1. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 1. The Building Blocks of DNA DNA has three types of chemical component: DNA has three types of chemical component:  Phosphate  a sugar called deoxyribose, and  four nitrogenous bases:  Adenine (A)  Guanine (G)  Cytosine (C)  Thymine (T).  Two of the bases, adenine and guanine, have a double-ring structure characteristic of a type of chemical called a purine. The other two bases, cytosine and thymine, have a single-ring structure of a type called a pyrimidine. The chemical components of DNA are arranged into groups called nucleotides, each composed of a phosphate group, a deoxyribose sugar molecule, and any one of the four bases. The chemical components of DNA are arranged into groups called nucleotides, each composed of a phosphate group, a deoxyribose sugar molecule, and any one of the four bases.

2. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 2. The four nucleotides Chemical structure of the four nucleotides (two with purine bases and two with pyrimidine bases) that are the fundamental building blocks of DNA. The sugar is called deoxyribose because it is a variation of a common sugar, ribose, which has one more oxygen atom

3. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 3. The double helix DNA is composed of two side-by-side chains ("strands") of nucleotides twisted into the shape of a double helix. The two nucleotide strands are held together by weak associations between the bases of each strand, forming a structure like a spiral staircase DNA is composed of two side-by-side chains ("strands") of nucleotides twisted into the shape of a double helix. The two nucleotide strands are held together by weak associations between the bases of each strand, forming a structure like a spiral staircase In three dimensions, the bases form rather flat structures, and these flat bases partly stack on top of one another in the twisted structure of the double helix. This stacking of bases adds tremendously to the stability of the molecule by excluding water molecules from the spaces between the base pairs.In three dimensions, the bases form rather flat structures, and these flat bases partly stack on top of one another in the twisted structure of the double helix. This stacking of bases adds tremendously to the stability of the molecule by excluding water molecules from the spaces between the base pairs.

4. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 4. Strand polarity The arrangement of the components of DNA. A segment of the double helix has been unwound to show the structures more clearly. The diagram shows the sugar-phosphate backbone and the hydrogen bonding of bases in the center of the molecule. The sugar-phosphate bonds are called phosphodiester bonds. The carbons of the sugar groups are numbered 1’ through 5’ (next slide). One part of the phosphodiester bond is between the phosphate and the 5’ carbon of deoxyribose, and the other is between the phosphate and the 3’ carbon of deoxyribose. Thus, each sugar-phosphate backbone is said to have a 5’-to- 3’ polarity, and understanding this polarity is essential in understanding DNA properties. In the double-stranded DNA molecule, the two backbones are in opposite, or antiparallel, orientation. One strand is oriented 5’  3’; the other strand, though 5’  3’, runs in the opposite direction, or, looked at another way, is 3’  5’

5. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 5. Base pairing The bases of DNA interact according to a very straightforward rule, namely, that there are only two types of base pairs: A·T and G·C. The bases in these two base pairs are said to be complementary. This means that at any "step" of the stair like double-stranded DNA molecule, the only base-to- base associations that can exist between the two strands without substantially distorting the double- stranded DNA molecule are A·T and G·C. Note that because the G·C pair has three hydrogen bonds, whereas the A·T pair has only two, one would predict that DNA containing many G·C pairs would be more stable than DNA containing many A·T pairs. In fact, this prediction is confirmed. Heat causes the two strands of the DNA double helix to separate (a process called DNA melting or DNA denaturation); it can be shown that DNAs with higher G+C content require higher temperatures to melt them

6. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 6. DNA forms giant molecules Although hydrogen bonds are individually weak, the two strands of the DNA molecule are held together in a relatively stable manner because there are enormous numbers of these bonds. It is important that the strands be associated through such weak interactions, since they have to be separated during DNA replication and during transcription into RNA Although hydrogen bonds are individually weak, the two strands of the DNA molecule are held together in a relatively stable manner because there are enormous numbers of these bonds. It is important that the strands be associated through such weak interactions, since they have to be separated during DNA replication and during transcription into RNA The sugar-phosphate backbone, being connected by covalent bonds, is also stable; bacterial DNA form a single giant molecule; in eukaryotes, each chromosome is composed by a single giant molecule of DNA The sugar-phosphate backbone, being connected by covalent bonds, is also stable; bacterial DNA form a single giant molecule; in eukaryotes, each chromosome is composed by a single giant molecule of DNA

7. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 7. How much DNA per genome? Almost all cells of all organisms contain at least one copy of the entire genome of the species (most cell are diploid, i.e. they contains two copies). Almost all cells of all organisms contain at least one copy of the entire genome of the species (most cell are diploid, i.e. they contains two copies). Genome sizes are measured in units of thousands of nucleotide pairs (called kilobases, kb) or millions of nucleotide pairs (megabases, mb), or sometimes in picograms (10 -12 gr) Genome sizes are measured in units of thousands of nucleotide pairs (called kilobases, kb) or millions of nucleotide pairs (megabases, mb), or sometimes in picograms (10 -12 gr) In general, the total amount of chromosomal DNA in different animals and plants does not vary in a consistent manner with the apparent complexity of the organisms. In general, the total amount of chromosomal DNA in different animals and plants does not vary in a consistent manner with the apparent complexity of the organisms. Yeasts, fruit flies, chickens, and humans have successively larger amounts of DNA in their haploid chromosome sets, in keeping with what we perceive to be the increasing complexity of these organisms. Yet the vertebrates with the greatest amount of DNA per cell are amphibians, which are surely less complex than humans in their structure and behavior. Yeasts, fruit flies, chickens, and humans have successively larger amounts of DNA in their haploid chromosome sets, in keeping with what we perceive to be the increasing complexity of these organisms. Yet the vertebrates with the greatest amount of DNA per cell are amphibians, which are surely less complex than humans in their structure and behavior.

8. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 8. Genome sizes Amount of DNA in the genomes of various organisms

9. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 9. Lenght of a DNA molecule The single chromosome of Escherichia coli is about 1.3 mm of DNA. To enable a macromolecule this large to fit within the bacterium, histone-like proteins bind to the DNA, segregating the DNA molecule into around 50 chromosomal domains and making it more compact. Then an enzyme called DNA gyrase supercoils each domain around itself forming a compacted, supercoiled mass of DNA approximately 0.2 µm in diameter, called nucleoid.

10. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 10. The nucleoid The nucleoid is one long, single molecule of double stranded, helical, supercoiled DNA. In most bacteria, the two ends of the double- stranded DNA covalently bond together to form both a physical and genetic circle. The chromosome is generally around 1000 µm long and frequently contains as many as 3500 genes. E. coli, that is 2-3 µm in length has a chromosome approximately 1400 µm long.

11. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 11. Eukaryotic Nuclear Genomes A human cell contains about 2 meters of DNA, packed into 46 chromosomes, all inside a nucleus only 6  m in diameter. A human cell contains about 2 meters of DNA, packed into 46 chromosomes, all inside a nucleus only 6  m in diameter. Thus, in order to pack the DNA into the nucleus, there must be several levels of coiling and supercoiling. Thus, in order to pack the DNA into the nucleus, there must be several levels of coiling and supercoiling. These levels of DNA structure cannot be resolved by the optical microscope, under which interphase nuclei stained with DNA-specific dyes appears composed of a dense, dark-staining material called heterochromatin, and is scattered throughout the nucleus, and a very light- staining flocculent material which fills the rest of the nucleus, called euchromatin. Nucleus Nucleolus Cell wall

12. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 12. DNA in interphase It is thought that most part of each chromosome in an interphase nucleus (chromatine) has the form of the “30 nm fiber” It is thought that most part of each chromosome in an interphase nucleus (chromatine) has the form of the “30 nm fiber” The figure on the left shows the tangled chromatin fibers obtained after disrupting a nucleus. Shearing forces can be used to further uncoil and stretch these fibers and the beaded filaments appear. The strands between the beads are segments of double stranded DNA (right panel).

13. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 13. The 30 nm fibre Different levels of chromosome uncoiling. The bottom of the figure shows the DNA helix (which is DNA stripped of its histones). In the normal, unstripped chromosome, the double stranded DNA is wrapped around sets of 8 macromolecules of histones (proteins) to form a 10 nm filament. These sets of histones are separated by spacer regions of 4 nm DNA filament. They are the 10 nm nucleoprotein fibrils or "beads on a string" seen in electron micrographs which are called nucleosomes. The next level of coiling produces the 30 nm nucleoprotein fibers.

14. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 14. Compacting factors Stuffing the long strands of chromosomal DNA into a eukaryotic nucleus requires that the DNA be compacted in length approximately 10,000 to 50,000 -fold. Incredibly, cells achieve this tight packing of the DNA while still maintaining the chromosomes in a form that allows regulatory proteins to gain access to the DNA to turn on (or off) specific genes or to duplicate the chromosomal DNA (replication). Stuffing the long strands of chromosomal DNA into a eukaryotic nucleus requires that the DNA be compacted in length approximately 10,000 to 50,000 -fold. Incredibly, cells achieve this tight packing of the DNA while still maintaining the chromosomes in a form that allows regulatory proteins to gain access to the DNA to turn on (or off) specific genes or to duplicate the chromosomal DNA (replication).

15. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 15. The average lenght of coding regions Estimates of the average length of polypeptide chains coded by genes of various organisms; these value have to be multiplied by 3 in order to obtaing the lenght of the corresponding coding DNA. Tipical values are 1,000 to 1,500 bp.

16. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 16. A “gene” is not only coding sequence Definition: A gene is a discrete unit of DNA (or RNA in some viruses) that encodes a nucleic acid or protein product that contributes to or influences the phenotype of the cell or the organism. Definition: A gene is a discrete unit of DNA (or RNA in some viruses) that encodes a nucleic acid or protein product that contributes to or influences the phenotype of the cell or the organism. Genes are the functional units of chromosomal DNA. Each gene not only encodes the structure of some cellular product, but also bears control elements (short sequences) that determine when, where, and how much of that product is synthesized. Most genes encode protein products; special classes of genes encode for RNA molecules. Genes are the functional units of chromosomal DNA. Each gene not only encodes the structure of some cellular product, but also bears control elements (short sequences) that determine when, where, and how much of that product is synthesized. Most genes encode protein products; special classes of genes encode for RNA molecules.  The way genes encode proteins is indirect and involves several steps. The first step is to copy (transcribe) the information encoded in the DNA of the gene as a related but single-stranded molecule called messenger RNA. Subsequently the information in the messenger RNA is translated (decoded) into a string of amino acids called a polypeptide. The polypeptides, on their own or by aggregating with other polypeptides and cell constituents, form the functional proteins of the cell.

17. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 17. Introns and exons Trying to pinpoint precisely what genes are is complicated by the fact that many eukaryotic genes contain mysterious segments of DNA, called introns, interspersed in the transcribed region of the gene. Introns do not contain information for functional gene product such as protein. They are transcribed together with the coding regions (called exons) but are then excised from the initial transcript. Trying to pinpoint precisely what genes are is complicated by the fact that many eukaryotic genes contain mysterious segments of DNA, called introns, interspersed in the transcribed region of the gene. Introns do not contain information for functional gene product such as protein. They are transcribed together with the coding regions (called exons) but are then excised from the initial transcript. Since correct sequence in the introns (as well as in the regulatory region) is necessary in order to generate a properly sized transcript at the right time and place, introns (along with coding and regulatory regions) should be considered part of the overall functional unit, in other words, part of the gene Since correct sequence in the introns (as well as in the regulatory region) is necessary in order to generate a properly sized transcript at the right time and place, introns (along with coding and regulatory regions) should be considered part of the overall functional unit, in other words, part of the gene

18. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 18. Schematic gene structure Generalized gene structure in prokaryotes and eukaryotes. The coding region (dark green) is the region that contains the information for the structure of the gene product (usually a protein). The adjacent regulatory regions (lime green) contain sequences that are recognized and bound by proteins that make the gene's RNA and by proteins that influence the amount of RNA made.

19. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 19. Number of introns-exons per gene Many eukaryotic genes contain mysterious segments of DNA, called introns, interspersed in the region of the gene. Introns do not contain information for functional gene product such as protein. Distribution of the number of exons among genes of three organisms

20. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 20. Average gene length Intron/exon statistics for various organisms

21. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 21. Genomes and genes The number of genes increases with genome size, but the trend is complicated due to repetitive DNA and introns.

22. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 22. Most eukaryotic DNA does not include genes Between genes there is DNA, mostly of unknown function. The size and nature of this DNA vary with the genome. Between genes there is DNA, mostly of unknown function. The size and nature of this DNA vary with the genome. In bacteria and fungi there is little, but in mammals the intergenic regions can be huge. In bacteria and fungi there is little, but in mammals the intergenic regions can be huge. Sequences of DNA that exist quite distant from a given gene can affect the regulation of that gene. They could thus be considered part of the functional gene unit, even though separated by long segments of DNA having nothing to do with the gene in question. Sequences of DNA that exist quite distant from a given gene can affect the regulation of that gene. They could thus be considered part of the functional gene unit, even though separated by long segments of DNA having nothing to do with the gene in question. In many eukaryotes some of the DNA between genes is repetitive, consisting of several different types of units repeated throughout the genome. Some of the repetitive DNA is dispersed; some is found in contiguous "tandem" arrays. Repetitive DNA is also found in some introns. The extent of this DNA is different in different species, and indeed there is variation of repeat number within species. In many eukaryotes some of the DNA between genes is repetitive, consisting of several different types of units repeated throughout the genome. Some of the repetitive DNA is dispersed; some is found in contiguous "tandem" arrays. Repetitive DNA is also found in some introns. The extent of this DNA is different in different species, and indeed there is variation of repeat number within species.

23. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 23. Comparing gene densities Schematic diagram of gene topography in four organisms. Light green = introns; dark green = exons; white = intergenic regions

24. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 24. A small fraction of total eukaryotic DNA is coding In mammals, only a few percent of the DNA is actualy coding:

25. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 25. Coding sequences are needles in the haystack It is apparent that the coding sequences are only a small part of the genome in most eukaryotes, particularly in human. Finding these regions is like finding a needle in the haystack. It is apparent that the coding sequences are only a small part of the genome in most eukaryotes, particularly in human. Finding these regions is like finding a needle in the haystack. In addition, the genes are not uniformly distributed. There are regions in the genome where the genes are packed together, and regions where they are sparse, where finding genes is like finding water in a desert. In addition, the genes are not uniformly distributed. There are regions in the genome where the genes are packed together, and regions where they are sparse, where finding genes is like finding water in a desert.