Organization Of Genetic Material In Eukaryotes PDF

Organization of genetic material in eukaryotes DNA in eukaryotes Location: In nucleus In mitochondria  Nuclear DNA in eukaryotes Long, linear molecules; Chromatin chromosomes; 10% of DNA – in genes, 90% - non-coding; Gene activity depends on: Ontogenetic p...

Organization of genetic material in eukaryotes DNA in eukaryotes Location: In nucleus In mitochondria  Nuclear DNA in eukaryotes Long, linear molecules; Chromatin chromosomes; 10% of DNA – in genes, 90% - non-coding; Gene activity depends on: Ontogenetic period; Type of cell; Environment  Nucleus Nuclear envelope: Inner membrane Outer membrane Nuclear lamina Nuclear pore complex Chromatin Euchromatin Heterochromatin Nucleoli Karyoplasm Nuclear skeleton Nuclear juice  Nucleus Functions: Contains ~ 98% of DNA; Assures functional packing of DNA: euchromatin – active DNA; heterochromatin – inactive DNA. Differential control of gene expression: Transcription and processing of RNA; Control of cell division: Replication of DNA and mitosis Biogenesis of ribososmes Nuclear pore  Nuclear pore 14:23:28 http://biologiemoleculara.usmf.md 11 http://e.usmf.md  Transport through nuclear pore mediated by karyopherins= importins and exportins Import (cytosol to nucleus): Histones Non-histones dNTP, NTP Ribosomal proteins Export (nucleus to cytosols): mRNA tRNA scRNA RNP 40S; RNP 60S  prophase CHROMATIN telophase CHROMOSOME ◼ Chromatin – genetic material (DNA) with different levels of packing during interphase; ◼ Chromosome – genetic material at the highest level of packing during cell division. ◼ Chemical organization: – 30% DNA + 40% histones + 25% non- histones + 5% RNA  Histones ◼ Globular proteins ◼ Basic proteins, rich in Lys and Arg; ◼ 5 types: H1, H2A, H2B, H3, H4 ◼ Function: – DNA packing at first levels – Non-specific control of gene expression Type of Basic amino No. of amino Shape of proteine histone acids acids H1 Liz 215 NH2 COOH H2A Leu, Liz 129 NH2 COOH H2B Ser, Pro, Liz 125 NH2 COOH H3 Arg, Cis 135 NH2 COOH H4 Arg, Liz 102 NH2 COOH Histones DNA + histones = nucleosome a. Histone core: 2H2A 2H2B Octamere 2H3 2H4 b. 200 b.p. Nucleosomal DNA c. H1 – stabilizes DNP complex 20  Non-histones Heterogeneous proteins; Nuclear enzymes for: replication; repair; transcription; processing of RNA; biogenesis of ribosomes Site specific proteins; Proteins of chromosomal scaffold Helix-Turn-Helix Zinc finger  Chromosomal RNAs Primary transcripts microRNA – parts of nuclear enzymes. Nucleus in different stages of cell cycle  Levels of packing of nuclear DNA Ist level – IInd level – IVth level – nucleosome = solenoid = 30 IIIrdlevel – metaphase 10 nm nm chromatin loops chromosome chromatin fiber; fiber; Characteristics of levels of DNA condensation: - Degree of condensation; - Thickness of chromatin fibers; - Mechanism of condensation; - Level of transcriptional activity; - Period of the cell cycle.  Ist level - nucleosome: 11 nm polynucleosome filament; x 6 condensation; Mechanism: DNA- interacts with basic proteins+ Transcription is possible.  Histone’s modification Methylation H3 (Lys4) – activation of expression Methylation H3 (Lys9) – attenuation of expression Acetylation Chromatin relaxation – active transcription Chromatin condensation – inactivation of Deacetylation transcription Phosphorylation H1 Chromatin supercoiling Dephosphorylation Chromatin decondensation H1 29  2nd level – solenoid 30 nm chromatin fiber; x 40 condensation; Mechanism: Phosphorilation of H1  folding of polynucleosome fiber – 6 nucleosomes per tour; Transcription - blocked. Solenoid  3rd level – loops Large or small loops , 300nm; x 600-1000 condensation; Mechanism: 30 nm fiber attaches to scaffold; interaction between SAR (MAR) and scaffold Transcription - blocked !!! SAR (MAR) - (Scaffold /Matrix Associated Region) – specific sites in DNA responsible for binding to scaffold; !!! scaffold interacts with: - chromatin; - lamins / proteins attached to nuclear envelope. 37  4th level – metaphase chromosome The highest level of condensation – x10000; Chromatid ~700nm; mechanism: Pfosphorilation of lamins → dissociation of nuclear envelope; Total phosphorilation of H1 – condensation of chromatin, loops are folded around scaffold. Transcription – blocked 2 nm 11 nm 30 nm 300 nm 700 nm 1400 nm  Satellite Secondary constriction Centromere Primary constriction Sister chromatids ◼ centromere = primary constriction ◼ telomer Centromere  Centromere A/T-rich DNA repetitive sequences Constitutive heterochromatin Histone H3 is substituted with CENP-A Ensures sister chromatid joining until anaphase of cell division (P. cohesin) Ensures kinetochore attachment Interacts with the spindle of division 43  Telomere Tandemly repeated 3-20 kb long G/C-rich nucleotide sequences (in H. sapiens - TTAGGG) Forms loops at chromosome ends (P TFR1, TFR2) Prevents chromosome shortening during replication Prevents chromosome fusion (individualizes chromosomes) Ensures resistance to exonuclease attack 44 Telomere Telomere DNA condensation related to the stages of the Cell cycle ◼ Interphase: Level I – G1 – pre-synthetic; Level II – S – synthetic; Level III – G2 – post-synthetic; ◼ Mitosis: – Prophase – Metaphase Level IV – Anaphase – Telophase 47  Types of chromatin Euchromatin: Heterochromatin: Active genes; Non-active regions of DNA is transcribed; DNA, Contains coding Contains non-coding regions. regions; Contains informational regions, unused in a specific cell; Could be constitutive and facultative.  Heterochromatin Constitutive: Facultative: Contains non-coding, Contains coding, but repetitive sequences non-active of DNA; sequences; Represents May be transformed centromeres, into euchromatin; telomeres, satellites, Assures: spacers between Cell differentiation; genes. Sexual differentiation; Control of ontogenesis  The same fragment of chromosome in three cells (A, B, C). E – euchromatin, Hc – constitutive heterochromatin, Hf – facultative heterochromatin  Nucleolus – part of nucleus responsible for biogenesis of ribosomes: Transcription of ribosomal genes and synthesis of precursor of rRNA (45S); Processing of 45S rRNA and formation of 3 types of rRNA: 5,8S + 18S + 28S; Assembling of RNP: rRNA 18 S + 33 ribosomal proteins = 40S – small ribosomal subunit; rRNA 28 S + rRNA 5,8 S + rRNA 5 S + 49 ribosomal proteins = 60S – large ribosomal subunit  Structure of nucleolus: Nucleolar organizer – sequences of DNA containing ribosomal genes; rRNAs: primary transcripts 45S; rRNA 5S, rRNA 5,8S, rRNA 18S, rRNA 28S; Ribosomal proteins; Enzymes required for synthesis of rRNA; RNP 40S and 60S. Steps in expression of ribosomal genes DNA P +1 18S 5,8S 28S T Transcription Pro-rRNA Processing 18S 5,8S 28S rRNA Assembling 40S + 60S 18S + 33 proteins = 40S 28S + 5,8S + 5S + 49 proteins = 60S 14:23:28 http://biologiemoleculara.usmf.md 55 http://e.usmf.md  Prophase Prometaphase Interphase Metaphase Telophase Anaphase  Human chromosomes Represent the morphologic substrate of H & V; Super-molecular organization of genetic information (DNA + histones + non-histones + RNA) Have different shape, size, level of condensation, gene activity Single- or bi-chromatidian; Chromatin or chromosome; Active or non-active. 58 Structure of metaphase chromosomes. Chromosomal landmarks Satellite Secondary constriction Primary Centromere constriction Sister chromatids The shape of chromosome depends on the position of centromere 59  landmarks of karyotype: relative and absolute length of chrs position of centromere = primary constriction - c presence of secondary constrictions - h presence of satellites - s chromosomes could be analyzed during: metaphase (homogenous painting or banding) prometaphase (banding) interphase (hybridization with fluorescent probes) 60  p x100 CI = p + q 61 Human karyotype 62 Classification of chromosomes By length: By shape: By type: -Large -Metacentric -Autosomes -Medium -Submetacentric -Gonosomes -Small -Acrocentric By presence of Groups: other landmarks: A 1-3 E 16-18 -h on p arm B 4,5 F 19,20 -h on q arm C X, 6-12 G 21, 22, Y -satellites D 13-15 63  Karyotype Normal Polymorphism Aneuploid Polyploid Chromosomal aberrations 46,XX 46,XX,9qh+ 47,XX,+21 69,XXX 46,XX,1q- 46,XY 46,XY,16qh- 45,XY,-12 69,XXY 46,XY,16p+ 46,XX,14s++ 48,XXXY 46,X,r(X) 45,X 46,XY,del(5p) 46,XX,t(12,22)  Human karyotype and chromosomal formula 46,XX – normal karyotype 46,XY – normal karyotype 47,XXY – gonosomal trisomy (Klinefelter sdr.) 45,X – gonosomal monosomy (Turner sdr.) 47,XY,+21 – autosomal trisomy (Down sdr.) 45,XY,-21 – autosomal monosomy (lethal) 46,XX,5p- - deletion in chrs. 5 (crie du chat) 65 Human karyotype and chromosomal formula 46,XX,13ps- – polymorphism 46,XY, 15ph++ – polymorphism 46,XX,15qh+ – abnormality 46,XY,16ph++ – abnormality 46,XY,16qh++ – polymorphism 46,XX,16q- – abnormality 46,XX,16p+ – abnormality 46,XX,13p- – polymorphism 66