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Fiona Rawle

Complete genetic linkage no crossing over only parental forms Incomplete genetic linkage more common mixture of parentalrecombinant Recombination frequency rno of recombinant gametestotal no of meioses varies between different pairs of genes depending on distance between the genes 101 Sickle cell disease SCD autosomal recessive SCD is caused by structural abnormality of Hb affects in carrying capacity of O Hb has hundreds of variants most are rare Symptoms include severe muscle pain when sickleshaped blood cells 2in the circulation are numerous enough to reduce blood flow in small vessels leading to Odeprivation in tissues causing pain and longterm damage Sickled RBCs are easily damaged and are removed from the circulatory systemcannot be replaced 2 quickly enough leading to chronic anemia Hb are tetramers with two globintwo globin Most common form is HbA Each carries one heme iron which reversibly binds to O141aa 146aa are structurallyfunctionally similar 3 2exons 2 introns SCD is caused by a single base pair substitution in the globin Two abnormalplus two normal abnormal Hb instability can cause Hb to collapse to linear crystallike molecules this causes deformation of RBCs Heterozygotes some Hb are abnormal most normal so no anemia have mild symptoms 102 Gel electrophoresis separates the Hb molecules of each type in an electric field based on differences on size shapecharge Migrate from negative to positive end Rates of migration are determined by the characteristics of the proteins Most common material used are agarosepolyacrylamide as they dont interact with proteins or nucleic acids Different globin genotypes have different electrophoretic mobility the characteristic rate of migration of a protein Densitometry measures how much light is blocked from passing through the gel by the protein in a band Twostep approach called peptide fingerprint analysis Hb is broken down into many fragmentsseparated by Gel electrophoresis and are next separated in chromatography Analysis of the peptide fragment of mutant and normal Hb showed one aa difference Val on position 6 instead of Glu The most common type of difference between individuals of the same species is SNPs single nucleotide polymorphisms SNPs are important genetic markers are transmitted genetically usually in unexpressed gene no phenotypic effect Restriction endonucleases multiple cloning sitespolylinker Inherited variability in the number or length of restriction fragments produces is called RFLP restriction fragment length polymorphism RE are palindromes produce either blunt no ssDNA or sticky ends short ssDNA SNPs can either destroy or create a restriction sequence can also change fragment length Ethidium bromide EtBr allows detection of DNA or RNA fragments in gels but is not specific to one gene it binds to DNA or RNA molecules exposure to UV makes it emit fluorescence General protein stains are nonspecific too Southern northern and western blotting The pairing of complementary sequences between the probe and target nucleic acid is called hybridization The SNP mutation causing SCD destroys a restriction sequence s leading to an RFLP that can be detected by southern blotting two instead of three for RE Ddel Northern blotting wont be useful since same length mRNA is produced but western blotting is useful 103 most Hb variants are rare arose C thEthindependently affects same codon as S in adjacent nucleotide 6 position Glu to Lys in west Africa and 26 position Glu to Lys in Southeast asia and pacific islands Malaria and SCD heterozygote advantage causes populations to keep both normal and mutant allele balanced by homozygote disadvantage while leads to equilibrium frequency gain of mutant alleleloss Heterozygotes of SCD advantage is based on the shorter average life span of RBCs which interrupts the development cycle of the Plasmodium larvae 111 Bacteria C are circular They are variable in size carry essential genes Bacteria and archaeal C are densely packed to form a small region called the nucleoid Bacteria C are packed in two ways protein helps organize the bacteria into loops that pack C into the nucleoidthe circular DNA undergoes supercoiling Negative supercoiling most common twists DNA in opposite direction to the helical twist and positive supercoiling twists the DNA in the same direction as the double helix Topoisomerases partially unwind supercoiled DNA to relieve stress 112 Eukaryotes The DNA and associated proteins of a C is called chromatin Each C isproteinDNAof the proteins are histones There are 5 types of histones H1 H2A H2B H3 and H4 Highly conserved Octamer 2 of H2A H2B form dimers H3 and H4 form dimers A span of DNA core DNA 146bp wraps around each octamer to form a nucleosome first level of condensation10nm beadsonstring The variable length of DNA between nucleosomes is called linker DNA Solenoid structure 30nm forms due to 10nm coiling 68 nucleosomes per turn and H1 stabilizing it Scaffold 300nm composed of filamentous nonhistone proteins Chromatin loops are anchored to scaffold by nonhistones at MARs matrix attachment regions C compaction allows for efficient separation of c at anaphase Active transcription takes place in segments of loops distant from MARs larger loops have more active transcription Replication fork passes nucleosomes breaks down H3H4 tetramers reassociate randomly with one of the sister chromatids H2AH2B dimers disassemble and are reassembled from new and old histones Chromatin remodeling nucleosomes must displace to expose promoter and other regulatory sequences Chemical modifications to histones in nucleosomes are epigenetic marks takes place at Nterminal consist of additionremoval of chemical groups and these control how tightly DNA and nucleosomes bind together 113 Centromeres divide C into arms of unequal length short arm is p armlong arm iscentromere near the middle of C submetacentricacrocentric close to one endq arm Metacentrictelocentric at the tip of the C no p arm Chromosome banding identifies each c in a karyotype G giemsa banding standard for human c banding Chromosome condensation varies from one part of a chromosome to another region that contain actively expressed genes are less condensed during interphase are called euchromatin Regions that remain condensed during interphase and contain fewer expressed genes are called heterochromatin facultative variable levels of condensation related to transcription levels and constitutive permanently condensed found in centro and telomeres Repetitive DNA sequence in centromeres facilitate binding of kinetochore proteins and spindle microtubules S cerevisiae 16 centromeres different sequences called CEN sequences CDEI 8bp RTCARTG CDEII 90 AT microtubule attachment site CDEIII 26bp mainly AT CENPA variant form of H3 takes it place in centromeric nucleosomes its Nterminal allows the binding of kinetochore proteins to the centromere In situ hybridization uses molecular probes labeled with fluorescence or radioactivity to detect their target sequence 114 The chromatin of eukaryotes is a prominent difference between eukaryote and bacterial genomes Structure of chromatin is imp in controlling gene transcription C are not uniformly distributed in nucleus C are partitioned into specific regions c territoriesrelated to gene activity during interphase C dont occupy the same territory in each nucleus but once confined to a territory doesnt leave until the M phase is initiated c are active within their territories C appear to be anchored in their territories by centromeres Interchromosomal domains are regions between territorieschannels for movement of proteins and RNA Early replicating parts of chromosomes are near the center of the nucleus and late replicating parts are near the periphery due to greater access to needed proteins and enzymesfaster dispersal of RNA transcripts Changes in level of compaction regulate access to DNA by proteins Position effect variegation PEV Gene expression can be silenced by the genes chromosomal positionsilencing is a feature of chromatin structure that can be transmittedMutations modifying PEV led to identification of proteins that play a role in establishing and maintaining chromatin structures two types of mutations Evar enhanceencourage the spread of heterochromatin beyond its normal boundaries Suvar suppressors restrict the spread of heterochromatin or interfere with its function Important Features of Epigenetic Modification1Alters chromatin structure 2Transmissible 3Reversible 4Directly associated with gene transcription 5Does not alter DNA sequence Specific patterns of histone modification correlate with the formation of chromatin structure Chromatin state is regularly remodeled by processes that activate and silence chromosome regions and genes HP1 is encoded by a Suvar gene it binds to nucleosomes that have Lys 9 of histone H3 methylatedmost common epigenetic modifications of h
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