Appunti di Genetica in inglese

Overview genetics!

• Lessons 1+2: Mendel’s experiments!
• Lessons 3+4: The hereditary material is DNA!
• Lessons 5+6: DNA replication!
• Lessons 7+8: Chromosomes, mitosis and meiosis!
• Lessons 9+10: More Mendel, Chi-Square test!
• Lessons 11+12: Interactions between the alleles!
• Lessons 13+14: Pedigree and chromosome theory!
• Lessons 15+16: Sex determination and linkage!
• Lessons 17+18: Chromosome mapping I!
• Lessons 19+20: Chromosome mapping II!
• Lessons 21+22: Transcription and gene function!
• Lessons 23+24: One gene one enzyme!
• Lessons 25+26: Gene interactions!
• Lessons 27+28: Bacterial conjugation!
• Lessons 29+30: Recombinant DNA technology!
• Lessons 31+32: Bacteriophage genetics!
• Lessons 33+34: Translation!
• Lessons 35+36: Genes and mutations I!
• Lessons 37+38: Genes and mutations II!
• Lessons 39+40: Chromosome mutations I!
• Lessons 41+42: Chromosome mutations II!
• Lessons 43+44: Regulation of gene transcription I!
• Lessons 45+46: Regulation of gene transcription II!
• Lessons 47+48: Population genetics I!
• Lessons 49+50: Population genetics!

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Lessons 1+2

Mendel’s experiments

Mendel is the father of genetics, the founder of the science of genetics: he was born in the district of Moravia and he performed his experiments in the monastery St. Thomas in the city of Bruenn.

He proposed the concept of the gene in 1865 but the importance of his work was not recognized until about 1900.

His experiences are still a good example of scientific practices and scientific technique.

He did research with materials which were suited to the problem at hand, such as peas (or Drosophila nowadays: even now scientists use models to study gene functions).

Mendel designed his experiments carefully and collected large amounts of data and was aware that he needed large amounts of data to make sense out of it.

After having done his experiments he used mathematical analysis to show that his results were consistent with his explanatory hypothesis: this was not done before.

The predictions of the hypothesis were then tested in a new round of experimentation: if the hypothesis didn’t fit he turned back to reformulate the hypothesis and did the experiments again (clear example of scientific method).

He did his research using the Garden pea (Pisum sativum). Many were the reasons why he chose peas:

• They were easily available in a wide array of distinct shapes and colors;
• They were also easy to self or cross-pollinate;
• They were inexpensive;
• They need a little space to grow: that means he could have many peas in a small area (same reason why Drosophila was and is used).
• They have a short generations time;
• They produce many offspring, many seeds per plant (offspring = progenie, prole).

He looked at seven characters which were localized on the first seven chromosome (one for each chromosome, each character was localized on a different chromosome):

• Round or wrinkled ripe seeds;
• Yellow or green seed interiors;
• With purple or white petals;
• Inflated or pinched ripe pods (bacelli);

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• Green or yellow unripe pods;
• Axial or terminal flowers;
• Long or short stems.

Mendel’s method

1. He didn’t look at all the seven characters together: he analyzed a single phenotypic character at a time. This is one of the rules of genetic’s analysis.
2. He created a pure line for each character: a pure line is a breed or strain of animals, or plants in this case, in which certain characters appear in successive generations as a result of inbreeding or self-fertilization. It means that if you self a pure line you always get the same phenotype. A pure line is also called parental line and indicated with “P”.
3. He counted the observed phenotypes and their proportions through mathematical analysis: nobody has ever done that before (quantitative approach).

From one pollination you can get many seeds.

He obtained the pure lines of each character by selfing each one: for example he used the round shape seeds and after having selfed it he always got round shape peas, so he got his pure line. The same thing was done with the wrinkled seeds and he always got wrinkled shape peas.

After having obtained a pure line Mendel used to cross two different pure lines: for instance he cross-pollinated the pure line of red flowers with the pure line of white flowers (by transferring the pollen from the red flower to the white one).

What he found out was that if he crossed the pure line of red flowers with the pure line of white flowers all the offspring was all red, he got all red flowers and there were no white flowers: red is the dominant character, while white is the recessive one but it is there since genes come in copies.

The red flowers obtained were not a pure line, they were a mixture: they have both the red character and the white character but the white is masked by the red one which is dominant.

He did the same procedure with the round shape seeds and the wrinkled ones and found out that all the seed of the first filial generation were round. That means one character was dominant over the other: round is dominant and wrinkled is recessive.

He did this experiment with each one of the seven characters and found out which phenotypes was dominant and which one recessive.

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Mendel liked to work with seed colour or shape phenotypes because in peas these phenotypes (round or wrinkles, yellow or green) are depending on the genetic constitution of the seed itself and not the maternal parent. He could already see these phenotypes on the mother plant where he did the cross.

This means that you can observe the ‘next generation phenotype’ without having to grow the seeds, you can look on the mother plant without growing the plant itself, if you work with seed color or shape phenotypes.

If you look at flower color you have to grow these seeds to see the color of the flower generation.

As we said before the first filial generation is not pure anymore because it’s a result of the cross between the round and wrinkled pure lines.

All the peas of the F1 were round, so round was the dominant character and wrinkled the recessive one (but the wrinkled character was there, masked by round one).

In fact, selfing the peas of the first generation, Mendel found out that there were also wrinkled peas: he considered a big number of peas (7324) and found out that 5474 were round (dominant) and 1850 wrinkled (recessive): this is a segregation ratio which was 3:1 and whichever characters he considered the result was always 3:1.

In the F1 you see just the dominant phenotype but in the F2 the recessive phenotype became visible again (3/4 is dominant and 1/4 is recessive —> 3:1 phenotypic ratio).

He analyzed the round peas from F2 (which were 3/4 of all the peas obtained), grew plants from them and selfed them showing that there were pure lines. What he found out was that:

• 1/4 of the peas obtained were pure-breeding round because 1/4 of the plants from the F2 gave only round;
• 2/4 of the peas obtained were impure round because 2/4 of the plants from the F2 gave a mixture of round and wrinkled peas in 3:1.

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Then he analyzed the wrinkled peas from F2 (which were 1/4 of all the peas obtained), grew plants from them and selfed them: he obtained just wrinkled peas demonstrating that they were pure-breeding wrinkled.

• P round x wrinkled;
• F1 all round;
• Grow F1 plants from the F1 peas: ¼ pure-breeding round;
• F2 ¾ round 2/4 “impure” round;
• ¼ wrinkled ¼ pure-breeding wrinkled;
• Mendel took 519 F2 round peas and grew plants from them and selfed them.
• 166 plants gave only round peas;
• 353 plants mixture round and wrinkled (ratio 3:1);
• F2 wrinkled were sown and plants were selfed: all wrinkled.

Mendel always observed this 1:2:1 ratio which is not a phenotypic ratio but a genotypic ratio.

He explained this as follows:

1. The existence of genes: there are hereditary determinants of a particular nature. We now call these determinants genes.
2. Genes are in pairs: alternative phenotype of a character are determined by different forms of a single type of gene (alleles). In adult pea plants, each type of gene is presented twice in each cell (gene pair).
3. The principle of segregation: the members of the gene pairs segregate (separate) equally into the gametes, or eggs and sperm.
4. Gametic content: consequently, each gamete carries only one member of each gene pair.
5. Random fertilization: the union of one gamete from each parent to form the first cell (zygote) to form a new progeny individual is random. Gametes combine without regard to which member of a gene pair is carried.

The round peas have two genes as well as the wrinkled peas. Each round peas is homozygous dominant while each wrinkled peas is homozygous recessive.

Crossing them you obtain an F1 which is all round because R is dominant over r. His gametes will be 50% dominant and 50% recessive. Each peas from the F1 is heterozygous.

Crossing the peas of the F1 you get the F2:

• The phenotypic ratio is 3:1, since 75% of peas is round while 25% of peas is wrinkled.
• The genotypic ratio is 1:2:1, because 1/3 of the 3/4 round peas of the F2 are pure-breeding round and 2/3 of the 3/4 round peas of the F2 are impure round because they gave a mixture of round and wrinkled peas as we said before.

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Mendel’s laws

1. First law (Law of Segregation): during the productions of gametes the two copies of each hereditary factor segregate so that offspring acquire one factor from each parents.
2. Second law (Law of Independence): the laws of chance govern which particular characteristics of the parental pairs will occur in each individual offspring. It states that the combination of the alleles are casual.
3. Third law (Law of Dominance): one factor in a pair of traits dominates the other in inheritance unless both factors in the pair are recessive.

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Lessons 3+4

The hereditary material is DNA

Mendel’s experiments (1866) indicated the existence of genes that control certain characters. But what is the basis of heredity? Are these hereditable genes formed by DNA or by proteins?

In 1869 Friedrich Miescher discovered a new type of weak acidic chemical that is present in large quantities in nuclei of leucocytes and he suggests that this is the substance of which genes are made of. We know now that this chemical is deoxyribonucleic acid or DNA.

After 1870 the importance of the nuclei becomes evident. Using a microscope it was observed that during fertilisation the nuclei of sperm and egg cells fuse together. The next step was the discovery of chromosomes that are present in the nuclei.

Around 1900 it was generally known that the number of chromosomes are specific for species, that their number is relatively constant in cells and are separated equally during divisions.

These observations supported the idea that these chromosomes are responsible for the heredity. Around 1920 there was several indirect evidence that chromosomes contain DNA.

Is the genetic material DNA or proteins?

Chromatin in cells is composed roughly of 50% nucleic acids and 50% proteins.

• Most of the scientists were convinced that the genetic material was made up of proteins. Proteins were already studied: they are macromolecules with known functions and they have a highly variable structure.
• Other were convinced that genetic material was made up of DNA, since it was such a stable molecule, the amount is the same in all cells and gametes have halve the amount of somatic cells.

The scientists that were intending to demonstrate the function of DNA had two problems: they had not only to show that DNA is the genetic material but they also had to demonstrate that proteins do not have this function.

The following experiments demonstrated that DNA is the hereditary material.

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Experiment of Griffith

The first scientist trying to demonstrate that DNA is the hereditary material was Griffith who used the pneumococcus. The pneumococcus, growing on a plate, started dividing by binary scission making up a colony of pneumococci.

Looking at these colonies he studied the phenotypes of the pneumococci.

There are two phenotype:

• S-type (smooth), which have a capsule;
• R-type (rough) without the capsule.

The bacteria which were enclosed in a polysaccharide capsule (S-type) can resist the immune system of human and mice and they are usually virulent. Virulent strain (specie) causes pneumonia in humans and is normally lethal in mice.

The bacteria which don’t have a capsule (R-type) cannot resist and are destroyed by the immune system of human and mice.

The experiment done by Griffith followed these steps:

Griffith found out that:

• If you put virulent bacteria (S-type bacteria) inside a mouse, he dies, since these bacteria can resist the immune system of human and mice;
• If you kill the virulent bacteria with warm and then inject them into the mouse he doesn’t die.
• If you inject the non-virulent bacteria (R-type bacteria) into the mouse he doesn’t die.
• If you mix virulent bacteria killed (S-type bacteria) with living non-virulent bacteria (R-type) and then inject them into the mouse, the mouse dies.

This fact suggested that something which was contained in the S-type bacteria had been transferred in the R-type cells making them S-type cells which caused the death of the mouse.

But that experiment doesn’t prove that DNA was the hereditary material: it only proved that hereditary information can be transferred from one bacteria-type to the other.

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Experiment of Avery, Mac Leod and McCarty

In 1944 Avery, Mac Leod and McCarty took an extract taken from the S-cells-killed and they incubated it in a RNAase (enzyme which destroys RNA) and in a protease (which destroys proteins).

Later they took another extract from the S-cells-killed and incubated it in a DNAase.

After that, they incubate the RNAase, DNAase and protease (in which they had incubated the extract taken from S-cells-killed) and mixed them with a large number of R-type-bacteria which were put in different plates.

Later they looked which one of the plates containing R-cells gave S-cells. What they found out was that:

• The colonies of R-cells became colonies of S-cells just when mixed with the RNAase and protease: even RNA and proteins were destroyed R-cells became S-cells meaning that the hereditary material which moved from the S-cells to the R-cells making them S-cells was not made up of RNA or proteins.
• The colonies of R-cells didn’t become colonies of S-cells when mixed with the DNAase: destroying DNA didn’t allow the R-cells to become S-cells.

Since R-cells became S-cells just when DNA is not destroyed this experiment proved that DNA was the transforming principle.

The demonstration that DNA is the transforming principle was the first demonstration that genes are composed of DNA and that are not made up of proteins/RNA.

Experiment of Hershey and Chase

In 1952 Hershey and Chase did an experiment using phage T2, a virus which is made up of genetic material protected by a capsule made up of proteins: it is not able to reproduce so it injects his DNA in the bacteria in order to reproduce itself.

Hershey and Chase marked the phages with Sulphur and Phosphate: the Sulphur was used to mark the proteins while the Phosphate was used to mark DNA.

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They incubated the phages marked with S in bacteria, later they did the same thing with the phages marked with P.

They wanted to find was where the radioactivity was and what they discovered was that:

• Injecting the phages marked with S, most of the radioactivity was in ghosts phages which have not entered the bacteria; that meant that proteins have not entered the bacteria and since phages usually injects they hereditary material in bacteria, that meant that their hereditary material was not made up of proteins.
• Injecting the phages marked with P they found out that most of radioactivity was contained in the bacteria itself and not in the ghosts phages; that meant that since phages usually injects they hereditary material in bacteria, their hereditary material was made up of DNA.

These experiments made clear that DNA is the hereditary material but why was there such reluctance to accept this conclusion? How could all the information of an organism’s features be stored in such a simple molecule?

• How could such information be passed on from one generation to the next?
• Genetic material must have both the ability to encode information and to duplicate that information precisely.

What kind of structure could allow this?

The structure of the DNA

The difference between RNA and DNA lies in the sugar: both are made up of nucleotides but while RNA-nucleotides contains the Ribose, the DNA-nucleotides contains the Deoxyribose the (in second position we have an -H in the Deoxyribose while we have an -OH in the Ribose).

The basic components of each DNA-nucleotides are:

• Phosphate;
• Deoxyribose sugar;
• Nitrogen base;

B