genetica per scienze naturali a.a. 03-04 prof s. presciuttini 1. the building blocks of dna dna has...

Genetica per Scienze Naturalia.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:

PPhosphatehosphate a sugar called a sugar called deoxyribosedeoxyribose, and , and four nitrogenous basesfour nitrogenous bases::

AdenineAdenine (A) (A) GuanineGuanine (G) (G) CCytosineytosine (C) (C) TThyminehymine (T) (T). .

Two of the bases, adenine and guanine, have a double-ring structure characteristic of a Two of the bases, adenine and guanine, have a double-ring structure characteristic of a type of chemical called a type of chemical called a purinepurine. The other two bases, cytosine and thymine, have a . The other two bases, cytosine and thymine, have a

single-ring structure of a type called a single-ring structure of a type called a pyrimidinepyrimidine.. The chemical components of DNA are arranged into groups called The chemical components of DNA are arranged into groups called

nucleotidesnucleotides, each composed of a phosphate group, a deoxyribose , each composed of a phosphate group, a deoxyribose sugar molecule, and any one of the four bases. sugar molecule, and any one of the four bases.


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. The double helix DNA is composed of two DNA is composed of two

side-by-side chains side-by-side chains ("strands") of nucleotides ("strands") of nucleotides twisted into the shape of twisted into the shape of a double helix. The two a double helix. The two nucleotide strands are nucleotide strands are held together by weak held together by weak associations between the associations between the bases of each strand, bases of each strand, forming a structure like a forming a structure like a spiral staircasespiral staircase

• In three dimensions, the bases form rather flat structures, and these flat bases 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. 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 This stacking of bases adds tremendously to the stability of the molecule by excluding water molecules from the spaces between the base pairs. excluding water molecules from the spaces between the base pairs.


4. Strand polarityThe 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. Base pairingThe bases of DNA interact according to a very The bases of DNA interact according to a very

straightforward rule, namely, that there are only straightforward rule, namely, that there are only two types of base pairs: two types of base pairs: A·TA·T and and G·CG·C. The bases in . The bases in these two base pairs are said to be these two base pairs are said to be complementarycomplementary. . This means that at any "step" of the stair like This means that at any "step" of the stair like double-stranded DNA molecule, the only base-to-double-stranded DNA molecule, the only base-to-base associations that can exist between the two base associations that can exist between the two strands without substantially distorting the double-strands without substantially distorting the double-stranded DNA molecule are A·T and G·C.stranded DNA molecule are A·T and G·C.

Note that because the G·C pair has Note that because the G·C pair has three three hydrogen bondshydrogen bonds, whereas the A·T pair has only , whereas the A·T pair has only two, one would predict that DNA containing many two, one would predict that DNA containing many G·C pairs would be more stable than DNA G·C pairs would be more stable than DNA containing many A·T pairs. In fact, this prediction containing many A·T pairs. In fact, this prediction is confirmed. Heat causes the two strands of the is confirmed. Heat causes the two strands of the DNA double helix to separate (a process called DNA double helix to separate (a process called DNA melting or DNA denaturation); it can be DNA melting or DNA denaturation); it can be shown that DNAs with higher G+C content require shown that DNAs with higher G+C content require higher temperatures to melt themhigher temperatures to melt them


6. DNA forms giant molecules

Although hydrogen bonds are individually weak, the two Although hydrogen bonds are individually weak, the two strands of the DNA molecule are held together in a strands of the DNA molecule are held together in a relatively stable manner because there are enormous relatively stable manner because there are enormous numbers of these bonds. It is important that the strands be numbers of these bonds. It is important that the strands be associated through such weak interactions, since they have associated through such weak interactions, since they have to be separated during DNA replication and during to be separated during DNA replication and during transcription into RNAtranscription into RNA

The sugar-phosphate backbone, being connected by The sugar-phosphate backbone, being connected by covalent bonds, is also stable; bacterial DNA form a single covalent bonds, is also stable; bacterial DNA form a single giant molecule; in eukaryotes, each chromosome is giant molecule; in eukaryotes, each chromosome is composed by a single giant molecule of DNAcomposed by a single giant molecule of DNA


7. How much DNA per genome? Almost all cells of all organisms contain at least Almost all cells of all organisms contain at least one copy of the entire one copy of the entire

genomegenome of the species (most cell are diploid, i.e. they contains two of the species (most cell are diploid, i.e. they contains two copies).copies).

Genome sizes are measured in units of thousands of nucleotide pairs Genome sizes are measured in units of thousands of nucleotide pairs (called kilobases, kb) or millions of nucleotide pairs (megabases, mb)(called kilobases, kb) or millions of nucleotide pairs (megabases, mb), or , or sometimes in picograms (10sometimes in picograms (10-12-12 gr) gr)

In general, tIn general, the total amount of chromosomal DNA in different animals he total amount of chromosomal DNA in different animals and plants does not vary in a consistent manner with the apparent and plants does not vary in a consistent manner with the apparent complexity of the organisms.complexity of the organisms.

Yeasts, fruit flies, chickens, and humans have successively larger Yeasts, fruit flies, chickens, and humans have successively larger amounts of DNA in their haploid chromosome sets, in keeping with amounts of DNA in their haploid chromosome sets, in keeping with what we perceive to be the increasing complexity of these organisms. what we perceive to be the increasing complexity of these organisms. Yet the vertebrates with the greatest amount of DNA per cell are Yet the vertebrates with the greatest amount of DNA per cell are amphibians, which are surely less complex than humans in their amphibians, which are surely less complex than humans in their structure and behavior.structure and behavior.


8. Genome sizes

Amount of DNA in the genomes of various organisms


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. The nucleoidThe nucleoid is one long, The nucleoid is one long, single molecule of double single molecule of double stranded, helical, supercoiled stranded, helical, supercoiled DNA. In most bacteria, the DNA. In most bacteria, the two ends of the double-two ends of the double-stranded DNA covalently stranded DNA covalently bond together to form both a bond together to form both a physical and genetic circle. 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. Eukaryotic Nuclear Genomes A human cell contains about 2 meters of DNAA human cell contains about 2 meters of DNA,, packed into 46 packed into 46

chromosomes, all inside a nucleus only 6 chromosomes, all inside a nucleus only 6 m in diameter.m in diameter. Thus, in order to pack the DNA into the nucleus, there must be Thus, in order to pack the DNA into the nucleus, there must be

several levels of coiling and supercoiling. several levels of coiling and supercoiling.

These levels of DNA structure cannot be These levels of DNA structure cannot be resolved by the optical microscope, under resolved by the optical microscope, under which interphase nuclei stained with which interphase nuclei stained with DNA-specific dyes appears composed of DNA-specific dyes appears composed of a a dense, dark-staining material called dense, dark-staining material called heterochromatinheterochromatin, and is, and is scattered scattered throughout the nucleus, andthroughout the nucleus, and a a very light-very light-staining flocculent material which fills the staining flocculent material which fills the rest of the nucleus, called rest of the nucleus, called euchromatineuchromatin..

Nucleus

Nucleolus

Cell wall


12. DNA in interphase It is thought that most part of each chromosome in an It is thought that most part of each chromosome in an

interphase nucleus (chromatine) has the form of the “30 nm interphase nucleus (chromatine) has the form of the “30 nm fiber”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. The 30 nm fibreDDifferent levels of chromosome ifferent levels of chromosome uncoiling. uncoiling. TThe bottom of the he bottom of the figure figure showsshows the DNA helix the DNA helix (which is DNA stripped of its (which is DNA stripped of its histones). In the normal, histones). In the normal, unstripped chromosome, the unstripped chromosome, the double stranded DNA is wrapped double stranded DNA is wrapped around sets of 8 around sets of 8 macromolecules macromolecules of of histoneshistones (proteins) (proteins) to form a to form a 10 nm filament. These sets of 10 nm filament. These sets of histones are separated by spacer histones are separated by spacer regions of 4 nm DNA filament. regions of 4 nm DNA filament. They are the 10 nm nucleoprotein They are the 10 nm nucleoprotein fibrils or "beads on a string" seen fibrils or "beads on a string" seen in electron micrographsin electron micrographs which which are called are called nucleosomesnucleosomes. The next . The next level of coiling produces the 30 level of coiling produces the 30 nm nucleoprotein fibers .nm nucleoprotein fibers .


14. Compacting factors Stuffing the long strands of chromosomal DNA into a Stuffing the long strands of chromosomal DNA into a

eukaryotic nucleus requires that the DNA be compacted in eukaryotic nucleus requires that the DNA be compacted in length approximately 10,000 to 50,000 -fold. Incredibly, length approximately 10,000 to 50,000 -fold. Incredibly, cells achieve this tight packing of the DNA while still cells achieve this tight packing of the DNA while still maintaining the chromosomes in a form that allows maintaining the chromosomes in a form that allows regulatory proteins to gain access to the DNA to turn on (or regulatory proteins to gain access to the DNA to turn on (or off) specific genes or to duplicate the chromosomal DNA off) specific genes or to duplicate the chromosomal DNA (replication). (replication).


15. The average lenght of coding regions

Estimates of the average length of polypeptide chains Estimates of the average length of polypeptide chains coded by genes of various organisms; these value have to coded by genes of various organisms; these value have to be multiplied by 3 in order to obtaing the lenght of the be multiplied by 3 in order to obtaing the lenght of the corresponding coding DNA. Tipical values are 1,000 to corresponding coding DNA. Tipical values are 1,000 to 1,500 bp.1,500 bp.

Organism Average length of gene product (aa)

Vibrio cholerae (bacterium) 304 Saccharomyces cerevisiae (yeast) 477 Drosophila melanogaster (fruit fly) 492 Cenorhabditis elegans (nematode) 436 Arabidopsis thaliana (weed) 435 Homo sapiens 497


16. A “gene” is not only coding sequence Definition: A gene is a discrete unit of DNA (or RNA in some Definition: A gene is a discrete unit of DNA (or RNA in some

viruses) that encodes a nucleic acid or protein product that contributes viruses) that encodes a nucleic acid or protein product that contributes to or influences the phenotype of the cellto or influences the phenotype of the cell or the organism. or the organism.

Genes are Genes are the functional units of chromosomal DNAthe functional units of chromosomal DNA. Each gene not . Each gene not only encodes the structure of some cellular product, but also bears only encodes the structure of some cellular product, but also bears control elements (short sequences) that determine when, where, and control elements (short sequences) that determine when, where, and how much of that product is synthesized. Most genes encode protein how much of that product is synthesized. Most genes encode protein products; special classes of genes encode for RNA molecules.products; special classes of genes encode for RNA molecules. The way genes encode proteins is indirect and involves several steps. The first The way genes encode proteins is indirect and involves several steps. The first

step is to copy (step is to copy (transcribetranscribe) the information encoded in the DNA of the gene as ) the information encoded in the DNA of the gene as a related but single-stranded molecule called a related but single-stranded molecule called messenger RNAmessenger RNA. Subsequently . Subsequently the information in the messenger RNA is the information in the messenger RNA is translatedtranslated (decoded) into a string of (decoded) into a string of amino acids called a polypeptide. The polypeptides, on their own or by amino acids called a polypeptide. The polypeptides, on their own or by aggregating with other polypeptides and cell constituents, form the functional aggregating with other polypeptides and cell constituents, form the functional proteins of the cell.proteins of the cell.


17. Introns and exons Trying to pinpoint precisely what genes are is complicated by the fact Trying to pinpoint precisely what genes are is complicated by the fact

that many eukaryotic genes contain mysterious segments of DNA, that many eukaryotic genes contain mysterious segments of DNA, called called intronsintrons, interspersed in the transcribed region of the gene. , interspersed in the transcribed region of the gene. Introns do not contain information for functional gene product such as Introns do not contain information for functional gene product such as protein. protein. They are transcribedThey are transcribed together with the coding regions together with the coding regions (called (called exonsexons) but are then ) but are then excisedexcised from the initial transcript. from the initial transcript.

Since correct sequence in the introns (as well as in the regulatory Since correct sequence in the introns (as well as in the regulatory region) is necessary in order to generate a properly sized transcript at region) is necessary in order to generate a properly sized transcript at the right time and place, introns (along with coding and regulatory the right time and place, introns (along with coding and regulatory regions) regions) shouldshould be considered part of the overall functional unit be considered part of the overall functional unit, , in in other words, part of the gene other words, part of the gene


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


19. Number of introns-exons per gene

MMany eukaryotic any eukaryotic genes contain genes contain mysterious segments mysterious segments of DNA, called of DNA, called introns, interspersed introns, interspersed in the region of the in the region of the gene. gene. IntronsIntrons do not do not contain information contain information for functional gene for functional gene product such as product such as protein.protein.

Distribution of the number of exons among genes of three organisms


20. Average gene length

Intron/exon statistics for various organisms


21. Genomes and genesGenome Group Size (kb) Number of genes

Eukaryotic nucleus

Saccharomyces cerevisiae Yeast 13,500 6,000

Caenorhabditis elegans Nematode 100,000 13,500

Arabidopsis thaliana Plant 120,000 25,000

Homo sapiens Human 3,000,000 100,000

Prokaryote

Escherichia coli Bacterium 4,700 4,000

Hemophilus influenzae Bacterium 1,830 1,703

Methanococcus jannaschii Bacterium 1,660 1,738

Viruses

T4 Bacterial virus 172 300

HCMV (herpes group) Human virus 229 200

Eukaryotic organelles

S. cerevisiae mitochondria Yeast 78 34

H. sapiens mitochondria Human 17 37

Marchantia polymorpha

chloroplast Liverwort 121 136

The number of genes increases with genome size, but the trend is complicated due to repetitive DNA and introns.


22. Most eukaryotic DNA does not include genes BBetween genes there is DNA, mostly of unknown function. The size etween genes there is DNA, mostly of unknown function. The size

and nature of this DNA vary with the genome.and nature of this DNA vary with the genome. IIn n bacteria and bacteria and fungi there is little, but in mammals the fungi there is little, but in mammals the intergenic intergenic

regionsregions can be huge. can be huge. SSequences of DNA that exist quite distant from a given gene can equences of DNA that exist quite distant from a given gene can

affect theaffect the regulation regulation of that gene. They could thus be considered of that gene. They could thus be considered part of the part of the functional gene unitfunctional gene unit, even though separated by long , even though separated by long segments of DNA having nothing to do with the gene in question.segments of DNA having nothing to do with the gene in question.

In many eukaryotes some of the DNA between genes is In many eukaryotes some of the DNA between genes is repetitiverepetitive, , consisting of several different types of units repeated throughout the consisting of several different types of units repeated throughout the genome. Some of the repetitive DNA is dispersed; some is found in genome. Some of the repetitive DNA is dispersed; some is found in contiguous "tandem" arrays. Repetitive DNA is also found in some contiguous "tandem" arrays. Repetitive DNA is also found in some introns. The extent of this DNA is different in different species, and introns. The extent of this DNA is different in different species, and indeed there is variation of repeat number within species.indeed there is variation of repeat number within species.


23. Comparing gene densities

Schematic diagram of gene topography in four organisms.

Light green = introns; dark green = exons; white = intergenic regions


24. A small fraction of total eukaryotic DNA is coding

In mammals, only a few percent of the DNA is actualy coding:


25. Coding sequences are needles in the haystack It It is apparentis apparent that the coding sequences are only a small part that the coding sequences are only a small part

of the genomeof the genome in most eukaryotes, particularly in human in most eukaryotes, particularly in human. . Finding these regions is like finding aFinding these regions is like finding a needle in the needle in the haystackhaystack..

In addition,In addition, the genes are not uniformly distributed. There the genes are not uniformly distributed. There are regions in the genome where the genes are packed are regions in the genome where the genes are packed together, and regions where they are sparsetogether, and regions where they are sparse,, where finding where finding genes is like finding water in genes is like finding water in aa desert. desert.

genetica per scienze naturali a.a. 03-04 prof s. presciuttini 1. the building blocks of dna dna has...

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