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What is the material within a cell that contains the genetic information?
Isolation of the transforming principle by Oswald Avery, Colin MacLeod y Maclyn McCarty (1944)
Phage T2
1953 Watson and Crick determined the structure of DNA DNA and its Building blocks: Guanine (G), Adenine (A), Cytosine (C), Thymine (T). Polarized strand, 5->3
Genes are carried by Chromosomes. Two plant cells visualized by light microscope. DNA was stained with DAPI.
(Interphase)
Cells in G1, S or G2 are said to be in Interphase, where the chromosomes are in a more dispersed conformation
Chromosomes
Chromosomes are discrete units used by the cells to organize its nuclear DNA. Each chromosome is a single DNA molecule. The DNA is not bare (not naked). It is coated and organized by a wide variety of proteins. Collectively, the DNA and protein assemblage that constitutes the chromosomes are referred as chromatin.
Plant Species
Arabidopsis thaliana (diploid) Rye (diploid)
Chromosomes Number
10 14
Maize (diploid)
Einkorn wheat (diploid) Durum wheat (tetraploid) Bread wheat (hexaploid)[
20
14 28 42
Potato (tetraploid)
Cultivated tobacco (diploid) Adder's Tongue Fern (diploid)
48
48 approx 1,400
Chrom. Num.
8
78 36
Species
Guinea Pig
Garden snail Tibetan fox
Chrom. Num.
64
54 36
Domestic cat
Laboratory mouse Rabbit Hares Gorillas, Chimpanzees Elephants Donkey Dog Goldfish
38
40 44 48 48 56 62 78 100-104
Domestic pig
Laboratory rat Syrian hamster Human Domestic sheep Cow Horse Kingfisher Silkworm
38
42 44 46 54 60 64 132 56
example mFISH
Chromosome Genes
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X (sex chromosome) Y (sex chromosome) Total 4,220 1,491 1,550 446 609 2,281 2,135 1,106 1,920 1,793 379 1,430 924 1,347 921 909 1,672 519 1,555 1,008 578 1,092 1,846 454 32,185
Total bases
247,199,719 242,751,149 199,446,827 191,263,063 180,837,866 170,896,993 158,821,424 146,274,826 140,442,298 135,374,737 134,452,384 132,289,534 114,127,980 106,360,585 100,338,915 88,822,254 78,654,742 76,117,153 63,806,651 62,435,965 46,944,323 49,528,953 154,913,754 57,741,652
Sequenced bases[47] 224,999,719 237,712,649 194,704,827 187,297,063 177,702,766 167,273,993 154,952,424 142,612,826 120,312,298 131,624,737 131,130,853 130,303,534 95,559,980 88,290,585 81,341,915 78,884,754 77,800,220 74,656,155 55,785,651 59,505,254 34,171,998 34,893,953 151,058,754 25,121,652
3,079,843,747 2,857,698,560
Euchromatin: transcribed and less condensed Loops of 30-nm fibers seen at interphase Heterochromatin: more condensed, genes silenced, replicated later in S phase.
A mitotic chromosome
Euchromatin: transcribed and less condensed Loops of 30-nm fibers seen at interphase Heterochromatin: more condensed, genes silenced, replicated later in S phase.
Facultative heterochromatin
Specifically inactivated specific times
H3
H4
13
14
10
11
Highly conserved
Highly conserved
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
1) Organization of chromatin 2) Transcriptional regulation 3) DNA replication 4) DNA repair 5) Recombination 6) Chromosome segregation
Assembly of nucleosome
The 30nm fibers are gathered into loops or domains These loops begin & end with AT-rich sequences anchored to a fibrillar network called the nuclear matrix Organization beyond this level is seen only during mitosis A fully compacted mitotic chromosome is packed 10 000 fold
SWI/SNF & HATs recruited by TF open DNA for RNAP & GTFs
RNAP & GTFs assembled on a promoter recruit SWI/SNF & HATs for elongation
LCR, cis-elements required for maximal transcription of -globin genes Multiple sequences with enhancer-like properties Flanked by insulators ensuring a euchromatin state
1) Replication origin.
2) Centromere.
Schematic and electron micrograph of X chromosome. Telomeres protect the ends. Centromere is at the primary constriction. It mediates chromosome cohesion, spindle attachment and chromosome segregation.
Human karyotype
X
C
D Y F
Centromere (DNA segment) is at the primary constriction. The kinetochore is a huge, complicated protein complex with several layers. The outer layer provides attachment sites for microtubules.
Mitosis
Meiosis
Centromers
Protenas no histonas proveen un andamiaje estrucutural para las hebras grandes de cromatina
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
RNA synthesis in Chromosome puffs Red: newly synthesized BrUTP; Blue: old ones diffused
Chromatin decondensation appears to require two types of protein complexes each made of several polypeptide subunits: 1. Histone acetylase complexes. These are often referred to as HATS for histone acetylases. 2. Chromatin remodeling factors. These are often refered to as Swi/Snf factors because they were first identified as yeast mutants defective in mating type switching and in the ability to metabolize sucrose (sucrose nonfermenting).
Chromatin remodeling factors use energy from ATP hydrolysis to rearrange the packing of nucleosomes in higher order chromatin structures There are several different chromatin remodeling complexes in cells. Some of these bind to activation domains and de-condense the associated chromatin. Some bind to repression domains and condense the associated chromatin.
TELOMERES
What are they? Why are they important? Telomere shortening and the end-replication problem Telomerase Telomere hypothesis of aging
Telomeres
Telomere
Telomere
Repetitive DNA sequence (TTAGGG in vertebrates) Specialized proteins Form a 'capped' end structure
Homologous recombination
(error free, but need nearby homologue)
Why are telomeres important? Prevent chromosome fusions by NHEJ (non-homologous end joining)
NHEJ
FUSION BRIDGE
Mitosis
Genomic instability
Cell death OR neoplastic transformation
Proliferative capacity
5'
3'
DNA replication is bidirectional Polymerases move 5' to 3' Requires a labile primer
Ori
Each round of DNA replication leaves 50-200 bp DNA unreplicated at the 3' end
Telomere also provide a means for "counting cell division: telomeres shorten with each cycle
20
Telomeres shorten from 10-15 kb (germ line) to 3-5 kb after 50-60 doublings (average lengths of TRFs)
Cellular senescence is triggered when cells acquire one or a few critically short telomeres.
10
(Telomerase Negative)
Cellular (Replicative) Senescence
Number of Doublings
TELOMERASE: Key to replicative immortality Enzyme (reverse transcriptase) with RNA and protein components Adds telomeric repeat DNA directly to 3' overhang (uses its own RNA as a template) Vertebrate repeat DNA on 3' end: TTAGGG Telomerase RNA template: AAUCCC
TELOMERASE:
+ TELOMERASE
20
10
+ Telomerase
(Telomerase Negative)
Number of Doublings
* = Hyperacetylated
TELOMERE STRUCTURE
5 3
Telomeric t loop
5'
3'
NUCLEAR MATRIX
Telomeric proteins: TRF1 TRF2 TIN2 RAP1 TANKS 1,2 POT1 etc
H2A.z
Important role boundary elements (SAR/MAR/LCR/) Heterochromatic structures are inherited during cell division !! Role in differentiation and reprogramming.
Mammals and plants also DNA methylation -> inherited, maintained during replication.
10.7 Repressors and activators can direct histone deactylation at specific genes
Por mucho tiempo se haba pensado que las modificaciones en las histonas poda alterar la estructura de la cromatina por la influencia de los contactos histona-DNA o histona-histona.
Se cree que este dominio de la cola de las histonas es un lenguaje que ahora se refiere como cdigo histona
Acetilacin
Fosforilacin
Metilacin
Mechanism of chromatine based silencing in other regions yeast and in other eukaryotes is different! Enzymes containing histone deacetylase (HDAC) and histone acetyltransferase (HAT) activity have been found in protein complexes with repressing and activating activity respectively