מבנה הקשר הפוספודיאסטרי 1) קשר '3 – '5 פוספו די-אסטרי 2) כיווניות 3) קצה ‘5 קצה ‘3 4) במעלה או במורד הזרם (upstream or downstream)

זיווגי בסיסים - basepairing בסיס משלים

מבנה הסליל הכפול dsDNA *הסליל מפותל סביב ציר מרכזי *הבסיסים פונים למרכז הציר

dsDNA קשרים תוך מולקולרים לחיזוק המבנה בסלילי

Helix axis DNA road

מגרעת

Groove חריץ/מסילה

A:T and G:C כיווניות קשר פוספו A = 10-10

A = 10-10 Mili (m) = 10-3 Micro (m) = 10-6 Nano (n) = 10-9 Pico (p) = 10-12 Fenta (f) = 10-15 A = 10-10

שאלת תירגול: מדוע בחרת ללמוד רפואה 1. אני מעוניין לעזור לאנושות 2. הנושא מעניין 3. קידום אישי 4. המשפחה לחצה יש לך 30 שניות לענות תשובה!

דנטורציה ורנטורציה And Denaturation

דנטורציה ורנטורציה של DNA

דנטורציה

ספקטרופוטומטר

DNA Denatures at High Temperatures

חד-גדילי דו-גדילי

1. לאדם 2. לחיידק 3. אין מספיק מידע 4. לשניהם Tm זהה שאלה: אורך הגנום ההומני הוא 3 טרליון זווגי בסיסים, לעומתו אורך הגנום של חיידק ה-e. coli הוא 1.5 מיליון זיווגי בסיסים. הגנום ההומני מורכב מ-47% זיווגי בסיסים של GC ולעומתו הגנום של החיידק בעל 52% של GC. למי טמפרטורת Tm יותר גבוה? 1. לאדם 2. לחיידק 3. אין מספיק מידע 4. לשניהם Tm זהה

רנטורציה

Less complex DNA renatures faster For an equal mass/vol:

Over 40% of the human genome corresponds to interspersed repeats Census of human repeats Over 40% of the human genome corresponds to interspersed repeats IHGSC. Nature (2001) 409 860-921

Types of DNA in each kinetic component Human genomic DNA

מיקרוסטליט 1.4 M

Humans (H. sapiens) and bonobos (P Humans (H. sapiens) and bonobos (P. paniscus) share a cryptic microsatellite element in the 5' regulatory region of AVPR1A, which is absent in the chimpanzee (P. troglodytes).

פטריות אצות 30-40,000 genes

Fundamental properties of genes Genes are heritable units, arranged linearly along chromosomes. Complementation analysis of a large number of mutants defines genes that determine a function. E.g., biosynthetic pathway or DNA replication. Genetic techniques in microorganisms were used to determine the fine structure of a gene. Genes encode polypeptides Codons are triplets of nucleotides that encode an amino acid.

מבנה הכרומוזום בשלב חלוקת התא Gene 3 גן – אזור ב-ד.נ.א מימנו מתורגמת מולקולת ר.נ.א בעלת פעילות ביולוגית

אינטרונים נמצאים רק באוקריוטים אינטרונים – רצפים שמוסרים בגרעין ולא נשארים במולקולת הרנא הבוגר אקסונים – רצפים שנשארים במולקולת הרנא הבשלה אינטרונים נמצאים רק באוקריוטים

אקסון – רצף שנשאר ברנא לאחר העיבוד ועובר לציטופלזמה אינטרון – רצף המוסר מהרנא בגרעין פרומוטר אקסון אינטרון

מהם הכרומוזומים? 1. אתרי עיגון של הדנא בגרעין 2. מבנה הגרעין 3. מיכלי אריזה של הדנא 4. מיכלי אריזה של החלבונים

Human genome 2.91 billion base pairs 24,000 protein coding genes (~32,000 non-coding genes) 1.5% exons (127 nucleotides) 24% introns (~3,000 nucleotides) 75% intergenic (no genes) Average size of a gene is 27,894 bases Contains an average of 8.8 exons Titin contains 234 exons.

List of 68 eukaryotes, 141 bacteria, and 17 archaea at Sample genomes Species Size Genes Genes/Mb H.sapiens 3,200Mb 35,000 11 D.melanogaster 137Mb 13.338 97 C.elegans 85.5Mb 18,266 214 A.thaliana 115Mb 25,800 224 S.cerevisiae 15Mb 6,144 410 E.coli 4.6Mb 4,300 934  List of 68 eukaryotes, 141 bacteria, and 17 archaea at http://www.ncbi.nlm.nih.gov/PMGifs/Genomes/links2a.html

What does the Genome tell us? Estimated number of genes ~32,000 Only 1 to 1.5% of the genome encodes proteins 75% of the genome is not transcribed 46% is repetitive DNA JUNK DNA –the fodder/history of evolution Recombination Diversity “genomic” disease

We humans are 99.9% identical at the DNA sequence level There are still ~3 million nucleotide differences among us called SNPs (60,000 within the exons)---that presumably account for differences in disease susceptibility, drug responses, etc. Polymorphic variation between and within populations Implications for concepts of “race,” “individuality”

24,000

Duchenne Muscular Dystrophy ניוון שרירים MOLECULAR BIOLOGY OF THE DISEASE Duchenne Muscular Dystrophy is one of more than twenty different types of muscular dystrophy. The Duchenne type affects only boys and is known to result from a defect in a single important protein in muscle fibers called Dystrophin. The muscle fiber will break down if the Dystrophin is missing and is unable to function properly. As a result, the reduction in the number of good muscle fibers and the whole muscle becomes weak.

כמה מחלות גנטיות יכולות להיות? 1. לא יותר מ-100 2. כ-1000 מחלות 3. כ-10000 מחלות 4. כמספר הגנים.

Summary DNA, Chromosome Centromere, telomere, replication origin Nucleosome, Chromatin, Histone: H1, H2A, H2B, H3, H4 Histone octamer, DNA packaging DNA binding proteins, Histone modifications

The Global Structure of Chromosomes Some rare cases of interphase chromosomes, certain features maybe universal Representative forms forming typical interphase chromosome Chromosome at mitosis

Lampbrush chromosomes (amphibian oocyte, immature eggs)

A model for the structure of a lampbrush chromosome Chromomeres: highly condensed and in general not expressed until unfolding

A polytene chromosome from Drosophila salivary gland Dark bands and interbands

Electron Microscope image of Drosophila polytene chromosome

Folding and refolding at a time course of 22 hours Chromosome puffs Folding and refolding at a time course of 22 hours

RNA synthesis in Chromosome puffs Red: newly synthesized BrUTP; Blue: old ones diffused

RNA synthesis in Chromosome puffs

RNA synthesis in Chromosome puffs

Model of RNA synthesis in Chromosome puffs

A model for the structure of an interphase chromosome

Position Effects on Gene Expression Heterochromatin: condensed Euchromatin: loose

Speculative Model for the heterochromatin at the ends of yeast chromosomes Sir: Silent information regulator binding to unacetylated histone tails

Speculative Model for the heterochromatin at the ends of yeast chromosomes DNA-binding proteins recognize DNA sequence close to telomere, recruit Sir proteins and cause histone tail modification, forming heterochromatin

Two speculative models for how the tight packaging of DNA in heterochromatin can be inherited during chromosome replication

The specialized nucleosome formed on centromeres Also belongs to heterochromatin

The structure of a human centromere Alpha satellite DNA sequence Kinetochore inner plate Kinetochore outer plate Spindle microtubules

The plasticity of human centromere formation

A typical mitotic chromosome at metaphase

SEM of a region near one end of a typical mitotic chromosome

EM of a mitotic chromosome

Condensin plays important roles Chromatin Packing Condensin plays important roles

The SMC (Structural Maintenance of Chromosomes) proteins in condensins

Selective localization of two interphase chromosomes Chromosome 18 (red) and 19 (turquoise)

Specific regions of interphase chromosomes in close proximity to the nuclear envelope Two different regions of chromosome 2 (yellow and magenta) close to the nuclear envelop (green)

Summary Chromosomes are decondensed during interphase and hard to visualize Lampbrush chromosomes of vertebrate oocytes and polytene chromosomes in the giant secretory cells of insects are exceptions, revealing the global organization of chromosome Gene expression needs the decondensation of chromosome loops Euchromatin and heterochromatin Telomere and centromere are general heterochromatin Chromosomes are spatially organized and deposited in nucleus Mitotic chromosomes are condensed and organized.

DNA Molecules are highly condensed in chromosomes Nucleosomes of interphase under electron microscope Nucleosome: basic level of chromosome/chromatin organization Chromatin: protein-DNA complex Histone: DNA binding protein A: diameter 30 nm; B: further unfolding, beads on a string conformation

חרוזים ע"ג חוט

Histone היסטונים

HDAC HDAC activity HAT

מבנה הנוקלאוזום סיכת ביטחון

Structure of the nucleosome How can you control attachment to histones?

Nucleosome Structures Histone octamer 2 H2A 2 H2B 2 H3 2 H4

Histones binding to DNA

The bending of DNA in a nucleosome 1. Flexibility of DNAs: A-T riched minor groove inside and G-C riched groove outside 2. DNA bound protein can also help

X-ray diffraction analyses of crystals Structure of a nucleosome core particle

Structural Organization of the Core Histones

The Assembly of the Core Histones

Notice the long tails of the octamer מה המשמעות של זנבות ההיסטונים?

Nucleosomes What is the problem in organizing DNA?

Higher order structure of nucleosomes A solenoid is a coil of insulated or enameled wire wound on a rod-shaped form made of solid iron, solid steel, or powdered iron

Irregularities in the 30-nm fiber Flexible linker, DNA binding proteins Structural modulators: H1 histone, ATP-driven Chromatin remodeling machine, covalent modification of histone tails

H2A H2B H3 H4

The function of Histone H1

The function of Histone tails

Chromatin Remodeling

Cyclic Diagram for nucleosome formation and disruption

Covalent Modification of core histone tails Acetylation of lysines (K) Mythylation of lysines Phosphorylation of serines (S) Histone acetyl transferase (HAT) Histone deacetylase (HDAC) HDAC activity אפיגנטיקה Acetylation Mythylation

שאלה: איזה מבין ההיסטונים עובר הכי הרבה מודיפיקציות? H2a H2b H3 H4

נוקלאוזום במבט על Protein Gel

Histone 3D

Zigzag model of the 30-nm chromatin fiber

מנוקלאוזום לסלנואיד solenoid

פיגום

מבנה הכרומוזום

מבנה הכרומוזום בשלב המטפזי

אופן אריזת הDNA בתא: מהנוקלאוזום לכרומוזום.

Histon assembly movie DNA to chromatin

מבנה של כרומוזום במיקרוסקופ אלקטרוני מבנה של כרומוזום במיקרוסקופ אלקטרוני

4 46 Human 32 Yeast 42 Macaque 40 Mouse 38 Cat 48 Potato 26 Frog 20 Total number of chromosomes/somatic (body) cell 4 Penicillin mold 46 Human 32 Yeast 42 Macaque 40 Mouse 38 Cat 48 Potato 26 Frog 20 Algae 16 Planaria Corn 8 Fruit fly Species There is no connection between the number of chromosomes and the genome size, gene content, or any other feature of genomes. It is and essentially independent characteristic. Dog 78

Myrmecia pilosula Smallest number of chromosomes known in a eukaryote: 1 pair (2 total per somatic cell) Myrmecia pilosula

Ophioglossum reticulatum, a fern Largest number of chromosomes known in a eukaryote: 630 pairs (1260 total per somatic cell) Ophioglossum reticulatum, a fern

שרך

Chromatine compartment

Cockayne syndrome group B (CSB) cells that fail to express CSB protein which causes profound neurological and developmental defects צביעת DAPI של הDNA- עם סמן לגן (נקודה לבנה)

Chromosome fragility

Chromosomal fragile sites are loci that are especially prone to forming gaps or breaks on metaphase chromosomes when cells are cultured under conditions that inhibit DNA replication or repair. The relationship of "rare" folate sensitive fragile sites with (CCG)n expansion and, in some cases, genetic disease is well established.

Fragile X syndrome What is Fragile X syndrome?  Fragile X syndrome is the most common inherited cause of mental impairment, affecting approximately 1 in 2,000 males and 1 in 4,000 females worldwide. Cytogenetic analysis of metaphase spreads demonstrates the presence of the fragile site in less than 60% of cells in most affected individuals.). In 1991, the fragile X gene (FMR1) was characterized and found to contain a tandem repeated trinucleotide sequence (CGG) near its 5' end. The mutation responsible for fragile X syndrome involves expansion of this repeat segment. The number of CGG repeats in the FMR1 genes of the normal population varies from six to approximately 50. There are two main categories of mutation, premutations of approximately 50 to 200 repeats and full mutations of more than approximately 200 repeats. There is no clear boundary between the upper limit of normal and the lower limit of the premutation range. For this reason, alleles with approximately 45-55 copies of the repeat are said to be in the "grey zone." Some alleles in this size range are unstable and expand from generation to generation, while others are stably inherited. A premutation is susceptible to expansion after passage through a female meiosis. The larger the size of a woman's premutation, the more the risk of expansion to a full mutation in her offspring. .  

חלוקת התא

Mitosis animation