factors of Mendel’s experiments, but it has to be done, for many of the factors are still important for today’s experimentalists.

Firstly, before the experiment,Mendel spent a long time observing different traits of the peas and decided which traits he was going to focus on in the after experiments. He was prepared, had anticipation and, perhaps already held some hypothesis of what was going to happen.

Then it was the choice of his “lab-rats”. As the link page says,”Mendel picked common garden pea plants for the focus of his research because they can be grown easily in large numbers and their reproduction can be manipulated. ” Based on a large number of offspring, the resulting statistics can be assumed as very close to theoretic statistics. In this case, it’s way more convenient to study the traits of these peas than those of some fragile and rare pole plants.

More important, “pea plants have both male and female reproductive organs. As a result, they can either self-pollinate themselves or cross-pollinate with another plant. In his experiments, Mendel was able to selectively cross-pollinate purebred plants with particular traits and observe the outcome over many generations. This was the basis for his conclusions about the nature of genetic inheritance.”

Reproductive
structures of
flowers

drawing of a flower cross-section showing both male and female sexual structures — the picture is from http://anthro.palomar.edu

Last but not least, Mendel was a pioneer in applying Math(Statistics) to experiment analysis. He rounded the ratio of numbers of different traits to a whole number and discovered the astonishing similarity of all the results.

In high school that’s all the factors, but actually there’s more. For one, Mendel succeeded because all the genes that controlled traits he picked to observe happened to be on different chromosomes. Otherwise, the phenomena of “linkage” would have appeared (which we’ll talk about later)and he should never have had such a groundbreaking discovery.