Rawy Shaaban

BIOL*1090 Final

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The Nucleus

Functions of the nucleus: ! Storage, replication, and repair of genetic material ! Expression of genetic material (transcription and splicing). ! Ribosome biosynthesis Structure of the nucleus ! Nuclear envelope ! Nuclear membrane ! Nuclear pores ! Nuclear lamina ! Contents: chromatin, nucleoplasm, matrix, nucleolus The Nuclear Envelope: ! Consists of 2 parallel phospholipid bilayers ! The outer membrane binds ribosomes and is continuous with the rough ER. ! The inner membrane bears integral proteins, which connect to the nuclear lamina. The nuclear envelope separates transcription & translation. It acts as a selective barrier that limits movement of molecules. Its supported by the nuclear lamina, which is a thin meshwork of lamentous proteins bound to the inner membrane of the nuclear envelope by integral proteins. Its the attachment site for chromatin and support structure for NE.

Nuclear pores: ! Gateways between cytoplasm and nucleoplasm ! 3000 to 4000 pores per nucleus ! Formed when inner and outer membrane fuse. ! Contain a complex protein structure - the Nuclear Pore Complex. The Nuclear Pore Complex: ! Composed of nucleoporins ! Has octagonal symmetry ! Fits into the pore ! Projects into cytoplasm and nucleoplasm Functions of NPC: ! Passive di"usion of molecules smaller than 50 kDa (fast) ! Regulated movement of larger molecules (slow) ! Regulated movement of proteins into the nucleus requires a nuclear localization signal, a short stretch of positively charged amino acids within the protein. Cellular function is acutely dependent upon nuclear import and export - Nucleocytoplasmic tra#cking of nucleotides, structural proteins, and DNA packaging proteins. Factors needed for nuclear import are; a nuclear localization signal (NLS) in the cargo protein, karyopherins, energy, and Ran-Small G proteins that act as chemical messengers and triggers (when attached to 3 phosphate groups, they are turned on

and when attached to 2 phosphate groups they are turned o"). Nuclear exports are mostly protein and RNA molecules containing NES, exportins bound to the NES, and Ran-GTP - hydrolysis of which releases the cargo.

The nucleolus comprises of clusters of ribosomal DNA (rDNA) gathered together as one to several nucleoli that produce ribosomes. Functions of the Nucleolus: ! Ribosome biogensis ! Synthesis of rRNA ! Processing of rRNA ! Assembly of subunits (rRNA + proteins) ! Small 40S (18 RNA + 33 proteins) and large 60S subunits exported to the cytoplasm
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The Cytoskeleton

The cytoskeleton is a dynamic network of protein laments that forms the cellular sca"olding as well as transport system for organelles and vesicles. Its primary functions are: ! Structural support ! Intracellular support ! contractility and motility ! Spatial organization within the cell There are three major elements: Microtubules, micro-laments, and intermediate laments. Microtubules Largest cytoskeletal element comprising of polymers of a-tubulin and B-tubulin proteins. Hetero-dimers are aligned in the same direction which results in structural polarity which is important for growth/shrinkage and direction of movement of material. MTs have a fast-growing + end and slow-growing - end. The motor proteins use ATP to generate force and movement. ! Dynein (- end directed). ! Kinesin (+ end directed). Microtubules undergo dynamic assembly and disassembly - this leads to rapid turnover of most MTs within the cell - dynamic instability. Shrinkage can occur very rapidly at the plus end, this is termed catastrophe. Formation of MTs is regulated and controlled by MAPs (microtubule-associated proteins). Microtubule-organizing centers (MTOC) are the central sites of MT assembly.

Intermediate Filaments Exclusive to multicellular animals, they provide structural support and mechanical strength. Relative to MTs, they are stable brous proteins of which there are 5 classes. a-helical domains wrap around each other forming a rope-like dimer. Monomers are aligned in parallel and dimers are polar molecules with di"erent N and C termini. Dimers associate anti-parallel; Once assembled, the laments are not polar. Microlaments Smallest cytoskeleton element comprising of polyers of the protein actin. Several wellcharacterized functions are maintenance of cell shape, cell movement, cytokinesis, and muscle contraction. G-actin monomers have a polar structure as the monomors are incorporated in the same orientation. F-actin laments are polar with a + end and - end.

F-actin assembly is a result of G-actin polymerizing reversibly due to nucleation (slow) or elongation (fast). Polymerization and structure organization of F-actin laments are regulated by actinbonding proteins. Myosin is an F-actin associated motor protein. It must move towards the + end and is divided into conventional and unconventional myosins. Unconventional myosins generate force and contribute to motility in non-muscle cells. All motor proteins are involved in vesicular transport.

The Extracellular Space extends outwards from the surface of the plasma membrane and contains a variety of secreted materials (from the cell) that inuence cellular behaviour. ! It mediates cell-cell and cell-extracellular matrix (ECM) interactions ! Provides mechanical protection ! Serves as a barrier ! Binds regulatory factors Cells of bacteria, plants, and fungi are surrounded by a cell wall, which is considered an ECM. Plant cell walls: ! Composed of cellulose, hemicellulose, pectin, and proteins ! Provides structural support to the cell and to the organism as a whole (skeleton) ! Protects the cell from mechanical damage and pathogens ! Contain biochemical information for the cell --------------------------------------------------------------------------------------------------------

~ End of Pre-midterm material ~

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DNA

DNA is a polymer. Each subunit is a nucleotide comprised of: ! a phosphate group ! a ve carbon sugar ! one of four cyclic nitrogenous bases

The four nucleotides of DNA are:

purines

pyrimidines

! The purine and pyrimidine nucleotides in polynucleotide chains are connected by phosphodiester bonds. ! DNA is double-stranded and the strands are antiparallel. ! The strands are held together by hydrogen bonds between bases on opposing strands and by hydrophobic interactions between adjacent stacked bases. ! Each DNA strand has chemical polarity. The 5 end has a free phosphate group and the 3 end has a free hydroxyl group. ! Opposing strands are said to be complementary. ! Base pairing is specic and is mediated by hydrogen bonds

! The most common form of DNA is called B-DNA ! There are two grooves of di"erent width: the major groove and minor groove

DNA in living cells is supercoiled.

This makes DNA very highly compact

! Similar amounts of supercoiling exist in the DNA of bacterial and eukaryotic chromosomes ! The DNA found in mitochondria and chloroplasts exists in circular chromosomes that resemble those of prokaryotes ! Both prokaryotes and plastids have circular genomes

First level of condensation - packaging DNA as a negative super coil into nucleosomes. This produces an 11 nm bre.

The linker region is susceptible to digestion by an endonuclease. DNA is wrapped around a nucleosome core of 8 histone proteins, and anchored by a 9th.

Second level of condensation - an additional folding or supercoiling of the 11 nm bre to produce a 30 nm bre. This is driven by nucleosomal interactions (histone H1 involved). To describe these structures, we have two models; solenoid and zig-zag. The conformation of the 30 nm bre depends on the methods used to visualize it.

The 30 nm bre is the basic structural unit of the metaphase chromosome (DNA in its most condensed form

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Third level of condensation - attachment of the 30 nm bre at many positions to a non histone protein sca"old.

This completes the condensation and packaging of DNA into full chromosomes.

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Chromosomes

Chromosome ends are protected by telomeres. Telomeres resist degradation by DNases, prevent fusion of chromosomal ends, and facilitate replication of the ends of the linear DNA

Centromeres provide the point of attachment of chromosomes to microtubules in the mitotic spindle. Yeasts centromeres are 110-120 base pairs long. There are three essential regions;

! Region I and III are conserved sequences that bind proteins involved in spindle attachment. Region II is ~90 bp and over 90% of those are A or T bases. ! Centromeres in multicellular eukaryotes are much larger and more complex. ! i.e. 5000 to 15000 copies of the 171 bp alpha satellite sequence. Centromeres are also binding sites for a protein called CENP, which is related to histone H3.

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Functions of DNA: ! The genotypic function - replication ! The phenotypic function - gene expression ! The evolutionary function - mutation The transformation principle (Gri#th, 1928) Mice injected with R bacteria --> remain alive Mice injected with S bacteria --> died Mice injected with dead S bacteria --> remain alive Mice injected with R + dead S --> somehow died The genetic material is DNA - part 1 (Avery, Macleod, McCarty, 1944) R cells + S cell DNA + Protease --> S cell colonies (transformation) R cells + S cell DNA + RNase --> S cell colonies (transformation) R cells + S cell DNA + DNase --> No transformation The genetic material is DNA - part 2 (Hershey & Chase, 1952) Part 1: Labeled proteins of page --> infect bacteria --> phage produced inside were unlabeled Part 2: Labeled DNA of phage --> infect bacteria --> phage produced inside were labeled.

Some viruses use RNA as their genetic material (Fraenkel-Conrat, 1957)

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Mitosis

Cellular organelles and cytoplasmic contents get divided more or less equally between daughter cells. The endoplasmic reticulum and golgi complex are fragmented at the time of division and are reformed in the daughter cells. Mitochondria and chloroplasts are randomly divided between daughter cells. However, Nuclear chromosomes must be duplicated exactly and distributed equally to daughter cells. Cell division goes through a set of stages called the cell cycle. G1 phase (Gap 1) - growth, metabolism S phase (Synthesis) - DNA replication (chromosome duplication) G2 phase (Gap 2) - prepare for mitosis M phase (mitosis) - divide + cytokinesis Cells that are not actively cycling may exit the cell cycle from G1 and enter a state called G0. These cells are said to be quiescent.

There is no invariant clock that regulated the cell cycle timing in eukaryotic cells. The centrosome cycle, in which centrioles are duplicated, progresses along with the cell cycle. In animal cells, the centrosomes also function as microtubule organizing centers (MTOCs Finally, when mitosis begins, each chromosome has already been duplicated.

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Duplicated chromosomes at metaphase condense under the inuence of condensin. Model for the role of condensin and cohesion in the formation of mitotic chromosomes;

Remember these numbers good multiple choice question

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1. Interphase ! Duplicate chromosomes called sister chromatids joined at the centromere by cohesion. ! The centrosome is duplicated. 2. Prophase ! Initiation of spindle formation
! Condensation of duplicated chromosomes ! Fragmentation of ER and Golgi ! Nucleolus disappears ! Nuclear membrane breaks down ! Spindle MTs invade nuclear space 2.5 Pro-metaphase Chromosomal microtubules attach to kinetochores, which are on the outer surface of centromeres. Chromosomes move towards the equator of the spindle.

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3. Metaphase Duplicated chromosomes are aligned midway between spindle poles. This equatorial plane is called the metaphase plate.

4. Anaphase ! Centromeres split and chromatids separate. ! Chromosomes move towards opposite spindle poles, which are moving apart. 5. Telophase ! Chromosomes cluster at opposite spindle poles. ! Chromosomes become dispersed and decondense. ! Nuclear envelope assembles around chromosomes ! Golgi and ER reform. ! Daughter cells form by cytokinesis

! In plants, a membranous cell plate forms the sca"old for a new, cellulose-containing cell wall.

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Meiosis

Homologues Heterologues

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Leptonema

Zygonema

Pachynema

Diplonema

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Oogenesis in mammals I

Oogenesis in mammals II

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In owering plants, the sporophyte is the conspicuous part of the life cycle. The gametophyte is much reduced and consists of just a few haploid cells in the anther and ovary of the ower.

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Mendelian Genetics

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Mendels Principle of Dominance: In a heterozygote, one allele may conceal the presence of another. Mendels Principle of Segregation: Neither allele is typically changed by coexisting with the other in a heterozygote Two di"erent alleles segregate from each other during the formation of gametes A dihybrid cross - are two traits inherited independently?

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Mendels Principle of Independent Assortment The alleles of di"erent genes assort/segregate independently of each other.

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Mendelian Predictions

Predicting the outcomes of a monohybrid cross with a Punnett square

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The chromosomal basis for Mendels principle of segregation

The chromosomal basis for Mendels principle of independent assortment

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Predicting the outcomes of a dihybrid cross with a Punnett square

So, for 23 di"erent genes, all on a di"erent human chromosome, 2^23 = 8,388,608 di"erent haploid gamete genotypes.

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The further apart two genes are on a chromosome, the more likely that they will assort independently

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Since a heterozygote may have the same phenotype as the homozygous dominant, a test cross may be performed to determine the individuals genotype. In a test cross, the individual of unknown genotype must be crossed with a homozygous recessive individual. i.e. a test cross involving an individual of the genotype DD Gg Ww would involve crossing this individual to one with the genotype dd gg ww

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Pedigrees

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Transcription & Translation

An overview of Gene expression:

! A gene is a transcribed region of DNA. Genes encode one of ve known types of RNA;

! RNA uses the pyrimidine uracil instead of thymine ! The RNA pentose sugar is ribose, not a deoxyribose ! The transfer of information from DNA to protein is a two step process in all organisms

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! General features of RNA synthesis from a DNA template:

! During transcription, the DNA double helix is locally unwound:

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A typical promoter in E.coli:

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A transcription terminator sequence in E.coli:

In prokaryotes, genes are closely spaced and several can be encoded on a single RNA molecule

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Eukaryotes have additional levels of complexity; the primary transcript is processed and exported to the cytoplasm for translation:

The promoter of a eukaryotic protein coding gene

! Unlike prokaryotic RNA polymerases, eukaryotic pols cannot initiate transcription on their own. A Transcription Factor is required to bind to the promoter to help assemble the transcription machinery.
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! Introns (intervening sequences) are non-coding sequences located between coding sequences. Introns are removed from the pre-mRNA and are not present in mature mRNA. Introns are variable in size and may be very large. ! Exons (both coding and non-coding sequences) are composed of the sequences that remain in the mature mRNA after splicing. ! mRNA is an intermediate between DNA and protein. ! In prokaryotes, an RNA sequence positions the ribosome to begin translation at the beginning of a coding sequence or open reading frame.

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About the genetic code;

There are no spaces between codons; codons are adjacent The genetic code is non-overlapping; each nucleotide is part of a codon The genetic code is degenerate; most amino acids are specied by related codons The genetic code is ordered; amino acids with similar properties are specied by related codons The genetic code is ~universal; with minor exceptions, each triplet/codon has the same meaning in all organisms

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Mutations

Mutations can arise spontaneously, as a result of an error during DNA synthesis . This can be due to the incorporation of rare isoforms of the four bases that have altered base pairing properties, or due to the inherent fallibility of replication proteins. The nitrogenous bases of DNA exist in two isoforms (tautomers). These rare isoforms have altered base pairing properties. Incorporation of a rare isoform during DNA replication can lead to a change in DNA sequence. Mutations of DNA in the germ line (i.e. during the mitotic divisions of spermatogonia or oogonia) will be inherited. Hot spots for spontaneous mutations during DNA replication include; simple repeats, symmetrical repeats, and palindromes.

! Mutations can also be induced by exposure to chemical mutagens. Chemical mutagens can be divided into two groups; ! Those that are mutagenic only to replication DNA (base analogues, acridine dyes), and those that are mutagenic to both replication and non-replicating DNA (akylating agents). ! Mutations can also be induced by exposure to radiation. Adsorption of UV energy by pyrimidines results in their dimerization. ! Gene mutations can a"ect the encoded proteins. The e"ects of a single base mutations vary enormously.

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A single base change in the beta globin gene causes sickle cell anemia:

Acridines intercalate between adjacent base pairs and distort the double helix.

When these molecules replicate, additions and deletions of one to a few base pairs occur.

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Expanding genes - At least 15 human inherited disorders result from expanding triplet/ trinucleotide repeats. Usually <40 copies of the triplet repeat are stably inherited. Larger numbers of copies are unstable and prone to expansion.

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Allele Variation & Gene Function

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Incomplete Dominance: heterozygotes (with one copy of the dominant allele) have half the functional gene dosage.

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From Genotype to Phenotype

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Gene Regulation

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~Fin

References: 1. Dr. A. Bendall BIOL*1090 Lecture Notes 2. P. Snustad and M. Simmons Principles of Genetics Textbook

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