euk DNA packaged w proteins to form chromatin nucleosom e nucleosomes contain DNA wrapped around protein core of 8 histone mlcs Reg gene expression in Euk 30 nm chromatin fiber transcriptional machinery deal w DNA packing: Chromatin remodeling, histone mod Histone mod: Histone N- term tails can interact w: DNA, other histones, other proteins in nucleus Reg gene expression: mlclr basis determination Reg Euk transcription more complicated by packing DNA into chromatin , complex DNA w proteins. basic unit chromatin nucleosome , strand of DNA wrapped 1.7 x (147 nucleotide base pairs) around octomeric histone core 2 copies each 4 distinct histone proteins. basic struct can be packaged into more complex struc, ie 30 nm fibers in interphase nucleus & highly packed metaphase chromosome. State packing chromatin will affect accessibility transcriptional machinery to DNA. Euk evo mech to mod condensation state chromatin to reg gene expression. include ATP-dependent multi-protein machines chromatin remodeling complexes , histone mods (methylation , acetylation phosph ). Mod funct as complex codes to direct proteins to particular regions DNA. depend code, (de)condense (pack) chromatin in region. Transcriptional activators interact directly w transcriptional machinery but recruit histone-mod enz or chromatin-remodeling complexes to genes they reg to facilitate
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euk DNA packaged w proteins to form
chromatin
nucleosome
nucleosomes contain DNA wrapped around protein core of 8 histone mlcs
Reg gene expression in Euk
30 nm chromatin fiber
transcriptional machinery deal w DNA packing: Chromatin remodeling, histone mod
Histone mod: Histone N-
term tails can interact w: DNA, other
histones, other proteins in
nucleus
Reg gene expression: mlclr basis determination Reg Euk transcription more complicated by packing DNA into chromatin, complex DNA w proteins. basic unit chromatin nucleosome, strand of DNA wrapped 1.7 x (147 nucleotide base pairs) around octomeric histone core 2 copies each 4 distinct histone proteins. basic struct can be packaged into more complex struc, ie 30 nm fibers in interphase nucleus & highly packed metaphase chromosome. State packing chromatin will affect accessibility transcriptional machinery to DNA. Euk evo mech to mod condensation state chromatin to reg gene expression. include ATP-dependent multi-protein machines chromatin remodeling complexes, histone mods (methylation, acetylation phosph). Mod funct as complex codes to direct proteins to particular regions DNA. depend code, (de)condense (pack) chromatin in region. Transcriptional activators interact directly w transcriptional machinery but recruit histone-mod enz or chromatin-remodeling complexes to genes they reg to facilitate transcriptional (in)activation. Cells remember history thru inherited patterns of gene expression (mlclr basis determination) Inherited patterns gene expression: epigenetic inheritance contribute: chromatin state via heritable histone mods, DNA methylation, pos feedback loops.
Histone mod: histone tails
MM methylationMA acetylation MP phosphorylation
Histone mod enz: add/ remove each mod; all reversible
newly exposed DNA
chromatin remodeling
How histone mod ∆ chromatin struct? prevent assoc taiils w DNA & core histones, making DNA more accessible to transcription factors or remodeling enz recruit other proteins: chromatin remodeling complexes & general transcription factors (TFIID) closed vs. open chromatin
transcriptional activators can direct assembly
transcription complex
Histone mod enzlong-lasting ∆ in gene expression thru inherited histone mod
transcriptional activators can direct local alterations in
chromatin struc Mlclr basis determination: how cells remember devel decisions
Epigenetic inheritance: heritable diff phenotype cell not from ∆ nuc seq DNA. chromatin state (via heritable histone mod), DNA methylation, pos feedback loops
DNA methylation turns genes off by attracting proteins that block gene expression
long-lasting ∆ in gene expression thru inherited DNA methylation
long-lasting ∆ in gene expression thru pos feedback
loops
Protein A: transcriptional regulator activates own
transcription
Pattern formationin Drosophila
Drosophila melanogaster: Easy to culture, grows in bottles w yeast for food rapid devel 24 hrs to hatch Simple genetics 4 chromosomes 14,000 genes sophisticated genetic and molecular biology tools > century genetics combined w tools for isolating DNA corresponding to genes identified by mutation. Completely sequenced genome.
segments of Drosophila larva
syncitium multiple nuclei in common cytoplasm blastoderm insect blastula
receptor tyrosine kinaseBicoid acts as graded morphogen
PHENOTYPES MUTANTS
GAP GENES
EXPRESSIONGENE/S
PAIR RULE GENESSEGMENT POLARITY GENES
mutations that affect
anterior/posterior patterning
Most these genes encode transcription regulators
EGG POLARITY protein bicoid binds GAP gene enhancers
GAP bindPAIR RULE
PAIR RULE bindSEGMENT POLARITY
GAP GENE EXPRESSION
PAIR RULE GENE EXPRESSION
if mutated
if mutated
if mutated
normal embryo (or wild type)
embryo mutant for Hunchback
embryo mutant for Giant
embryo mutant for Kruppel
pair rule gene expression in gap mutants
Broad overlapping
bands
7 stripes
14 stripes
Anterior gradient
gene regulatory cascade specifies segments
body of larvae made up 14 segments, each can ID by particular pattern of hairs. early devel of Drosophila (& other insects) differs most vert: embryo doesn’t cleave into individ cells. period during which nuclei proliferate in common cytoplasm. multiple nuclei share common cytoplasm syncytium. certain # divisions, most of nuclei move to periphery of cell, form single layer nuclei, each barely separated from neighbors by a slight invagination of the plasma membrane. stage of development syncytial blastoderm. After a few additional nuclear divisions, each nucleus becomes completely enclosed by membranes in cellularization. stage of devel after cellularization and before gastrulation is called the cellular blastoderm stage. Animal development relies on: genome of mother, which directs the production of the oocyte, and the embryonic genome that results from fusion of haploid sperm & egg nucleus. Depending on animal, maternal genome can direct more or less of early devel. flies, maternal component significant mammals maternal component is very minor. Genetic analysis combined w newly developed molecular biology tools made it possible to learn molecular mechanisms that underlie Drosophila devel. 3 groups maternally-expressed genes alter anterior/posterior pattern: anterior, posterior, & terminal group. For each mutant class, key gene. Torso, encodes receptor tyrosine kinase activated only at poles of egg during oogenesis by signals sent from subset of follicle cells that surround egg. After activated: cytoplasmic determinant. 2 other key genes encode cytoplasmic determinants: Bicoid anterior Nanos for posterior group. mRNAs for 2 genes loaded into egg in polarized way: Bicoid at anterior, Nanos at posterior tip. After egg fertilized, mRNA translated but stays in place. protein forms gradient, highest at site where mRNA localized. gradient persists thru most of syncytial blastoderm stage. Bicoid encodes transcriptional activator functions as cytoplasmic determinant & morphogen gradient. Evidence functions graded fashion first & from observation protein forms gradient from anterior to posterior & 2 nd, more important, when shape of gradient D by ^/ dec # copies bicoid gene, D spatial pattern embryo in coord manner. 3 groups embryo-expressed genes alter anterior/posterior pattern: gap genes, pair rule genes segment polarity genes. Mutations in gap genes caused large gaps in pattern. other classes mutants surprising bc instead of alterations in contiguous regions embryo, mutations affected alternating regions. pair rule mutants, of 14 segments, 7 missing, alternated w ones remained. Some mutations skipped odd-# segments, others skipped even-# (one called even-skipped). segment polarity mutants also showed an alternating pattern, but differed from pair rule genes: half-segments missing/ duplicated in reverse polarity. Each gene unique pattern expression mirrored mutant phenotypes. Gap genes expressed in broad bands w each gene unique pattern partially overlapped w that of other gap genes; pair rule genes expressed in 7 stripes, some overlapped w other pair rule genes; segment polarity genes expressed in pattern 14 stripes. 9 gap genes respond to gradients of bicoid & nanos & consequences of torso signaling by becoming activated in broad & overlapping regions of syncytial blastoderm stage embryo. gap genes direct the expression of pair-rule genes, & pair-rule genes, in turn, direct expression of segment polarity genes. transcriptional regulatory cascade: very efficient way pattern embryo during syncytial blastoderm stage bc nuclei exposed to common cytoplasm allowing transcription factors to move by diffusion & enter nuclei near nucleus that transcribed them