Appunti di Genetics

Mendel’s experiments

Gregor Mendel

• Born in the district of Moravia (part of Austro-Hungarian empire)
• Performed experiments in the Augustinian monastery St. Thomas in the city of Bruenn, now Brno of the Czech Republic.
• He is the founder of the science of genetics.
• He proposed the concept of the gene (not the word) in 1865. The importance of his work was not recognized until about 1900 (after his death).
• Interested in making hybrids, worked with different vegetables and crossing breeds.
• Came from a poor family but he studied in Vienna, trained in physics and maths. He taught in the monastery.
• We don’t know a lot about him: published 1 article but no info on how he came up with the idea because there are no notes or documents because after he died, the next monk burned all his things.

His approach was very new and still used now (scientific technique): how the characters are inherited in the next generation. The inheritance at that time was a mystery. There was the blending theory popular in his time: cross 2 animals, the offspring has the mixture of the 2 individuals (ex red paint + white paint = pink). So you can never get back to the original (never will have white paint). Mendel saw this from the physics point of view (particles) so it’s not a blend because you can get back what you had in the original generation. He used a model system (garden peas) to study: small and easy to get (like the mouse and drosophila-exp for genetics, now). Model system for genetics: large offspring, easy to handle, easily crossed, clear phenotypes, cheap, different characters available. So he used peas because he selected them as model system. He made a hypothesis on what the outcome would be. He collected large data set (statistic) to see that the results were consistent with his hypothesis. Hypothesis – experiment – result.

Why peas?

Peas were easily available in a wide array of distinct shapes and colours; Easy to self or cross-pollinate; Inexpensive; Little space needed; Short generation time; Produce many offspring (many seeds per plant). He studied 7 phenotypes:

• Round vs wrinkled seeds
• Yellow vs green seeds
• Purple vs white flowers
• Inflated vs pinched pods
• Green vs yellow unripe pods
• Axial vs terminal flowers
• Long or short stems

Each character is in a different chromosome. Method: analysis of a single character at a time (it’s better to analyse them independently even in the exercises); he used plants that differ for only one single character; use parental/pure line (gives always the same offspring for that specific character ex always white flowers, with no exception); he counted the observed phenotypes and their proportions (quantitative approach) – he vs the others who studied genetics: they observed small amount of numbers, he observed large quantity.

If the seed is round, the gene is active, otherwise mutated (silent). He crossed purple and white flowers by taking anthers (main reproductive organs before pollen) and taking the pollen in the white flower. The F1 (filial) generation was all uniform. He then crossed the round and wrinkled and saw that they were all round so round was dominant and wrinkled recessive. That happened for all 7 characters (slide). He studied wrinkled and round because they depended on both father and mother and not only on one and it’s easier to look in the seeds (you don’t have to wait) than flowers (wait till the next year, plant them).

Round vs wrinkled: F1 all round, F2 (3/4 round to 1/4 wrinkled) and that happened to all characters. So all the characters acted the same. He took 519 F2 round peas and grew plants from them by self-pollinating (166 only round, 353 impure mixture round and wrinkled, ratio 3:1). F2 wrinkled gave pure breeding wrinkled. It segregates in 1:2:1 ratio.

Mendel’s explanation

There are hereditary determinants of a particular nature, genes (he called them particles). 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). Cells were just discovered so he knew how everything worked from his friends who studied fecundation. The dominant (RR) (gives R gamete) and recessive (rr) (gives r gamete) give Rr heterozygous (round seeds because R is dominant). F1 gives 50%/50% of R gametes or r gametes: ¼ homozygous dominant, 2/4 heterozygous dominant, ¼ homozygous recessive. The principle of segregation: the members of the gene pairs segregate equally into the gametes, which carry only one member of each gene pair. The fertilization is random: 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.

Mendel’s laws

First law - Law of segregation

The principle stating that during the production of gametes the two copies of each hereditary factor segregate so that offspring acquire one factor from each parent.

Second law – Law of independence

The principle stating that the laws of chance govern which particular characteristics of the parental pairs will occur in each individual offspring.

Third law – Law of dominance

The principle stating that one factor in a pair of traits dominates the other in inheritance unless both factors in the pair are recessive.

When Mendel’s proposal is viewed in the light of what was known of cells in the mid-nineteenth century, Mendel was decades ahead of his time. His prescient deduction of paired elements that break up their association and separate into different daughter cells is what makes him the founder of genetics. Mendel died at the age of 61. At his death, no one officially recognized the significance of his scientific contribution except the Agricultural Society (in obituary). “His experiments opened a new epoch. What he did will never be forgotten”. The world eventually acknowledged his contribution to science 16 years later. To the present day, his ideas, modern attitude, and way of scientific critical thinking made his legacy ever living.

The hereditary material is DNA

It was believed that hereditary material were proteins at Mendel’s time. How it was discovered: Mendel’s experiments in 1866 indicated the existence of genes (elements as he called them) that control certain characters but he didn’t know what was the basis of heredity, DNA or proteins? In 1869 Friedrich Miescher discovered a new type of weak acidic chemical present in large quantities in nuclei of leucocytes and he suggested that this is the substance of which genes are made of. We now know that this chemical is deoxyribonucleic acid or DNA. After 1870 the importance of the nuclei becomes evident: a microscope was used and observed that during fertilisation the nuclei of sperm and egg cells fuse (The next step was the discovery of chromosomes that are present in the nuclei). Mendel was inspired by that discovery and based his findings on this. Then chromosomes were discovered. Around 1900 it was known that the number of those are specific for species and that their number is relatively constant in cells and are separated equally during a division. These observations supported the idea that these chromosomes are responsible for the heredity. Around 1920 there was several indirect evidence that chromosomes contain DNA. So is the genetic material DNA or protein? More or less 50/50 (chromatin). Scientific establishments were convinced that it was all protein: there were known functions and they were highly variable in structure. Those who suggested it was all DNA did that because: DNA is a highly stable molecule, its amount is the same in all cells except for gametes which have half of it. These people had to prove 2 things: that it is DNA and that it’s absolutely not protein.

Experiments: 1928 Griffith – bacteria in colonies (streptococcus pneumoniae) divide by binary scission. Each colony comes from one single cell. It has two strains: S III (smooth) and R II (rough). S contains a polysaccharide capsule that makes it smooth and virulent (causes pneumonia in humans and lethal in mice). R is not virulent because it doesn’t have the capsule, it’s genetically determined. Experiment: he took S strain and injected it in the mouse and the mouse died and then injected R and the mouse lived. If he took the S strain and killed them with heat, the bacteria all die and the mouse lived. When they mixed S strains dead and living R in the mouse, the mouse died. When they took samples, they saw S living bacteria. They saw that genetic material necessary for the capsule was passed to R strains. So the character passed from the heated S to R cells.

Avery, MacLeod, and McCarty in 1944 repeated this experiment: they added RNAases (enzyme that degrades RNA) or proteases (enzyme that degrades proteins) or DNases to the heated S cells. They used these cells on the colony and they found that S cells grew from the first 2, in the last there were only R cells (no transformation). It means that if you degrade DNA, there is no way R cells can become S cells, but without RNA or protein, it still works. That meant that DNA was the hereditary material (experiment asked in exam). They found out that genes are composed of DNA. 1952 Hershey and Chase used T2 phages (with S35 to label the proteins or P32 to label the DNA). T2 phage with S35, it injects its DNA in E coli and then put them in the blender so that all phages are released from the bacteria surface by the shaking (they are called ghosts). DNA here is not radioactive so most of radioactivity is found in the proteins. Using T2 phages with P32 on the phage, it showed that it is not protein that it injected into the bacteria but DNA and that had all the information for new phages (progeny), so it encodes for new proteins. That is because all the radioactivity was in the E coli and some of it even in the progeny of the phage. So now there’s proof that DNA is the hereditary material. There was still reluctance to accept this conclusion: how could a human derive from one cell? How could DNA encode all this and how could it be transmitted from one individual to another? Genetics material must have both the ability to encode information and to duplicate that information precisely.

So what kind of structure could allow this? There is the sugar (pentose) – RNA (one strand, OH; DNA 2 and H), phosphate group, nitrogen bases (purines with 2 aromatic rings – bigger so it paired with each other it doesn’t fit in the DNA structure, pyrimidines 1). After the evidence of the role DNA plays, its structure was studied and discovered in 1953 by Watson and Crick (didn’t do it alone, Rosalind Franklin and Maurice Wilkins who did an X-ray diffraction analysis: rays – sample DNA – screen: found out that DNA is long and skinny, has two similar parts that are parallel to each other and run along the length of the molecule and that the molecule is helical (spiral-like), when they presented their findings in the meeting in which Watson and Crick were present and it was a great help for their studies)). Earlier Chargaff in the end of the 40s analysed a large selection of DNAs from different organisms. He established rules about the quantity of each component of DNA: n purines = n pyrimidines, T=A, C=G, A+T not always equal to G+C. Watson and Crick published the “suggestion” of structure of DNA in 1953. The DNA has a sugar phosphate backbone held together by hydrogen bonds and phosphodiester bonds between the sugar molecule and the phosphate group. A-T two bonds, G-C 3. There are the major and minor groove (used by proteins which enter in the major grove). From diffraction analysis, there are 2 forms of DNA:

• A form (less hydrated, more compact; 11 bases per turn; observed under the condition of low humidity, present in certain DNA/protein complexes; RNA double helix adopts a similar conformation)
• B form (10 bases per turn, observed at high humidity, most closely corresponds to the average structure of DNA under physiological conditions; more hydrated, less compact, found most frequently in living cells)
• There is also Z form which is very rare.

The double helix is antiparallel: DNA is oriented from 5’ (prime) to 3’; each cell has 2m of DNA. Human DNA was sequenced in 10 years (600 pairs at a time), only 10-15% gives proteins. Extract DNA from cell – you have fragments but need it whole and distinguish different chromosomes. Take 1 chromosome, divide it in big pieces, divide it once again and sequence them.

DNA replication

The model was proposed by Watson and Crick. The 2 strands are separated (hydrogen bonds) and each strand becomes a template for a new daughter strand. This way it’s possible to copy the exact info. How it occurs? It could be (they didn’t know) semi conservative (each original strand is a template for a new strand), conservative (the original strand gives identical strands), dispersed (mosaic of pieces dispersed). How they understood that it’s semiconservative: in 1958 Meselson and Stahl performed experiments to understand which replication model is correct. They used CsCl (caesium chloride) gradients. They mixed DNA with CsCl and centrifuged (45 000 rpm) it till an equilibrium was reached and they got the gradient of density (used UV to observe it, 260nm). The DNA could be separated and extracted with a needle. They grew E coli cells in a medium in N15 (heavier than N14, so it would create a different band in the centrifuge than N14; which is incorporated in nitrogen bases which are part of DNA nucleotides) and grew them for many divisions, so that their DNA contained only N15. The cells were washed and put on the medium containing N14 and samples were taken after 1 or 2 cell divisions (different generations). Then the DNA was extracted and centrifuged on CsCl gradient. At time 0, all the DNA is N15. After one generation (1 division), one strand is N14 and one N15, so together it forms an intermediate band. After 2 divisions, you get 2 bands: one light band and one intermediate. Looking at this, they knew it was semi-conservative. If it were conservative, at time 0 it would all be N15, but after 1 division there would be one heavy band and one light band. If it’s semi-conservative, you will never get the heavy band back. (asks what bands you get on exams). RNAs can have one single strand or can form double strands (ex 16S rRNA E coli) by base pairing, miRNA have necessary loops to function.

DNA replication process

DNA replication is a very complex process and requires the participation of many components. There is also a quality control check (sometimes there is uracil, which is not good to have in DNA). Mutations often occur in replication but are usually removed. The repairing mechanism during and after replication are important because otherwise, they are transmitted to the next generation. It has basic components: you need the DNA polymerase (catalyses synthesis) which needs a primer (RNA primase) and that needs a free 3’ (5’-3’). The primer is a starting point: it’s RNA because DNA polymerase needs a primer, while RNA polymerase doesn’t. The template strand goes from 3’ to 5’. On the free 3’ the DNA polymerase starts and incorporates a nucleotide 3 phosphate (alfa, beta and gamma), so there is the release of the pyrophosphate (beta + gamma). To label DNA with P32, you label the alfa phosphate because it stays. It forms a phosphodiesterase bond. To start, it needs the replication fork. The DNA is unwound but the strands are antiparallel, so one strand goes from the inside to outside (lagging strand), while the other is continued while the fork opens (leading strand). The lagging needs many primers and the Okazaki fragments then have to be united. The RNA then is degraded (by the nuclease) and the DNA polymerase can elongate the primer with new DNA and then the ligase unites the pieces. The ligase uses phosphodiesterase bond by using 1 ATP. Overview: the DNA is opened by helicases and they have proteins (SSB) that destabilise the double helix to keep the double strands open. The double helix is wound like ropes so for the helicase there’s more and more tension to open the strands so to take tension off the helix topoisomerases are used. These complexes break one strand, unwind it and then bind them again and then moves upstream of the replication fork. In the cell there is never a double stranded breaks and they are very dangerous, while single strand break is no issue.

DNA denaturation and renaturation

DNA denaturation: it has a melting temperature Tm, then it renaturates at 20 degrees and forms a double strand again. If the DNA is heated and then cooled very quickly (4 degrees) it cannot fold back, so it needs a gradual cooling. If you heat it or change pH, in the renaturation it folds back on itself. You can also hybridize it with a small piece of DNA, cool it, and this piece of DNA will bind with its complementary, it finds it. If you label the hybrid (“probe”), you can find the complementary sequence to analyse it.

PCR (polymerase chain reaction)

You take a piece of genome and amplify it. You need primers for PCR so you need to know the sequence. Nowadays we have the complete genomic libraries. It has a limit in size (5000 bases) + it sometimes makes mistakes. It uses a thermal cycler (video). It goes in cycles (usually 36). From 1 single copy of DNA, you can get millions in just some hours. At every cycle you have to add new polymerases because they denaturate. Thermophilus aquaticus has DNA polymerases which isn’t affected by heat so it doesn’t have to be changed.

The structure of chromosomes

The chromosome consists of 1 DNA molecule and in human they are all different in size. The karyotype by giemsa staining can be used. All 46 chromosomes in humans make up to 2m of DNA. Prokaryotes have circular chromosomes. How is the DNA packed in small cells so that it’s also functional? It is winded in nucleosomes (8 histones). The DNA + protein is called chromatin, which structure is important for the function of living organisms. There is euchromatin and heterochromatin which are different in packaging, if eu is stained, the stain won’t be intense as it is in heterochromatin. Heterochromatin is 2 types: constitutive (always present) in centromeric and telomeric regions; facultative, where the chromatin can switch from eu to heterochromatin and vice versa and in heterochromatin most genes are inactive because it’s highly packed so proteins and factors can’t enter. Facultative regions are important to regulate genes so chromatin is