eukaryotic organization

Eukaryotic genomes: organization “The Inner life of the Cell” : 

Eukaryotic genomes: organization “The Inner life of the Cell” V. Baburao HOD Dept of Zoology P. B. Siddhartha college Vijayawada

DNA packing : 

DNA packing DNA + histones form nucleosomes (10nm fiber). Nucleosomes coil to form chromatin fiber (30nm fiber). Fiber folds into looped domains (300nm fiber), looped domains into condensed scaffolds (700 nm) Chromatin condenses further to form the metaphase chromosome (highly compacted 1400 nm)

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Nucleosome structure

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Terminology of DNA Coding DNA – Codes for proteins or RNA Inter-genic DNA - DNA region between the genes. Extragenic DNA – DNA other than coding regions Gene-related – functionally related to genes

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Eukaryotic gene

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Eukaryotic gene

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Genes Coding – protein coding and RNA coding Single copy genes Gene families Duplicated genes Gene clusters Tandem genes Eukaryotic DNA

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Coding genes – protein coding are transcribed and translated; RNA coding genes are only transcribed. Protein coding genes – these are mostly Single copy genes; some are also present as clustered genes in gene families Gene families – they contain single copy genes, clustered genes as duplicated genes and pseudogenes Duplicated genes – they may be identical or similar. Their gene products are similar in their function Gene clusters- these are present in gene families Tandem genes - some RNA genes and histone genes are present as tandem genes. They are arranged end to end. Eukaryotic Genes

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Gene-related Promoter Regulatory sequences Enhancers Gene fragments Pseudogenes Anti-leader Anti-trailer Introns Eukaryotic DNA

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Eukaryotic DNA Extra genic DNA Unique/low copy number - 70 to 80 % Moderate/ high repetitive - 20 to 30 % Disperser repeats – 40% SINEs, LINEs Clustered repeats – 60% Satellite DNA, Minisatellites, Microsatellites

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Eykaryotic Genes Genes are present as cistrons, mutons and recons Eukaryotic genes are split genes They transcribe monocistronic DNA Transcription occurs in cytoplasm nucleus and translation occurs in cytoplasm TIC (RNA pol II = basal transcriptional factors) forms at the TATA box. Gene expression is increased by enhancers The first amino acid in translation is non-formylated methionine. Regulation of gene expression is by hormones and cytokines

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Gene families Some Genes occur as gene families These are identical or similar genes present in multiple copies These gene families may present in single or multiple loci Gene families consists of pseudogenes and duplicated genes which are clustered genes or single copy genes

Gene family : 

Gene family

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Pseudogenes Gene fragments Pseudogenes These are diverged genes with inactivated mutatuions. These pseudogenes can not be transcribed Gene fragments Gene fragments are inactive genes that lack part of the parent genes These are formed by deletion mutations and recombination of original genes

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Gene-related Introns - non-coding regions of the cistron Promoter- an upstream region for RNA pol binding Regulatory sequences- an upstream region (cis acting elements) for the binding of transcriptional factors (trans acting elements) Enhancer – an upstream or downstream region for the binding of transcriptional factors. Anti-leader- at the 3’ of the cistron Anti-trailer- at the 5’ of the cistron

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Extragenic DNA This DNA region is neither genes not gene related Most part of eukaryotic DNA is extragenic DNA It has no known function except sat DNA 70 to 80% is extragenic DNA is unique or small copy number DNA 20 to 30% extragenic DNA is repetitive DNA

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Unique or Low copy DNA Unique DNA is single copy DNA region Low copy DNA is small number copy DNA

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Moderate or high repetitive DNA- Dispersed and clustered Clustered Repetitive DNA Three types - Satellite, minisatellite and microsatellite. These are tandem repeats. Satellite DNA -The first type identified in human genome is satellite DNA. Consists of repetitive DNA 105 to 107 kbp long. Present in centromere, telomere and satellites of chromosomes. This is the structural DNA Minisatellites – These are variable number tandem repeats (VNTRs). 102 to 105 bp long. Repeat unit is 10 to 100 bp long. GC rich. Microsatellites – These are repetitive sequences of 10 to 100 bp long containing repeating unit of 1- 4 base pairs. These variable tandem repeats are Short Tandem Repeats, STRs as repeat-unit is shorter than 10bp.

Simple sequence DNA : 

Simple sequence DNA Short, noncoding DNA sequences Tandemly repeated Prominent in centromeres and telomeres Play a structural role in the chromosome

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Functional Classification of DNA Functional DNA Coding genes, promoter, regulatory sequences, enhancer, Anti-leader, anti-trailer, satellite DNA and mobile elements Non-functional DNA Also called junk DNA or Spacer DNA Introns, unique /low copy DNA sequences, minisatellites and microsatellites

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Mobile Elements DNA mediated transposition (transposons) Inverted repeats flanking coding region with introns Excise DNA and transfer to the target site RNA mediated transposition (retro-transposons) SINEs and LINEs are retrotransposons 3’ AT rich regions. A full length copy encodes a reverse transcription. Transcription into RNA forms internal promoter. Folding of this transcript provides the primer for reverse transcriptions.

Transposable elements : 

Transposable elements Transposons Move within a genome via DNA intermediate Can move via: Cut-and-paste methods Copy and paste methods Retrotransposon Move within a genome via a RNA intermediate This is the most prevalent type

Gene expression:prokaryotic eukaryotic : 

Gene expression:prokaryotic eukaryotic Small genome, no specialization Most of their DNA codes for protein or RNA’s, very little “junk” Genome = DNA + few proteins in simple arrangement RNA processing not an option for controlling gene expression mRNA has a short life span (minutes) Both alter gene expression in response to environment; in both, transcription initiation is the most important control point Larger genome, cell specialization Most of the DNA does not code for protein or RNA’s Genome = DNA + many proteins in complex arrangement RNA processing allows for several opportunities to regulate genes mRNA is long lived (days to months)

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Gene Mapping Genetic Mapping

Gene maps : 

Gene maps Genetic maps or linkage maps Classical linkage maps Modern linkage maps Physical maps Cytogenetic maps Restriction hybridization maps Sequencing maps

Genetic maps maps : 

Genetic maps maps Classical linkage maps Pedigree analysis Recombination analysis Modern linkage maps RFLP mapping

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Gene Mapping determines order of genes & relative distances between them 1 map unit = 1 cM (centimorgan) = 1% recombination A B A B A & B are Cis arrangement (coupled) Trans arrangement (repulsed) a b a b

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Gene Distance recombination frequencies between alleles: determine relative distance between them proportional to their distance apart 1% recombination = 1 map unit = 1 cM A B A F a b a f 10% recombination 45% recombination

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% Recombination <50% recombination --> genes linked on same chromosome >50% recombination --> genes are far apart on chromosome acts like genes are unlinked (indep. assort.)

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Genetic Distance vs Physical Distance recombination “hot spots” overestimate physical length low rates in heterochromatin and centromeres underestimate actual physical length A a f F

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Genes on Separate Chromosomes + + a b a a b b +a X aa +a, +a, aa, aa 1/2 : 1/2 +b X bb +b, +b, bb, bb 1/2 : 1/2 +a+b : +abb : aa+b : aabb dihybrid pure recessive normal bent albino albino/bent expected 1/4 : 1/4 : 1/4 : 1/4 + = wt (normal for any trait) (A, B) a = albino (recessive mutant) b = bent (recessive mutant) + a + b X a a b b

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Diploid Mapping wildtype albino/bent bent albino 430 437 65 68 = 1000 total + + a b a a b b + a + b X a a b b To determine distance between 2 genes: total # of recombinants/total # offspring = freq. of recombination freq. x 100 = % recomb. = M.U. = # cM 65 + 68 / 1000 = 0.133 x 100 = 13.3 % or 13.3 cM X

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Genes can be used as genetic markers, but they are not ideal choices because they occur infrequently (ca. every 100 kb in humans). 4 major types of markers are used: RFLPs = substitutions at a restriction site. Minisatellites (VNTR) = repeated DNAs 5-10s bp long Microsatellites (STR) = short tandem repeats Single nucleotide polymorphisms (SNPs) Genetic markers

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RFLP mapping

Physical Mapping : 

A physical map is primarily based on the locations of landmarks along a DNA molecule and units of distance are expressed in base pairs. Physical Mapping

Low Resolution Physical Mapping : 

Low Resolution Physical Mapping Cytogenetic map In situ hybridization

Somatic Cell Genetics in Mammals : 

Somatic Cell Genetics in Mammals

Somatic cell hybridization : 

Somatic cell hybridization

Medium Resolution Physical Mapping : 

Medium Resolution Physical Mapping Restriction hybrid mapping

Radiation Hybrid Mapping : 

Radiation Hybrid Mapping

High Resolution Physical Mapping : 

High Resolution Physical Mapping Clone by clone sequencing Shot gun method

Microsatellites in mapping : 

Microsatellites in mapping Genome-wide mapping Genetic mapping was revolutionized by the discovery of abundant polymorphic genetic markers (microsatellites). By 1994, human genetic map had localized 5,264 microsatellites to 2,335 chromosome loci (average density of one marker every 599 kb) In the process, thousands of sequence tagged site (STS) identified. STS = couple hundred base pairs of known sequence

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Mechanically shear or partially digest genomic DNA with restriction enzymes and clone large 200-500 kb overlapping fragments to YACs or BACs. An entire genome or single chromosome can be represented in a YAC or BAC clone library (depends on starting point). Overlapping YAC/BAC clones can be assembled without sequencing by DNA fingerprinting using microsatellites. Large BAC clones are sequenced using shotgun approach. High Resolution-YAC/BAC Clone Contig Maps

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Human karyotype

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Cut DNA at each base:A,C,G,T Fragment’s migrate distance is inversely proportional to their size Sanger Sequencing TCGCGATAGCTGTGCTA Run gel and read off sequence Gene sequencing

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Chromosome walking

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Chromosome jumping

Shot gun sequencing : 

Shot gun sequencing

RH mapping : 

RH mapping

Top down mapping : 

Top down mapping