heredity and DNA
di-hybrid cross - 2 diff genotypes. 9:3:3:1 in F2 generation. F1 generation cross both are hetrozygous. mendel's first postulate - alleles present on homologous chromosomes. Second - dominant and recessive alleles. Third - generation of haploid gametes by meiosis. Fourth - independant chromosome assortment. Thomas hunt morgan - constructed genetic linkage maps only 1% coding DNA. used drosophila as short life cycle, large progeny size and mutatns are easily generated.
DNA complexed with positively charged proteins histones, 2 subunits each of 4 histones comprise a 'nucleosome'. evidence of DNA being material of hereditary - griffith's experiment - avirulent cells were transformed to virulent cells by material from the hear-killed virulent cells. IBS cells treated with protease, ribonuclease and deoxyrionuclease and only one treated with deoxyribonuclease stopped transformation so DNA was active factor.
crick and watson discovered double helix. 3' to 5' phosphodiester bond. nucleotide has phosphate and deoxyribose backbone. antiparrallel strands. metaphase - they align. anaphase - they divide and move to opposite poles. telophase - nuclear membrane reforms, spindle fibres disappear, chromosomes uncoil and become a tangle of chromatin. Semi-conserv replication.
DNA and genetic code
code is degenerate, non-overlapping and universal in codons. transcription - RNA polymerase main enzyme creates short RNA primer, T to U. Ecoli starts at origin of replication and goes both ways, has leading strand and lagging strand (short pieces in opposite direction). translation - elongation of amino acids joined by peptide bonds by a peptidyl transferase activity. Translation stopped by termination codons. 5' to 3' direction. Proflavin is mutagen which removes or adds nucleotides during replication.
recombination between 2 mutant genes that are close togehter can generate wild type gene and gene with 2 mutants. Both mutants could restore reading frame if one insertion and one deletion. evidence for RNA being messenger - artificial RNA polymers using polynucleotide phosphorylase, made poly-U poly-A and Poly-C to see if polypeptide could be made. put RNA polymer, TRNA coupled to radioactive amino acid, ribosome and enerygy dsource in tube. Measured amino acid conc in tube. UUU codes phenylalanine, AAA - lysine, CCC - proline.
stop codons - UAA, UAG and UGA.
3' is RNA like strand, 5' is template strand. RNA polymerase starts at promoter, The alpha factor increases affinity for promoter sequences. In prokaryotes RNA polymerase binds to promoter sequence at 5' end, promoter sequence start at -10 and -35 relative to start. transcribed rom 5' to 3' end, when RNA moves off promoter site another can attach. RNA sequences signal end of transcription (terminators), intrinsic terminators hairpin loops form in RNA. extrinsic terminators the rho protein required to stop transcription.
lac operon gene in E-coli has lac Z,Y and A genes and lacl repressor gene before to make repressor protein. allolactose inducer can bind to repressor to induce conformational change so will unbind to operator sequence and transcription can occur and generate polycistonic mRNA.
Eukaryotic genes - promoter sequence control - timing, location and amplitude of gene expression. RNA splicing to join exons together. transcriptional regulation involves cis and trans acting factors. Trans-acting gene products interact with cis-acting elements. Cis-acting elements include TF binding sites, promotors and enhancers. Enhancers can activate or repress and work when reversed in orientation. RNA polymerase II. post transcriptional modifications - capping - inverted G residue, linked through triphosphate and then methylated. 3' are polyadenylated (adding A's to 3' end).
tRNAs mediate translation. they carry an anticodon on one end - 3 nucleotides complementary to the mRNA codon. charge tRNA covalently cooupled too its amino acid. contains bases which are modified post-transcriptionally. CCA added to 3' end of each tRNA. Each 'charged' with own amino acid, attached to 3'.aminoacyl-tRNA syntetase catalyzses the attachment of tRNA to amino acid. tRNA molecule can be chemically modified e.g cysteine tRNA can be treated with nickel hydride to bind with alanine instead, but anticodon unchanged. tRNAs recognise more than one codon. Inosine in the anticodon can pair with U, C or A in the 3' of a codon.
ribosomes has Exit site, peptidyl site and aminoacyl site. pepitde bond links C of COO- to N or Nh2. Initiation in prokaryotes by AUG start codon and shine-delgamo box, 3' end of 30s rRNA binds to shine-delagmo sequence, and initiator tRNAfmet carries met with chemically modified N-terminus so 50s subunit can bind to complex. Initiation in Eukaryotes small subunit binds to 5' cap in mRNA, which then migrates to AUG codon, then initiator tRNAmet forms comple with 60s subunit. Elongation ribosome moves in 5' to 3' direction, addition of amino acids to C terminus, tRNA enters at A site. Termination nonsense codon recognised at 3' end, release factor proteins halt synthesis.
diff between pro and eu replication
following translatopn on the ribosome proteins can be post translationally modified e.g pro has fMet on N terminus removed. Proteolytic processing to cut protein into smaller functioning proteins is possible, various chemical modifications include phosphorylation and glycolysation.
diff in gene expression. Pro - No nucleus, all takes place in one compartment, translation and transcription usually coupled. No exons or intron. One RNA polymerase consisting of 5 subunits. primary transcripts are actual mRNA, have triphosphate start at 5' and no tail at 3'. unique initiator tRNA has formylmethionine. mRNA have multiple ribosome binding sites. small ribosomal subunits automatically bind to mRNA.
eu - nucleus separated from cytoplasm by nuclear membrane. transcription in nucleus, translation in cytoplasm, coupling not possible. exons and introns, involve splicing. several kinds of RNA polymerase each with 10 or more subunits. primary transcripts undergo processing to produce mature mRNA with methylated caps and poly-A tail at 3'end. initiator tRNA carrys methonine. mRNA has only one ribosome start site. small ribosomal subunit binds first to methylated cap end and then scans mRNA to find ribsome binding site.
number of genes in pro genomes is proportional to genome size. model organisms - C.elegans, drosophila, M.musculus (mouse) controleled genetic crosses, natural or induced variation, construction of genetic map, gene isolation. missense - changes one codon, nonsense adds stop codon, framshift. cystic fibrosis gene mapped to location of chromosome 7. trans-membrance protein that regulated Cl transport.
DNA makes up 30% chromosome mass. replication origins: site of initiation of DNA synthesis. Centromere - site of kinetochore formation. Telomeres- repeated DNA sequences that enable efficient replication of chromosome termini, in its absence chromosomes would shorten after each replication cycle. Euchromatin - gene rich regions of chromosomes. Heterochromatin - gene poor. autosomes and sex chromosomes shown in karyotype. chromosomes classified by: size, centromere location and giemsa-banding pattern.
comparative mouse and human genomes diverged c.85mya. comparative genomics helped to identify: protein coding genes, transcriptional control sequences and mechainsm of chromosomal evolution. In suntenic blocks gene order is highly conserved.
mitosis and meiosis
chromosomes replcate during 'S' phase. sister chromatids segregate during mitosis. interphase consists of G1, S and G2 phasses. Terminally differentiated cells arrest in Go. alpha satalite DNA sequences are repeated many times at centromeres and kintochore binds to it. Heterochromatin forms on alpha satalite DNA. Kinetochore has inner and outer plates.
mitosis - somatic cell division - each homolog replicates. homologous chromosomes dont associate. sister chromatids attach to spindle microtubules and are partioned to daughter cells. each daughter cell has full chromosome completment. Meiosis - gamete production with 2 cell division. homologs associate at prophase 1, recombination occurs, maternal and paternal chromosomes assort independantly, hapoloid cells are produced.
Each centrsome is made up of a pair of centrioles. in G1 2 centrioles separate into 2 mother centrioles. A new centriole forms at the base of each mother in S phase. centrosome pairs remain closely associated until M phase, centrosomes nucleate microtubule outgrowth. astral, kinetochore and overlap microtubules. 'motor proteins' at + ends have role in MT elongation. Motor proteins at - end anchor and organise MTS. MTs mainly compsied of tubulin subunits.
mitosis and meiosis 2
prophase - chromosomes condense and become visable, centrosomes move apart and generate new microtubules, nucleoli begin to disappear. prometaphase - nuclear envelope breaks down, microtubules invade nucleus, sister chromatids attach to microtubules. metaphase - chromosomes align with sister chromatids facing opposite poles. Anaphase - centromeres divide. segregation of genetic components in termed karyokinesis. telophase - nuclear membrane and nucleoli reform. spindle fibres disappear. chromosomes uncoil and become tangle of chromatin. cytokinesis - cytoplasm divides. contractile ring comprised actin and myosin.
meiosis - homologous chromosomes pair up during prophase 1 to form bivalents and reciprocal exchange of chromosomal sequences occur. prophase 1 has 5 stages. leptotene - threadlike chromosomes begin to condense and thicken. zygotene - chromsomes clearly visable and begin pairing along the synaptonemal complex to form a bivalent. pachytene - full synapsis of homologues, recombination nodules appear along synaptonemal complex. diplotene - bivalent remains connected at crossover sites called chiasmata. site of crossing over is chiasma. reciprocal exchange of chromosomal sequences. diakinesis - further condenasation of chromatids, nonsister chromatids that had exchanged parts remain closely associated at chiasmata. at anaphase 1 homologous chromosomes segregate i.e chromosome dysjunction and generate 2 dyads.
mitosis and meiosis 3
at metaphase 2 the chromatids in each dyad attach to kinetochore MTs and anaphase 2 they are segregated. independant assortment of maternal and paternal chromosomes - generates gametes with new combinations of chromosomes. recombination increases number of potential varients.
oogenesis - at 6 months post-conception foetal ovaries contain 5x10^5 primary oocytes. at puberty one primary oocyte is released each month. Asymmetrical cell division produces one large egg. meiotic division 2 commences following ovulation but is only completed after fertilisation. spermatogenesis - symmetrical meiotic division produce 4 sperm. each sperm contains 22 autosomes and either X or Y chromosome.
chromosomal nondisjunction - homologous chromsomes fail to segregate normally at division 1. get aneuploids. leads to down syndrome (47,+21), more likely during oogenesis than spermatogenesis. prenatal diagnosis acheived by amniocentesis.
P1 is pure breeding homozygous. F1 is first generation after mating. used peas as easy to grow and hybridise, analysed height, seed shape etc. established principles or transmisssion genetics. Le gene encodes protein required for gibberellin biosynthesis which controls internode elongation growth which effects length of stem. 3:1 phenotypic segregation. 1:2:1 genotypic ratio. dihybrid crosses need to have locus on different chromosomes. for genes located on diff chromosomes all possible gametic combinations are fromed due to independant assortment at meiosis.
2 point linkage analysis - thomas hunt morgan, first described X-linked inheritance. used mutagenesis and natural variation to identify drosphila genes. 4 chromosomes. genetica analysis can be used to determine the frequency of recombination between linked genes. distance between genes determines how frequently they recombine. for genes that are closely linked the frequency will be low, for further apart will be more frequent. frequency of recomb is termed linkage ratio. NCO classes will always be more abundant than CO gametes. recomb occurs in prophase 1. count how many CO and NCO offspring and put over total number of offspring x100 to get %. all female mums wild type. all dads mutant. NCO should be either completely wildtype or completely mutant, CO will be half mutant half crossover.
3 point linkage
order of genes can be reliably determined using 3 point crossing data. non-sister chromatids in bivalent may join by several chiassmata so more than one reciprocal exchange. can produce 8 distinct phenotypic classes. NCO (ABC and abc) SCO (ABc, abC and Abc) and DCO (AbC and aBc).
requirements for 3 point linkage: F1 genotype must be hetrozygous. Cross must be constructed so genotypes of all progeny can be seen from phenotype. Sufficent number of progeny. F1 females wild type, males mutant. DCO will always be least abundant so you can see which has the least and then you can tell the order from them as the odd letter out will be middle e.g Bac - aBc. SCO allow linkage ratio between the groups to be determined. to calculate genetic differences do SCO + DCO / number of offspring x100.
sex determination - in humans the SRY gene determines male differentiation. In drosophila the number of X chromosomes determines sex. P1 white eyed W/W female, red eyed w/y male. F1 males are white eyed and females are red eyed. occasionally you get white eyed females and red eyed males due to abnomal numbers of X chromosomes that arise through non-disjunction. Where nullo-X nondisjunctional egg means red eyed male and double W/W from female mixes with Y to make a white eyed female
mitotic non-disjunction also generate clones of aneuploid cells. mitotic non-disjunction following zygote formation generates clones containing 2 or 1 x chromosome. most aneuploid embryos are non-viable apart from 21,13,18 and sex chromosomes. turner syndrome (45,X) where no X or Y chromosome only 1 x means short, rudimentary ovaries, underdeveloped breasts, webbed neck. Klinefelter syndrome (47,**Y) tall, poor beard growth, female pubic hair patttern, testicular atrophy, minor breast development.
absence of any X chromosomes in both humans and flies is lethal. In mammals Y specifies male. In drosophila Y is required for fertility, Extra Y has little effect in both. X:Y ratio < 0.5 = male. ** means SXL expression = inactivation of autosomal activators of X chromosome genes. genes on X chromsome of male fly are transbribed x2. homodimers of sisterless-A and B proteins encoded on the X chromosome activate transcription of the sex lethal gene (sxl). its not transcribed in males as these proteins form heterodimers with 'denominator' proteins. in fly males the X chromo is hyperactivated, activation requires 5 genes on male specific loci. each MSL protein binds to single copy of X forming a protein complex with RNAs rox1 and 2 only produced in males. leads to chromo decondensation, histone acetylation, unique histone H4 composition. ectopic expression of sxl in XY zygotes is lethal as it supresses genes required for hyperactivation of X chromosome.
sex determination 2
X and Y chromos in humans are homologous only at psuedoautosomal regions which are essential for pairing in meiosis in males. Y chromo evolved from X. X and Y cant recombine except at the pseudoautosomal regions. rare illegitimate recombination between X and Y chromo identified region of Y essential for specifying male differentiation. confirmed it bu injecting DNA into the pronucleus of a fertilised mouse egg. Embryo is replaced, DNA integrates at random, all cells contain transgene in most cases, SRY gene wasintroduced into ** females to confirm function. SRY encodes a putative transcription factor, expressed in male genital ridge and present in ** males. transgenic ** mice expressing SRY are phenotypically male.
'Barr' bodies in nuclei of mammalian females and comprised of heterochromatin, XY male cells dont contain it but **Y klinefelter males do. XIST RNA from one X chromosome mediates silencing of its homologue. it never leaves nucleus and binds to X chromosome from which it was transcribed, modifying histones H3 and H4, binding to X chromo gene rich regions making it inactive. other examples of mosaicism of X-linked genes is tortoise shell cats where female is hetro for pigment genes. Patches are due to inactivation of diff X chromos, males are only orange or black.
human genetic disorders
X-linked recessive traits - primarily affect males, females act as carriers e.g haemophilia. X-linked dominant traits - affected males produce affected daughters but not sons, fmeale hetro trasnmit trait to half children e.g Hypophosphatemia. Y-linked traits - passes exclusively from father to son. Autosomal dominant traits - affects male and female equally only one defective allele required e.g Marfan syndrome. Autosomal recessive traits - 2 defective alleles required males and female equally effected e.g cystic fibrosis. heamophilia A is due to mutation in clotting factor VIII. female 'carriers' of recessive mutant alleles have 50% chance of passing trait to sons. if trait is lethal prior to reproductive maturation only males likely to be affected.
factor VIII was purified and its amino acid sequence determined. Information used to construct DNA sequence. Synthetic DNA used as probe to isolate corresponding genomic DNA from libary. Gene can be expressed in animal cells to produce recombinant protein. sickle cell anemia results from mutation in gene encoding B chain of human haemo. Glutamic acid to valine. form long linear crystals under low o2 conc and break easily. Requires transfusions, become trapped in capillaries easily, common in west africa and india as some resistant to malaria. hetro advantage. encodes plasma membrane protein CFTR controlling Cl influx of cells in secretory glands. 1 in 25 carriers. targeted gene disruptions in mouse can be used to model human diseases.
transgenic animals can be used to produce recombinant proteins. produced by transformation of zygote are useful. fuse donar egg with removed nucleus with cell from another sheep and implant embryo. produce human factor VIII for treatment of haemophilia A. because of universal nature of code, any gene can be expressed in E.coli using recombinant DNA technology in theory. use E.coli because well charactorised genetically, rapid generation time, can be manipulated and cloned in expression vectors (plasmids), easily and cheaply scaled up.
insert foreign DNA into plasmid, and transform into host cells, then select cells which have plasmid using ampicilin e.g. and then distinguish cells carrying recombinant molecules from cells carrying vectors without inserts as LacZ gene should be split so no blue pigment when X-gal is there. Cis-acting sequences required for expression from a transcriptional-fusion when using Lac regulatory systems as have 5' control sequence for inducible expression. 3' control sequeence for transcript termination. transcription factors bind to enhancer sequences that determine spatial and temportal patterns of gene expression.
insulin synthesised from the pro-insulin precursor by PC1 and 2 and carboxypeptidase E, which induces glycogen sunthesis and affects DNA replication and protein synthesis. can be produced in E.coli
synthetic genes for A and B subunits for optimal expression. translational fusions to LacZ. B-galatosidase/insulin A and B fusion protein accumulate in cells. Extract and purify B-gal/insulin fusion proteins, treat with cyanogen bromide to cleave A and B chains, purify and mix A and B to form insulin. Use of transgenic yeast to express hepatitis B virus surface epitope as vaccine. gene can be expressed in E.coli but doesnt give effective protection against virus. Expression in yeast generates correct conformation protein which is immunogenic and protective.
plant transformation strategies - Direct DNA uptake (not very effective), Biolistic transformation - shoot microscopic pellets coated in DNA directly into tissue. Agrobacterium - mediated transformation - good choice of vetors available, has broad host range and causes galls on wide range of dicotyledonous and monocot plants. The vector joins with plant cell and injects the immature T complex attached to Vir D2 which crosses VirB/D4T455 channel and follows radial microtubules to nucleus where it injects DNA into plant DNA. uses - herbicide resistance in soybean, maize etc - enzyme EPSP synthase located in the chloroplast and is important for aromatic amino acid biosynthesis by shikimic acid pathway. Mutant version of enzyme from agrobacterium strain CP4 is insensitive to glyphosphate. Agrobacterium transfer plasmid into plant chromo so high levels of EPSP synthasase made. plants regenerated from calluses are glyphosphate resistant
Bacillus thuringiensis is a bacteria that produces Bt toxin, which kills caterpillars as generates pore in the gut so bacteria can feed on nutrients but insect dies. Bt toxin gene introduced to several crop using agrobacterium. not considered toxic to humans, Some batches of non-Bt maize unfit due to fungal mycotoxins.
golden rice - animals use ingested B-carotene as precursor for the synthesis of vitamin A and retinoic acid. immatur rice endosperm makes the GGDP, then phytoene synthase converts GGDP to phytoene. enzymes 2 and 3 catalyse introduction of 2 double bonds inot the phytoene molecule to make lycopene. Enzyme 4 lycopene B-cyclase turns it to B-carotene.
transgenic plants to express surface epitopes as vaccines -gene from human pathogen cloned in agrobacterium - bacteria infects leaf segments - leaf segments sprout into whole plants carrying gene for human pathogen - eating raw patoatoes triggers immmune response to pathogen.
structure of DNA and RNA
DNA nucleotides: pentose sugar (deoxyribose), phosphate group, nitrogenous base. RNA: Ribose pentose sugar, phosphate group, nitrogenous base. Purines - adenine and guanine. Pyrimidines - cytosine, uracil and thymine. Nucleoside - base + sugar. Nucleotide = Base + sugar + phosphate. Ribonucleotides - AMP, CMP, GMP and TMP. deoxyribonucleotides - dAMP, dCMP etc. nucloetides can be mono, di or tri phosphates. in nucleic acids, nucleotides are linked 3' to5' by phosphodiester bond between sugars. structure of DNa determined by a crystal of DNA placed in a beam of x-rays and the diffraction pattern provided an electron density map which provided atomic model.
phosphates in backbone are negatively charged and repel the 2 strands. NaCl helps to neutralise this so strands bind more tightly. In cells the DNA is neutralised by positively charged proteins. Each base pair is 0.34nm from each other. 10 bps in complete turn of double helix.
mRNA - copy of DNA sequence gets translated to proteins. tRNA synthesised as single stranded molecule and folds to form double stranded structure, has anticodon, acceptor stem, variable and anticodon loop. and rRNA (ribsosomal rna) in pro you get 50s and 30s subunits in Eu you get 60s and 40s subunits which make up ribosome.
meselsons and stahl experiment to show semi conserv replicatin - N14 and N15 isotopes were used to label and separate DNA fragments by weight after centrifuging. Generation 1 had both N14 and 15 and generation 2 had half N14 and half a mix. daughter strands synthesised 5'-3'. DNA synthesis is carried out by DNA polymerase III and lesser extent I. Begins by RNA polymerase (primase) making a short RNA primer for nucleotides to join to.
In E.coli DNA replication begins at origin of replication and goes both ways to form replication bubble. is Bi-directional so 1 origin 2 replication forks. replication fork - leading strand is snythesised continuously in direction of fork. Laggging strand is synthesised discontinuously in short pieces (okazaki fragments) in opposite direction. 2 DNA polymerase III molecules used. DNA gyrase - removes supercoils, helicase - unwinds DNA, ligase - synthesises phosphodiester bonds joining okazaki fragments. DNA polymerase I - removes RNA primer and replaces with DNA.
in origin of replication - consequence - sequence thats repeated 3 repeats of 13bp and 4 repeats of 9bp. tandem repeat - all facing same way, inverted - facinf each other. initiation - DnaA proteins each with ATP bind at 4x9bp repeats and DNA wraps around this complex. 3x13bp repeats are denatured. Hexamers of DnaB bind to each strand and DnaB helicase unwinds DNA in prep for priming and DNA synthesis.
termination - on circular DNA eventually 2 replication forks meet at terminus. in E.coli this contains multiple copies of 20bp sequence callled ter. Ter sequences function as binding sites for Tus. tus-ter complexes arrest forks from 1 direction so are arranged in opposite orientations. Topoisomerases serparate the circular chromosomes.
'eye-form' replication of eu genomes - occurs from multiple origins, individual replicons are activated at specific times during S phase. replication of chromosome ends - Eu chromosome ends (telomers) have no 3' end to begin growing strand, not possible to use normal replication apparatus as would result in shortening chromosomes. Telomerase is an enzyme that has an RNA subunit and synthesises new DNA. Rna dependant DNa polymerase, RNA is used as template to direct synthesis of new DNA at 3' end of chromo. Ends of eu chromosomes contain many repeats, telomerase synthesises new repeats to prevent loss of DNA from ends.
signalling pathways e.g - low blood sugar means release of peptide hormone glucagon, binds to transmembrane protein that serves as receptor and is linke to G-protein, G-protein releases one subunit which activates adenylate cyclase which activates protein kinase A which liberates glucose from glycogen. 2 hybrid system - if bait and prey interact then reporter gene is expresssed, which typically codes for survival factor.
amino acids - R side chains, NH3 amino group, COO carboxylic group and H group with C in middle. when join a carboxylic acid condenses with an amino group to release water. Glu-val mutation causes self-association and polymerization and a hydrophobic interaction between haemoglobin tetramers causing sickle-cell haems.
protein modification - regulate activity, protein-protein interactions, subcellular localization (to acnhor to membrane), aging (to identify protein for degradation). Soluble proteins and proteins targeted to mitochondira, chloropasts and peroisomes are synthesised on free ribosomes. Integral membrane proteins, secreted proteins and proteins in the ER, golgi and ysosomes are synthsesised on ribosomes bound to ER membrane as has N terminal ER signal sequence causing ribosomes to bind to membrane, translocation of protein into lumen and cleavage of signal sequence. also get removable mictochondrial matrix signal and perminant peroxisisome and nucleus targeting signals.
ER targetting requires 2 special recpetor proteins - SRP which binds to emerging signal sequence from ribosome and SRP receptor with alpha and beta subunit. a initiates binding of ribsosomes-SRP to ER membrane. B is intrinsic membrane protein. ribsosome binds to translocon and the gate opens, signal sequence inserts into translocon central cavity with N-terminus towards cystol, signal peptidase celaves signal sequence (post translational modification), mature protein is extrudedinto ER and folds.
post translationial modifications - covalent - disulphite bridge formation, cleavage of peptide bonds and the N and C-terminus. Non covalent - addition of metal, cofactors etc. e.g Pairs of cysteine residues can form disulfide bond by becoming oxidised. e.g peptide bond cleavage - carried out by proteases, activation of proenzymes and prohormones, removal of signal sequences. e.g phosphorylation - responsible for activating and deactivating many enzymes and receptors. phosphorylation catalysed by protein kinases, dephosphorylation by phosphatases. Can occur on -OH groups of serine, theronine and tyrosine. E.g Glycosylation - addition of polysaccharide to protein molecule. Implication for protein folding and stability, N linked glycolysation - amide nitrogen of asparagine, O-linked - hydroxyl oxygen of serine and threonine.
E.g insulin, 2 polypeptides held together by disulfite bonds (oxidaation), cleavage of signal peptide, excision of C-peptide and truncation of B peptide.
protein structure + function
protein structure can be determined by x-ray crystallography. or by nuclear magnetic resonance. globular proteins - water-soluble or 'globular' form a hydrophobic core with a more hydrophilic surface e.g lysozyme. Membrane proteins - domains consist of collection of a helixs, approximately parrallel to membrane, in porins the membrane is spanned by 16 strands of B-barrel making narrow pore. Fibrous and structural - e.g tropomyosin, collagen consist of an association of diff polypeptide chains.
structure stabalised by weak interactions - hydrogen bonds between donar and acceptor groups, ionic interactions between acidic and basic sidechain groups of opposite charge, hydrophobic interactions between alphatic and aromatic side chain groups, covalent disulfite bridges. N-1st C- last terminus. globular - amino acids with hydrophobic R groups hide in core, ones with polar R-groups on surface.
secondary structure - right handed alpha helicies. B sheet stabilised by inter-chain hydrogen bonds can get parrallel, anti-parrallel and mixed. domains are built from motifs (super secondary structure) mix of alpha and Beta, stabalised by disulfide bridges. tertiary - arrangement of domains in a single chain protein. quaternary - multi-subunit proteins.
protein structure and function
GFP = fluorophore formed from residues 65-67 (ser-tyr-gly) oxygen is required to form the final fluorophore. GFP has an 11-stranded B-barrel structure where 1st and last strand are joined together. Fluorophore is found inside barrell. Absorbs blue light and emit green light.
Imaginng by confocal microscopy - taggged cellular proteins can be detected by this. Excitation filter then sample then emission filter then detector. non-polar sidechains tend to be buried in the cores of proteins. globular proteins tend to fold to remove hydrophobic sidechains from exposure to water - entropy-driven. Charged and polar sidechains tend to map protein surgaces. clusters of conserved residues are called sequence motifs and are regions which carry out a particular function or form a particular structure that is important for protein.
purification - in vitro applications require purified proteins for protein characterization - assay to determine function, 3D structure; study protein regulation and interactions; use in drugs and diagnostics; produce antibodies. source of proteins: native; recombinant protein expression systems, gene cloned in vector and inserted into orgnaism which produces protein via consitiutive and inducible expression. proteins are separated by affinity and hydrophobic interactions.
protein purification + proteomics
affinity chromotogrpahy - use inert chromatographic resin which has bee covalently attached to a compound which has specific afinity for protein of interest. ligand affinity or immunoaffinity. can engineer proteins for purification and detection by adding signal sequence that causes secretion into culture medium. add protein that helps target protein fold and stay soluble. Add sequence that aids detection or add an affiinity tag e.g His tag.
immobilised metal interaction chromo - purification of recombinant proteins with polyhis tag. matrix surface is coated with a metal chelator to which Ni, Zn or Co is bound. His-tagged proteins bind specifically to immobilised metal, proteins displaced with imidazole. hydrophobic interactio chromo - GFP not very phobic so add salt which increases it. add bacterial lysate to column matrix in high salt buffer, phobic proteins stick to column, low salt buffer washes away less phobic, GFP eluted away by adding salt free buffer.
analyse by electrophoresis - gives approximate mass and mass spectrometry - gives accurate mass. PAGE - separation of mixtures of proteins by rate of migration, for denatured use SDS-PAGE (based on mass) for native use NATIVE-PAGE (will be based on mass and charge). electrophoresis - molecules with net -ve charge (anions) move towards anode and vice versa with cathode. rate of migration depends of mass:charge ratio.
SDS- polyacrylamide gel electro - unfolded proteins bind to sodium dodecyl sulfate which results in charge being proportional to mass in all proteins so proteins can be seived according to molecular weights. acrylamide is mixed with cross-linker and oxidised with persulfate to form matrix. proteins run and migrate smallest fastest. Gel stained with dye. Disulfide bonds must be reduced with a reductant to ensure protein fully denatured. Isoelectric focussing separates proteins by overall charge. 2D-PAGE of complex mixtures 1st by charge then 2nd by size.
mass spectrometry - protein identification, identification of protein posttranslational modifications that change mass, profiling of protein expression. ionise the sample e.g MALDI - subject charged molecule to an electric field - Detect ions and determine velocity. means peptide mass fingerprinting is available - using sequence proteases e.g trypsin the protein chain can be cut selectively at certain amino acids. the protein fragments have distinct masses which can be measured by MS. compare fingerprint to database and indentify protein.
complete genomes of organisms can be sequencesand proteome can be derived. extract mixture of protein from cells/tissue and analyse by 2D-PAGE, then extract spots from gel and digest with trypsin and then identify by mass spectrometry.
protein folding and misfolding
if folded native protein is refolded is aggregates -disease state, if degraded it forms amino acids/peptides. polypeptide exit channel in ribosomes is quite narrow so likely to be little co-translational protein folding in the channel. Anfinson's thermodynamic hypothesis - the 3D structure of native protein is the one in which the Gibb's free energy of the whole system is at its lowest. i.e the folded state of a protein represents its global free energy minimum.
protein folding in vitro - alkali-denatured haemoglobin spontaneaously recovers its biological properties when pH is returned to normal. Many simple proteins e.g RNases can be reversibly unfolded by temp or denaturant conc. Protein folding adopts a 2 state model. however folding accessory proteins are sometimes needed. e.g protein disulfide isomerases - form disulfide bonds as contain 2 cysteines that are easily interconverted between reduced SH form and oxidised S-S form. PPIs facilitate formation of cis-peptide bonds in proline by catalyzing the rotation about peptide bonds.
Molecular chaperones - prevent improper folding, aggreation and improper associations. DnaK/DnaJ/GrpE family which bind to growing polypeptide chains while being synthesised on ribosomes to prevent premature folding. Chaperonin family bind to solvent-exposed hydrophobic surfaces of an unfolded or aggregated protein and release them, assist correct folding post translationally.
folding and misfolding 2
GroES-GroEL complex - unfolded protein binds to hydrophobic residues of apical domain, ATP binds to equatorial domain. GroEL has high affinity for unfolded protein, GroEL-ATP has low affinity. ATPase activity set up cycles of peptide binding, folding and release. Misfolded proteins are either refolded by chaperones or targeted by proteosomes which are linked to ATPase and unfold translocate and degrade. in neuronal cells not 100% efficiency leads to neuro-degenerative disease.
heriditary diseases - results from mutation/proteolysis of protein destabilizes native structure. aggregated B-sheets whose Bstands are perpendicular to the fibril axis form amyloid fibrils e.g alzheimers (results in neuronal loss due to deposition of amyloid plagues) B and Y secretases are key proteases involved in formation of B-amyloid protein, aggregation of this leads to fibrils with extended B-sheets(amyloid plagues). Aquired - infectious prion diseases caused by exposure to misfolding priod proteins from another organism e.g vCJD. Prion proteins - in all vertebrates, membrane anchored proteins on neuron surfaces, flexible disordered N-terminus and a-helical C-terminal domain. foreign amyloid fibre-like form of prion (PrPsc) can catalyze the refolding of native prion protein (PrPc) and increased B-sheet content. spongiform encephalopathies (prion)
cytogenetics specifically relates to the technique for analysis of whole chromosomes for disease diagnosis. autosomal - trait not involving sex chromos. euploidy - 2n chromos. Aneuploidy - 2n + or - chromos e.g downs syndrome. genetic diseases can be: chromosomal abnormalities (aneuploidy); single gene disorders (sex-linked, autosomal dominant/recessive and non-mendelian inheritance); polygenic inheritance (number of genes operate together e.g cancer, coronary artery disease).
cytogenetics vs molecular. cyto - investigation at chromo level, smallest detectable abnormality is several Mb of DNA, can detect gross abnormalities without prior knowledge of chromos involved. molecular - investigation at gene level, allows detection of single DNA base changes, need to know genes involved before analysis possible. diff chromos can be distinguished by location of centromere, submetacentric short arm (p) shorter than long arm (q). metacentric p = q. acrocentric chromos have a dense appendage called a satalie at end of p. telocentric only p or q not normal.
tissues for cytogenetic analysis - blood, chorionic villi, skin, bone marrow, amniotic fluid and tumours. amniocentesis - abdomen first cleaned with iodine, ultrasound guides needle through uterine wall and 10ml of fluid is taken. chorionic villus uses catheter tip to get sample of baby. need dividing cells at metaphase, bone marrow and villi cells already dividing
how to prepare chromos: colcemid to arrest cells in metaphase. hypetonic to swell cells. fixative to kill and preserve cells. slide-making spot cell suspension onto slides and air dry. banding special staining techniques. G-banding - G bands are induced by treating chromos with trypsin followed by staining with giemsa. chromos take on charactoristic pattern of ligth and dark bands depending on chromatic amount. Pale bands are yrtpsin-sensitive regions and are gene rich with high G-C content and chromatin less tightly wound. Dark band opposite. then analysis them by counting and scoring (looking for abnormality in a cell).
fluorescent in situ hybridization: oligonucleotide DNA probes labelled with fluorochrome and hybridised to region of interest. centromeric probes - usually aplha-satellite repetitive probes that hybridise to the centromeres. whole chromosome paints, Locus-specific probes and telomeric probes. need to denature probe, denature chromo preparation, hybridise probe to target sequence, wash off unbound probe, counterstain chromos and visualise with fluorescence microscope. rapid prenatal interphase FISH probes for chromos 13,18,21 X and Y. Detects most prevalent chromo aneuploidies, results in 24-48 hrs, simple protocal. however, expensive, only detects numerical abnormalities of specific chromos, potential problems of signal misinterpretations: cross-hybridsation, non-specific hybridisation and mosaicism
types of chromosomal abnormality: numerical - aneuploid, gain or loss of an entire chromos. or structural: e.g translocation, inversion, deletion, isochromos, ring, duplication and insertion. translocation - can be balanced - where no loss of genetic info or unbalanced where theres loss. balanced can still cause problems are break may occur at an important gene and may disrupt it. like in chronic myelogenous leukaemia - reciprocal translocation between chromo 9 and 22 which results in cell regulatory gene being fused to the BCR gene resulting in overexpression in cells of the haematopoietc system resulting in cancer.
single gene disorders affect 1% of pop. autosomal dominant - heter has same phenotye as homo, no carriers. affected parents have affected offspring e.g familial breast/ovarian caner. Autosomal recessive - trait only expressed in homos, hetro are carriers e.g CF. Xlinked recessive inheritance - usually affects males whereas females are carriers e.g haemophilia. aneuploidy - where individual gains of looses chromos, loss of one is monosomy only X chromo for turner syndrome as autosome monosomy is lethal in most animals, segmental deletions of parts of chromos are lost. Cri-du-chat syndrome where part of chromo 5 is lost causing anatomic malformations including larynx. Trisomy is gain of chromo e.g downs syndrome is trisomy 21, shortened life expectancy and prone to respiratory and heart disease, caused by non-disjunction of chromo 21 during parental and maternal meiosis so not inherited
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some individuals have specific area of chromo that did not stain known as fragile sires which are susceptable to breakage when cultured in absence of folic acid. individual bearing folate sensitive site on X chromo exhibit fragile X syndrome which is inherited form of mental retardation. is dominant trait so female carrying one are affected however only 30% of fragile X females are retarded. gene which spans site FMR-1 may be responsible as has trinucleotide repeats. above 200 repeats leads to expression of disorder, repeats can increase in future generations called genetic anticipation.
X linked inheritance in humans. easilt identified in a pedigree because of the crisscross pattern of inheritance. appear more frequently in males as they are hemizygous. affected males usually born to unaffected mother. not passed from father to son.
mitochondrial DNA disorders - mitochondria contain own DNA and are transmitted from mother to offspring through cytoplasm of egg. as a result genetic disorders resulting from mutations in mitochondrial genes are maternally inherited. both males and females affected but only females trasmit. often associated with defects in energy conversion. e.g lever optic atrophy and MELAS syndrome.
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autosomal recessive traits - for rare traits most affected individuals have unaffected parents. all children of 2 affected will be affected. risk of afffected child from 2 hetro is 25%. e.g cystic fibrosis, sickle cell anemia.
autosomal dominant - every affected individual must have at least one affected parent. most affected are hetro with homo unaffected there is 50% chance child will get it. 2 affected individuals can ave an unaffected child as most are hetro. e.g huntingtons and porphyria.
disease-associated genes have been identified using : knowledge of the protein that is defective e.g hemophillia A factor VIII. Through compelemntation of a genetic defect in cells of some other organism e.g mice . Through positional cloning - depends on having genetic markers that can identify particular regions of chromos so patterns of inheritance can be followed.
markers and linkage - genes very close to gene of interest will be inherited together and so that gene can be used to identify the region of chromo containing the varient disease gene.If the marker and gene responsible for genetic disorder are on same chromo they wil show linkage. use lod scores and score of 3-4 is evidence that gene and marker are linked. haplotype - blocks of sequences with low recombination containing linked genes.
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positional clong relies on : southern blot analysis, restriction fragment length polymorphism analysis, PCR, microarray analysis, comparative genomic hybridisation, real-time PCR. RFLP analysis - digest DNA with restriction enzyme and southern blot fragments. fragments can be hybridised with radioactive probe to region of interest. can track inheritance of disease genes e.g B-thalassemia caused by partial deletion of B-globin gene. in mutant allele an exon is deleted, producing smaller fragments when cut with restriction enzymes.
PCR - DNA oligonucleotide primers needed. can screen by using allele specfic oligonucleotides attached to probes. gene screening using DNA microarray - DNA extracted and PCR'd, then spotted onto glass slide and each sample attached to the glass substrate and occupied diff position on microarray. fluorescent labelled oligonucleotide probes corresponding to normal and mutant versions are added. microarray scanned by a laser and analysed by software. means lots of people and lots of genes can be analysed. can also be used to find genes whose expression is altered in disease. can also be usde to look for gain or loss of specific region of chromosomes in cancer by using ratio of normal to cancer DNA.
RFLPs are actually single nucleotide polymorphisms where a single base change determines whether a restriction enzyme will cut or not. SNPS occur about one ever 1000 nucleotides. Some alter susceptibility to diseases and sensitivity to drugs. as distrubuted fairly evenly through genome they are excellent genetic markers.
short tandemly repeated sequences found in introns of some genes and the number of repeats called minisatellites varied between individuals. can be used for forensics and paternity tests as closer relations will have closer number of repeats. minisatellites (VNTRs) - repeat unit length 9-80 bases, thousands scattered through genome mostly near end of chromos (telomeres). Microsatellites (STRs) - 2-7 bases, 5 to 100 repeats each microsatellite thousands randomly scattered. amplify DNA and create fingerprint of diff lengths of STRs, VNTRs, amplified fragment length polymorphism and SNPs.
better methods for isolating DNA from older more decomposed tissue. Quantitative real-time PCR allows better sensitivity. Lase capture microdissection allows isolation of single cells from complex mixtures. real time PCR - uses 2 specific primers and an oligonucleotide probe which has fluorescent reporter group and quencher group which prevent fluorescence priot to and during binding of target sequence. hydrolysis of probe duing polymerization phase release the reporter generating a singla proportional to amount of target amplified
LCM - place cap on tissue, pulse laser at target cells, remove cap with adhered target cells and extract molecules from target cells.
construction of Haplotype map: SNPs are identified in DNA samples from multiple individuals. Adjacent SNPs that are inherited together are compiled into haplotypes. Tag SNPs with haplotypes are identified that uniquely idetify those haplotypes. By genotyping the tag SNPs researchers can identify which are present in each individual and can therefore discover disease associated genes.
'next generation sequencing revolution' - anchor single DNA molecules to solid surface and copy each molecule in situ by PCR. add 4 coloured labeled reversible terminators, polymerase and universal primer. remove unincorperated nucleotides, detect with lase and reverse termination chemically or enzymatically and repeat cycle 1-100 times.