Chapter 3 and 10: Genetics

Genetic modification

from interspecies gene transfer, technique developed by molecular biologists, possible bc. universal gen. code (=a. acid seq. --> same polypep. regardless of species, but some bacteria actually have stop codons UAG/UGA code for a. acids inst. of stop codons). Genes have been transferred euk. to bacteria (one early ex. = making hum. insulin in bacteria --> large quantities of hormones to diabetics). GM = has been used to introduce new characteristics to animal species (ex. goats secreting milk w. spider silk proteins - spider silk = immensely strong but we can't use spiders to produce it commercially). GM also used for new crop plant varieties (=GM crops), ex. snapdragons' genes transferred to tomatoes to produce purple fruits; golden rice production = 2 genes from daffodils + 1 from bacterium --> rice so gets yellow pigment BETA-CAROTENE Scientists attempt to assess risks w. GM crops/livestock, since 70s w. first gene transfer expt.s (Paul Berg planned expt. where DNA from monkey virus SV40, known to cause cancer, inserted in E. coli). GM bans imposed in some countries due to safety of research/using GMOs (--> so some useful applications are underdeveloped bc. of bans) GM crops = sev. potential benefits, widely publicised by GM-crop/seed-producing corps but promoted by othrs (ofc are disagreements bc. rel.ly recent procedures w. complex issues) - Environmental benefits w. GM crops: pest-resistant crop varieties produced by transferring gene for making toxin to plant --> less insecticides used --> fewer bees/othr insects harmed; GM variety use reduces ploughing/spraying-crops need --> less fuel for farm machinery; imprvd fruit/veggie shelf-life --> less wastage, reducing area of crops req.d - Health benefits w. GM: imprvd nutritional value (ex. incr.d vitamin content/ w. golden rice w. beta-carotene from 2 daffodils + bacterium); crop varieties lack allergens/ toxins nat.ly present in them; GM crops produce edible vaccines by eating crop - Agricultural GM benefits: produced varieties (by gene transfer) resistant to drought, cold, salinity --> expended range over which can be produced --> incr.d yields; gene for herbicide resistance transferred --> all unwanted plants die off when applied herbicide --> less weed competition --> higher yield, can create weed-free conditions for sowing non-GM crops but can't be used once crop grows; crop varieties can be produced resistant to viral diseases currently reducing crop yields (only current control method = reduce transmission by killing insect vectors of viruses w. insecticides, which is bad for biodiversity) Wide variety of concerns abt. GMs raised (e.g. effect on farmers' incomes, but only relevant if assessed expt.ally per species) - Health risks of GM crops: proteins produced by transferred genes could be toxic/ cause allergic reactions in hum.s/ livestock eating crop; antibiotic resistance genes used as markers in gene transfer could spread to pathogenic bacteria; transferred genes could mutate --> unexpected prob.s not risk-assessed during development. - Environmental GM crop risks: non-target org.s can be affected by toxins intended to control pests in GM crop plants; genes transferred to crop plants to make them herbicide-resistant spreading to wild plants --> uncontrollable super-weeds not fought off by herbicides; reduced biodiversity if lower sunlight proportion passes to weed plants/ plant-eating insects/ org.s feeding on them where GM inst. of non-GM crops have been grown - Agricultural GM risks: some seed from crop = is always split, germinates as unwanted volunteer plants that must be controlled --> could become hard if crop has herbicide-resistance gene; widespread GM crops w. toxin killing insect pests --> spread of toxin resistance in pests that were initial prob. (as ones w. gene mutations will survive), spread of secondary pests resistant to toxin but prev. being scarce; farmers not permitted by patent law to save/re-sow GM seed from crops they have grown do so anyways so strains adapted to local conditions cannot be developed Insect pests = can be controlled by spraying w. insecticides, but varieties recently produced by gen. engineering producing toxins to kill insects; gene from bacterium Bacillus thuringiensis coding for Bt (cursive) toxin, protein, killing memb.s of insect orders of butterflies, moths, beetles, flies, bees, ants. GM corn varieties produce Bt toxin in all part of plant, incl. pollen; are sev. Bt crop varieties produced, incl. Zea mays (=corn in Am., maize/corn on the cob in Eng.), crop attacked by sev. insect pests incl. corn norers (moth Ostrinia nubilalis larvae); are concerns expressed abt. Bt corn on non-target insect species, ex. monarch butterfly Danaus plexippus (its larvae feed on milkweed leaves Asclepioas curassaciva, sometimes growing close to corn crops to become dusted w. wind-dispersed corn pollen - risk butterfly may be poisoned from GM corn crops; risk as been expt.lly investigated)

Gregor Mendel

father of genetics, crossed varieties of pea plants (e.g. tall pea w. dwarf pea, white-flowered pea w. purple-flowered pea) --> deduced that diff.s between varieties he crossed tghr were due to diff. heritable factors, were alternative forms of same genes (=alleles) His paper "Experiments in Plant Hybridization", offered explanations blending theory (that we're just blendings of our parents) couldn't = published 1866, initially w. little impact and only cited ca 3 times next 35 yrs. Turn of cent. = incr.d recognition of his wrk. At same time, discrepancies arose between observations and Mendel's principle of ind. assortment. William Bateson nd Reginald Punnett conducted crosses w. sweet peas. One of parent plants had long pollen LL and purple flowers PP and othr had round pollen ll and red flowers pp. As expected all of F1 had long pollen purple flowers LlPp when crossed pure-bread flowers. Surprising result came F2 gen. of dihybrid cross (Punnet square w. 2 genes). Inst. of expected ratio 9:3:3:1 were far more individuals w. parental phenotype seen in F1 gen and much smaller no.s of non-parental phenotypes (recombinants). Even though realised results didn't conform to Mendel's ind. assortment principle didn't develop clear explanation of discrepancy. Thomas Hunt Morgan = observed similar in fruit fly Drosophila melanogaster (not first one to use this as research org. but he popularised its use in research; in this species crossing over only occurs in females!) discrepancies, discovery of sex linkage led him to develop theory of gene linkage that accounted for higher-than-expected no. of parental phenotypes and notion of crossing over to explain presence of recombinants. At start of investigation = critical of Mendel's theory of inheritance nd NOT convinced by aspects of emerging chromosomal theory of inheritance, believed variation observed in org.s = better xplained by environmental influence, but subsequent observations of pattern of inheritance of whites eyes --> reconsidered --> his results reinforced aspect of Mendel's conclusions nd identified exceptions to principle of ind. assortment. Noticed single fruit fly w. white eyes inst. of normal red colour in F1 --> mated it w. ordinary red-eyed fly. Although only 3/1,2k F1 offspring had whites eyes, in F2 became ratio 1:3 (white:red), as predicted by Mendel's idea of recessiveness/dominance - surprising thing = all white-eyed were males nd all females had red eyes. One of Drosophila's 4 chrom. pairs = thought to be sex. chrom.s. Morgan's own idea = sex determined by quantity of chromatin (since XY is smaller, technically correct) --> if factor for eye colour was located exclusively on X Morgan could use Mendelian rules for dominance/recessive trait inheritance to xplain observations. Chromosomal theory = could xplain why sex nd eye colour didn't assort ind.ly. Similar expt.s done in early 20th century; proposed crossing over, Morgan was first one to observe it and prop.d chiasmata When Mendel's work rediscovered mechanism causing ind. assortment of UNlinked genes = soon identified, observations of meiosis in grasshoper Brachystola magna had shown homologous chrom.s pair up during meiosis and then sep. to opp. poles (which pole dep.s on where each chrom. randomly faces, one pair's orientation = doesn't affect other ones' orientations = indep. assortment). Each allele has 50/50 to go to each pole. For anothr gene on othr chrom. is also 50/50 in where will end up --> chance of 2 alleles from diff. chrom.s coming tgthr to same pole = 25 % (0,5 x 0,5) (so genotype AaBb can therefore produce gametes w. AB, Ab, Ba, ab = equal probability of each being produced. Mendel = pioneer in research methods in biology. Discovered principles of inheritance w. expt.s in which large no. of pea plants (have either red or white flower colour so can easily be studied; can produce hybrids or self-pollinate - have both a-/sexual reproduction) were crossed. When living org.s reproduce pass on characteristics to offspring (--> all are memb.s of same species = offspring inherits parents' characteristics but ACQUIRED ones CAN'T be inherited). Mendel = used variety of pea plant (each which reliably had same characteristic when grown on its own), carefully crossed pea seeds formed as result and grew them to find out what their characteristics were, repeated each cross w. many pea plants, also did expt.s w. 7 diff. pairs of characters (=his results reliably demonstrated principle of inheritance in peas, not just isolated effect), published research 1866, yr 1900 sev. biologists rediscovered work --> did cross-breeding expt.s w. othr plants/animals = confirmed that Mendel's theory explained basis of inheritance in all plants/animals. Mendelian/classic genetics = Mendel's explanations of genetics (disregards e.g. epigenetics and phenomenon of linked genes) Mendel's laws: - law of segregation/first law = 2 alleles of a gene sep. when forming gametes, while combining randomly in forming offspring; - law of independent assortment/second law = alleles of genes located on diff. chrom.s are inherited ind.ly of each othr

PCR

(see ch. 2) --> possible to study DNA w.o. risk of using up limited sample, very small amounts of DNA from blood/ semen/ hairs are req.d = can be amplified for use in forensic investigations. PCR = used to copy specific DNA seq.s (selected for using primer binding to desired seq. by complementary base pairing). PCR selectivity = allows particular desired seq. (VNTRs are common, w. just individual non-coding regions) to be copied from whole genome/greater DNA mix. DENATURATION - DNA sample is heated to sep. it into 2 single strands (~95ºC for 1 min) nd break H bonds between purines (A nd G) + pyrimidines (T and C) ANNEALING - DNA primers attach to 3' ends of target seq. (~55ºC for 1 min) ELONGATION - heat-tolerant DNA polymerase (from Thermus aquaticus, Taq) binds to primer, copies strand (~72ºC optimum temp.) for 2 min

DNA profiling

/DNA fingerprinting; one test for presence of GM ingredients in food incl. use of primer binding to GM DNA nd any such DNA present = amplified by PCR but if none present PCR = w.o. effect DNA profiling incl. DNA sample obtained from known individual/othr source, ex. fossil/crime scene; seq.s in DNA varying considerably between individuals (VNTR, acts as inherited allele, but non-coding, so can be used for personal/ parental identification; repetitive seq.s) are selected, copied by PCR; copied DNA --> split into fragments using restriction endonucleases; fragments sep.d by gel electrophoresis --> produces band pattern consistent per individual (individual's DNA profile, like DNA fingerprint, as shows pers.'s amount of VNTR which may differ in length); profiles of diff. ppl can be compared to see which are same, which differ. Can be used for forensic investigations: suspect's clothing's e.g. blood stains could be shown to come from victim/unknown blood stains shown to come from suspect/single hair = suspect's?/semen from sexual crime = from suspect? can be checked --> DNA mat. from crime scene compared w. DNA sample from suspects/victims. If band pattern exactly matches = highly likely DNA's from same pers. --> CAN be strong evidence; some countries have DNA databases of DNA profiles (--> crim. cases solved) Paternity investigations: find fathr of child, are sev. reasons for test being requested - men may want to avoid paying for child so claims not to be fathr, women may have had sev. partners, child may want to show deceased man was fathr to prove is heir etc. DNA profiles of mothr, child, fathr = req.d, band patterns can be compared; if band pattern in child not in mothr nor claimed fathr must be false DNA profile analysis in forensic investigations = straightforward, paternity cases more complex as have to compare w. 2 sources, not just one

Meiosis

2n --> n (n = no. of chrom. pairs; haploid no. of chrom.s). Meiosis can happen at any time during sexual life cycle, but in animals happens during process of creating gametes (mitosis = somatic cells = diploid, have 2 copies of most genes) Meiosis: nucleus divides twice, first producing 2 haploid nuclei in meiosis I, then these 2 divide in meiosis II to produce 2 more haploid nuclei each (sperms: --> 4 unique sperm cells; in ovary: --> 1 ovum, egg, + 3 small polar bodies, not functional cells). Starting nucleus in meiosis = diploid w. homologous chrom.s, each of 4 nuclei produced in end are haploid (= meiosis halves chrom. no. = REDUCTION DIVISION), cells produced have one chrom. of each type so halving of chrom. no. happens in FIRST DIVISION, NOT 2nd. 2 nuclei produced in meiosis I have haploid no. of chrom.s (n) but each chrom. still consists of 2 sister chromatids that sep. in meiosis II, producing 4 nuclei w. haploid no. of chrom.s of single chromatids SOME plants have interphase II (=INTERKINESIS), per. of rest before entering meiosis II. In that case nuclear mbr. + nucleolus quickly reemerge (otherwise remain disassembled until process is complete)

Chromosomal recombination

= exchange of gen. mat. between non-sister homologous chromatids (prophase 1) (i.e. chromatids belonging to same homologous pair but on diff. chrom.s). First, homologous pairs pair up (are 4 DNA molecules associated w. each pair bc. each chrom. consists of 2 rep.d DNA strands); pair of homologous chrom.s = bivalent/tetrad (tetrad if chrom.s are X-shaped), pairing process = synapsis. Soon after synapsis we have recombination where JUNCTION is created on chromatids in exactly same points nd break in each of homologous chrom.s, are "cut" --> DNA of each chromatid gets joined up to DNA of non-sister chromatid --> chunks of DNA are swapped; process of fastening chromatids again = LIGATION; crossed over sections remain next to each othr (sida vid sida) until are sep.d later on. Once exchange is done non-sister chromatids continue to adhere to site where crossing over occurred (i.e. remain directly next to each other; these random connection points where gen. mat. is exchanged = CHIASMATA (sing. = chiasma), which look like knot-like structures that bind crossed over chromatids tgthr; acc. to evidence connections via chiasmata = essential for successful meiosis completion). Consequences of chiasmata formation = INCR.d STABILITY of BIVALENTS at chiasmata, incr.d GEN. VARIABILITY if crossing over occurs; results in DNA exchange between maternal nd paternal chrom.s, can decouple (sep.) linked allele combos so lead to indep. assortment of them (but is rarer, hence lower recombinant rate of linked traits), crossing over can occur sev. times in bivalent + between both non-sister chromatids, chiasma = X-shaped knot where crossing over has occurred, holds homologous chrom.s tgthr for while but then slide to end of bivalent, allowing chrom.s to move to diff. poles of cell in anaphase. Recombination occurs at random positions anywhere along chrom.s; at least 1 crossing over occurs in each bivalent (not being sex. chrom.s if XY) nd can be sev.; bc. crossover occurs at exactly same position on 2 chromatids is mutual exchange of genes between chromatids. As chromatids = NOT identical some alleles are likely do differ --> chromatids w. new allele combos. Crossing over --> recombinant (+ possibly also parental) gametes (which are ones affected by crossing over nd have had genes swapped), which maximises gen. variation.

Locus

A gene occupies specific position on one type of chrom., from expt.s in which diff. varieties of plants/animals are crossed show that genes are linked in groups (and makes sense as we have more genes than we do chromosomes) and each group correspond to one of chrom. in species. E.g. in fruit flies are 4 groups of linked genes (=chrom. pairs - are 4 types of chrom.), in maize 10 and hum.s 23. Maps showing gene seq. along chrom.s in fruit flies and othr org.s were produced by crossing expt.s, but more detailed maps can now be produced when genome of a species has been seq.d

Animal cloning

Animals can be cloned at embryo stage (when still pluripotent --> theoretically still possible to develop into all tissues) by breaking up embryo into more than 1 group of cells = process SPLITTING/FRAGMENTATION; coral embryos observed to clone themselves by breaking into smaller groups of cells/single cells (presumably bc. incr.s chance of 1 embryo surviving). Formation of identical twins = can be regarded as fragmentation (but most species don't do it nat.ly, but is possible to break up animal embryos artificially, in some cases for sep.d parts to --> multiple embryos). In livestock egg can be fertilised in vitro, allowed to develop into multicell. embryo; individual cells can be sep.d from embryo whilst still pluripotent nd transplanted into surrogate mothrs, but only limited no. of clones can be obtained this way bc. after certain no. of divisions embryo no longer pluripotent (splitting of embryo = typically most successful at 8-cell stage); has been little interest in artificial cloning this way bc. at embryo stage not possible to assess whethr new individual from sex. reproduction has desirable characteristics Methods have been developed for cloning adult animals using differentiated cells (preferable bc. then known if has desirable traits, but harder to clone as undifferentiated pluripotent cells are req.d). Biologist John Gurdon = made expt.s on cloning in long Xenopus frog as Oxford postgrad. 1950s, removed cells from nuclei from body cells in tadpoles, transplanted them into egg cells w. removed nuclei - all developed as if were zygotes, carried out mitosis, growth, differentiation to form all tissues of normal frog (--> Nobel Prize 2012) First cloned animal using differentiated cells = Dolly the sheep 1996, are reproductive + therapeutic uses for this type of cloning; if same procedure done w. hum.s embryo would comprise pluripotent stem cells which could be used to regenerate tissues for adult. Bc. cells would be gen.ly identical to those of adult from whom nucleus was obtained wouldn't cause rejection prob.s Production of Dolly = pioneering animal cloning development, w. method SOMATIC-CELL NUCLEAR TRANSFER (somatic cell = normal diploid body cell): adult cells taken from udder (spena) of Finn Dorset ewe (tacka), were grown in lab using medium w. low nutrient conc. (--> inactivated genes in cells so differentiation patterns lost); unfertilised eggs taken from ovaries of Scottish Blackface ewe cells, removed nuclei from these eggs. One of cult.d cells from Finn Dorset placed next to each egg cell inside zona pellucida around egg (protective gel coating around egg cell), then used small electric pulse to cause cells to fuse tghtr, ca 10% of fused cells developed like zygote into embryo; injected embryos when ca 7 days old into othr ewes' uteri (like surrogate mothrs), done in same way as IVF; only one of 29 embryos implanted successfully nd developed through normal gestation (--> Dolly, gen.ly identical to donor, at Uni. of Edinburgh)

Plasmid gene transfer

Are sev. possible techniques to transfer genes between species, tgthr known as genetic engineering; gene transfer to bacteria = typically incl. plasmids, restriction enzymes, DNA ligases Plasmid = small xtra DNA circle (smallest ca 1 kbp = 1000 base pairs; largest ca 1000 kbp), commonly in bacteria; most abundant ones have genes encouraging replication in cytoplasm + gene transfer from bacterium to othr --> are parallels w. viruses but plasmids = NOT pathogenic, nat.l selection favour plasmids giving advantage on bacterium inst. of disadvantage. Bacteria use plasmids to exchange genes so nat.ly absorb them nd incorporate into main circular DNA molecule using ex. pili --> plasmids useful in gen. engineering as vectors (means of transferring gene) Restriction enzymes = endonucleases, cut DNA molecules at specific base seq.s, can be used to cut open plasmids, cut out desired genes from larger DNA molecules, some have useful prop. of cutting 2 strands of DNA molecule at diff. points --> single-stranded sections STICKY ENDS, created by one particular endonuclease w. complementary base seq.s so can be used to link tgthr DNA pieces by H bonding between DNA bases; are part of bacteria's nat.l defence against viruses; are also "blunt ends" restriction enzymes which cut genes off bluntly, i.e. w.o. leaving sticky ends (--> less convenient) DNA ligase = enzyme joining DNA molecule tghr firmly by making SUGAR-PO4 BOND (COV. PHOSPHODIESTER BOND) between nucleotides, when desired gene has been inserted into plasmid using sticky ends are still small cuts in each sugar-PO4 backbone, DNA ligase can be used to seal these. Obv. requirement for gene transfer = copy of gene being transferred; usually easier to obtain mRNA transcripts of genes than genes themselves. Enzyme REVERSE TRANSCRIPTASE make DNA copies from RNA molecules into cDNA, COMPLEMENTARY DNA (can be used to make DNA req.d for gene transfer)

HUGO

Human Genome Project/Organisation: wanted to seq. entire hum. genome, began 1990 --> drove rapid techniques developments in base seq.ing techniques, allowed 1st draft seq. to be published 2000, complete seq. 2003 (much sooner than predicted!), ran projects all over wrld through public/private competition/collaboration --> biotech. developments (had to analyse ~3,2 billion base letters of hum. DNA) Led to seq.ing othr genomes --> gene seq.s can be compared between species; results can be used to determine evolutionary relationships, identify conserved seq.s to allow species to be chosen for exploring function of that seq. Wrk continues to find seq. variations between individuals. Vast majority of base seq.s = shared between all hum.s (=why same species) but are also many SNPs + VNTR/ STR contributing to hum. diversity. Since publication of hum. genome base seq.s of many othr species have been determined, comparisons between these reveal aspects of evolutionary hist. of living org.s prev.ly unknown Although knowledge of entire base seq. hasn't given us immediate nd tot. understanding of hum. genetics has given us vast mine of data to be wrked on by researchers for yrs to come. Ex. now possible to predict which seq.s are protein-coding genes (ca 23k of these in hum. genome; estimates originally much higher). Anthr impt. discovery = most of genome isn't transcribed, was originally called "junk DNA", although now more recognised that within these "junk" regions are elements affecting gene expression as highly repetitive seq.s satellite DNA (denser than normal DNA, hence name "satellite", make out great part of functional centromeres); HUGP outcomes: complete hum. DNA/ chrom.s seq.d, identification of all hum. genes/ found position/map of all hum. genes, discovered protein structures/ functions, found evidence for evolutionary relationships/ hum. origins/ ancestors, found mutations/base substitutions/ single nucleotide polymorphisms, genes causing/ incr.ing chance of diseases --> developed tests/screened for diseases, developed new drugs (based on base seq.s)/ new gene therapies, tailored medication to individual genetic variation (=pharmacogenomics), promoted internat.l coop. Genome seq.d consists of 1 particular set of chrom.s --> is A hum. genome, not the hum. genome

Recessive and dominant alleles

Late 18th cent. discoveries by Thomas Andrew Knight in pea plants: fe-/male parents gen.ly contribute equally to offspring (even though male and female gametes have diff. size and mobility - eggs are generally much bigger and immobile or less mobile); a white flower disappearing in F1 can emerge later on --> inheritance is discrete rather than continuous as blending theory suggested; one characteristic, like red flower, can show "stronger tendency" than the alternative characteristic (=be dominant) In each of Mendel's 7 crosses between diff. varieties of pea plant all offspring showed character of one parent (e.g. crossing tall and dwarf plant --> tall plant), diff. in height = due to TT or tt alleles in parents (were pure bred) --> pass on one allele each to offspring --> Tt in F1 bc. tallness gene is dominant; othr recessive allele is ineffective if dominant is present; are also co-dominant genes (e.g. for flower Mirabilis jalapa red + white flower parent --> pink flower). Typical reason for allele dominance = it codes for protein that is active, "masks" recessive protein, which may be non-functional/ doesn't code for protein at all

Genetic diseases

Meiosis = sometimes subject to errors, ex. when homologous chrom.s fail to sep. in anaphase (=non-disjunction, can happen in both meiosis I and II); can happen any of tetrads, both chrom.s move to 1 pole, neither to othr --> gamete w. xtra chrom./deficient in 1 (--> in hum.s = 45/47 chrom.s); abnormal chrom. no. --> syndrome (often, collection of physical signs/symptoms). Ex. trisomy 21 (Down's) = bc. non-disjunction leaving gamete w. 2 chrom.s 21 (so in fertilisation gets 3 of these); individuals may vary but features often incl. hearing loss, hearing/ vision disorders, mental + growth retardation. Trisomy birth rate rapidly incr.s as mothr reaches 40s (is correlation w. maternal age nd non-disjunction, we don't know why). Most othr trisomies = so serious offspring doesn't survive long in womb; sometimes trisomies 13 and 18 (=Edwards's syndrome) can lead to surviving baby, perhaps bc. don't have too "impt." genes on them. Non-disjunction can also result in too many/few sex chrom.s (Klinefelter's syndrome = XXY, Turner's = only X) Many gen. diseases (illness caused by gene) in hum.s = due to recessive alleles of autosomal genes; carriers = ppl w. dominant healthy allele nd recessive ill allele. Some gen. diseases = sex-linked, some due to dominant alleles (small proportion of gen. diseases are; ex. Huntington's disease) = you can't be carrier for these, only afflicted if has dominant allele so will develop disease. Very small proportion of gen. illnesses are due to co-dominant alleles (e. sickle cell anaemia, 6th SENSE codon GAG to GTG on chrom. 11). Some gen. diseases = sex-linked (typically carried by X chrom.). Othr well-known, gen. diseases = phenylketonuria (PKU, autosomal recessive), Tay-Sachs disease (autosomal), Marfan's disease, albinism (by recessive allele), vitamin D-resistant rickets (by dominant sex-linked) Med. researchers = have identified +4k gen. diseases, many more to be found --> might be surprising most don't suffer from one (bc. are so many), but is so bc. most gen. diseases = recessive, rare - chance of inheriting one is small so inheriting both = even rarer. Modern possibilities to rel.ly cheaply seq. hum. genome --> research reveals no. of gen. disease alleles someone. carriers. Current estimates = average person have 75-200 recessive alleles of ca 23k genes carried are for genetic diseases

Cells involved in meiosis

Ovum = egg cell, gamete Spermatozoa = sperm, gamete Germ (line) cells = cells in reproductive organs to go through meiosis to become spermatozoa/ovum + polar bodies, found in testes/ovaries and have 46 chromosomes Primary oocyte = present in females at birth, what are to develop into ova

Meiosis I

Prophase I = nuclear mbr./nucleolus dissolve, centrioles/ centrosomes move to poles, synapsis, crossing over, chrom.s supercoil (then spindle microtub.s grow from poles of cell, attach to centromeres after nuclear mbr. has disassembled; spindle microtub. attachment to centromeres = NOT same as in mitosis as each chrom. now is attached to 1 pole only (so spindles are attached to whole centromere so entire chrom.s move w. it) and 2 homologous chrom.s in each tetrad become attached to diff. poles, which pole dep.s on way chrom. faces (=orientation, is random so each chrom. has equal chance of attaching to each pole nd being pulled to it, orientation of 1 bivalent doesn't affect orientation of othr bivalents --> gen. diversity)), as soon have done so = clearly consist of 2 chromatids (bc. DNA was replicated in interphase, now visible; initially 2 chromatids in each chrom. = identical bc. DNA rep. = very accurate, xtremely few errors made); some of meiosis's most impt. events = in beginning of meiosis I while chrom.s still are elongated, can't be seen w. microscope. Unlike from mitosis, chrom.s pair up already in prophase 1 (synapsis --> tetrad bc. consists of 4 chromatids; in MANY euk.s protein-based structure SYNAPTONEMAL COMPLEX forms between homologous chrom.s to mediate synapsis + keep them tgthr), crossing over occurs (AFTER that we have condensation) (both of these 2 processes = BEFORE SUPERCOILING). Metaphase I = pairs line up in pairs in metaphase plate equator due to spindle microtub.s (orientation of paternal/ maternal chrom.s on either side of equator = random, independence of homologous pairs) Anaphase I = homologous pairs are sep.d by disjunction, chiasmata slide off, prob. synaptonemal complex too, one chrom. from each pair to each pole as microtub.s shorten Telophase I = haploid chrom.s uncoil (during following potential interphase, NO rep.; if interkinesis occurs nuclear mbr. quickly emerges), cleavage furrow emerges; cytokinesis occurs Sep. of bivalents in meiosis 1 halves chrom. no.; mov. of chrom.s = NOT same in meiosis I as in mitosis (mitosis: centromere divides, 2 chromatids making up chrom. move to opp. poles; in meiosis centromere doesn't divide yet, whole chrom.s move to each pole); initially, 2 chrom.s in each bivalent = held tgthr by chiasmata but these can slide to end of chrom.s, as does synaptonemal complex, then chrom.s can sep. (=disjunction, this sep. of homologous pairs before anaphase 1) --> 1 chrom. from each pair move to each pole. Sep. of whole chrom.s from each pair = halves chrom. no. in cell (=meiosis I = reduction division) bc. 1 chrom. type moves to each pole so both daughter cells --> haploid Differs from meiosis II nd mitosis as sister chromatids remain associated w. each other throughout; in prophase homologous chrom.s behave in coordinated fashion, homologous chrom.s exchange DNA (--> genetic recombination). Processes resulting in creation of genetic variety in gametes = initiated in meiosis I, seg. of homologous chrom.s occurs in anaphase I (--> 2 haploid cells each w. one copy of one chrom. in homologous pair)

Meiosis II

Prophase II = chrom.s condense nd become visible Metaphase II = chrom.s (of 2 chromatids) line up in middle Anaphase II = chrom.s sep., chromaTIDS move to opp. poles Telophase II = chromatids reach opp. poles, nuclear envelope + nucleolus form, cleavage furrow + cytokinesis occur Each gamete produced = new allele combo due to crossing over (may allow linked genes to be re-shuffled, incr.s no. of allele combos, is effectively infinite; each of 2 alleles on chrom.s have equal chance of being passed over; BEFORE SUPERCOILING)/random orientation of bivalents (process generating gen. variation among genes of diff. chrom. types - for every additional bivalent no. of possible chrom. combos to each cell doubles; for haploid no. n are 2^n possible combos of each chrom. in random orientation in gamete (from random orientation) for metaphase 1 - for hum.s w. haploid no. 23 are 2^23 = over 8 million combos (e.g. if we only had 2 chrom. pairs so 4 chrom.s in tot. could combine in 4 diff. ways in metaphase = 2^2 combos); random mating also incr.s genetic variation). Fusion of gametes to form zygote = highly sig. event both for individuals + species - is start of life for new individual, allows new alleles from 2 diff. ppl to be combined in 1 new individual; allele combo = unlikely to have ever existed before, gamete fusion --> gen. variation in species promoted (essential for evolution)

Monohybrid crosses

Punnett squares (diagrams abt. predictions of outcome of particular breeding event, is stable systematically combining every possible combo of maternal-paternal allele) of one character so only involves one gene, most crosses start w. pure-breeding parents (=are homozygous) --> each parent produces 1 types of gamete concerning that allele; offspring obtained by crossing = F1 (first filial) hybrids/F1 generation (have 2 diff. alleles of gene so can produce 2 types of gamete). If F1 self-pollinate/ F1 plants are crossed --> 4 diff. types of gametes can be produced in F2 gen. To make Punnett grid as clear as possible gametes should be labelled, both alleles of character of 4 possible outcomes should be shown on grid, often useful to give overall ratio below Punnett grid. Dihybrid crosses = inheritance of 2 genes = investigated tgthr (assuming diff. alleles seg. ind.ly). Mendel = performed dihybrid crosses like pure-breeding peas w. round yellow seeds w. pure-breeding peas w. wrinkled green seeds --> all of F1 had round yellow (as are due to dominant alleles), but when self-pollinated appeared round yellow, round green, wrinkled yellow, wrinkled green. If genotype of F1 hybrids = SsYy (as P1 = pure bred) possible gametes = SY, Sy, sY, sy nd if ind. assortment of this = chance of gamete containing S or s won't affect chance of containing Y or y. Chance of gamete containing each allele = 0,5 so chance of it containing 2 specific alleles = 0,5 x 0,5 = 0,25 --> theory that alleles of 2 genes can pass into gametes w.o. influencing othrs = ind. assortment. Has one allele (combo.) per gamete (=4combos per gamete in dihybrid)

SINGLE NUCLEOTIDE POLYMORPHISMS

SNPs ("snips") = position in gene where more than 1 base may be present, i.e. places varying between alleles; sev. SNPs can be present in gene but even then alleles differ by only few gene bases (more alike than diff.).

Red-green colour-blindness

X-bound recessive allele for 1 of PHOTORECEPTOR PROTEINS made by 1 of cells in RETINA, DETECT SPECIFIC RANGES of visible light - if son inherits mother's allele w. colour-blindness will develop it (parts of northern Europe: 8% of males have illness, girls have 0,08 x 0,08 =0,64 %, w. actual % being 0,5% so fitting prediction well)

Allele

are various specific forms of gene, can be many diff. alleles for same gene; one of first ex.s of multiple alleles discovered in mice for coat colour, can be "yellow", grey, black; and 3 alleles for ABO blood group system; e.g. in fruit flies eye colour can be very many diff. alleles Alleles of same gene have same locus, only one allele can occupy locus of gene on chromo. at one time. Most animal/plant cells have 2 copies of each type of chrom.s so 2 copies of a gene can be present that could be same or diff. Diff. alleles have slight variations in base seq., usually very small no. of diff.s (like that an A is on anti-sense strand in one allele and a C on another, in that case BASE SUBSTITUTION MUTATION has once occurred at this SNP, SINGLE NUCLEOTIDE POLYMORPHISM)

Test cross

breeding/mating between dominant phenotype (homo-/ heterozygous) w. recessive (homozygous) to deduce genotype of dom. (if homozygous --> only dom. offspring, if if heterozygous --> 25% recessive ones) (Yy x yy OR YY x yy); Batson/ Punnett/ Saunders did one to investigate crossings w. unexpected results (got 7:1:1:7 w. most of offspring looking like parents, not being recombinants) --> hypothesised was coupling/connection between parental alleles for flwr colour nd pollen grain shape. If are UNliked genes --> Mendelian ratios, if on same chrom.s nc very close --> very few recombinants; if on same chrom. but far apart --> still many recombinants due to crossing over (but not necessarily Mendelian ratios) (LINKED if RECOMBINATION RATIO less than 50% recombinants)

Reproduction

can be asexual (offspring have same chrom.s as parent = gen.ly identical)/ sexual (are diff.s between chrom.s of offspring nd parents --> genetic diversity); in euk.s sex.l reproduction involves process of fertilisation (=union of sex cells, gametes, usually from 2 diff. parents, w.o. meiosis this would cause doubling of chrom. no. every fertilisation if no. wasn't also halved at some stage of life cycle in meiosis). Meiosis = complex process, at moment not yet clear how developed (what is clear = critical in euk. origin as w.o. meiosis can't be fusion of gametes nd euk.s' sexual life cycle couldn't occur). Fertilisation = gametes' nuclei fuse tgthr, become diploid. SEPARATION OF ALLELES INTO DIFF. CELLS = SEGREGATION (Mendel's first law) of alleles so that each gamete only carries 1 copy of allele, they sep. in gamete formation (meiosis), breaks up existing allele combos in parent, allow for new combos in offspring

Chi-squared tests

can be used for genetic crosses to see if results are due to chance or not, if diff. in observed nd expected freq.s = statistically sig. - Identify hypotheses (null H0 and alternative H1) - Construct table of freq.s (observed in first row for each trait, w. expected frequencies in row below so are 2 numbers per column nd 4 per row) - Apply chi-squared formula - Determine degree of freedom (df) - Identify p value (should be <0.05 if want 5% uncertainty) Null hypotheses = H0 = NO correlation between 2 species/ traits, occurrence tgthr is RANDOM - traits assort INDEP.LY Alternative hypothesis H1 = sig. diff. between distribution of species - species/traits ARE ASSOCIATED, DON'T ASSORT INDEP.LY - ARE LINKED 1. Draw table of freq./contingency of observed freq.s, which are no.s of individuals observed of each phenotype 2. Calc. expected freq.s, assuming ind. assortment (i.e. freq.s from dihybrid crosses multiplied by tot. no. of all observed traits) 3. Determine no. of degrees of freedom (mathematical restriction designating what range of values fall within each significance level) w. one less than tot. no. of classes (if are 4 phenotypes, take (m-1)(n-1); for all dihybrid crosses degree of freedom should be (no. of phenotypes - 1) = 3; m = number of rows, n = number of columns = 1 x 3; When distribution patterns for SPECIES are compared, df should always be 1 4. Find critical region for chi-squared from table of x^2 values, using degrees of freedom that have been calc.d nd sig. level (p) of 0.05. CRITICAL REGION = any value of chi-squared LARGER than value in table. 5. Calc. chi-squared using x^2 = ∑[(Obs-Exp)^2/Exp] for each trait = all phenotypes in columns added tgthr of (observed frequency - expected frequency)^2 divided by expected frequency 6. Comparison of calc.d value to critical region - if is in reg. (= ABOVE VALUE) is evidence at 5% level for association between traits (that traits are linked) --> reject H0, ASSUME H1 (=traits are linked as only 5% claims aren't so); if calc.d value = NOT in crit. reg. bc is equal to/below table value H0 is kept = is NO evidence at 5% uncertainty level for association of traits

Homologous combination

carry same seq. of genes but not necessarily same alleles (vary in SNPs) of those genes. If 2 chrom.s have same seq. of genes are homologous, are homologues, which are usually not identical as some of genes carry diff. alleles (=HYBRID VIGOUR, protects us that we have 2 alleles, against dangerous mutations as most gen. diseases are recessive). If 2 euk.s are memb.s of same species we can expect each of chrom.s in 1 of them to be homologous w. at least 1 chrom. in othr (=allows for interbreeding). Some euk.s have fewer, larger or more, smaller chrom.s. As all euk.s have at least 2 diff. types (1 sex chrom. + 1 w. common characteristics of species) of chrom.s diploid no. is at least 4, in some cases over 100. Technically we can have only 22 homologous pairs if you're male (bc. XY is NOT homologous)

COX-2 gene

codes for enzyme CYCLOOXYGENASE, gene comprises +6k nucleotides; 3 SNPs have been discovered in COX-2 associated w. gastric adenocarcinoma (stomach cancer). 1 of SNPs occur at nucleotide 1195, base at this seq. can be A or G on anti-sense strand; large Chinese survey involved seq.ing both copies of COX-2 gene in 357 patients who developed gastric adenocarcinoma, in 985 ppl w.o. illness, all were asked if had ever smoked cigarettes (=is pos. correlation between smoking and gastric adenocarcinoma)

Cystic fibroris

commonest gen. disease in parts of Europe, bc. recessive allele of CFTR gene on CHROM. 7; gene product = Cl- ion channel involved in sweat, mucus, digestive juices secretion; recessive allele --> Cl- channels malfunction (--> SWEAT w. EXCESSIVE NaCl, DIGESTIVE JUICES/MUCUS w. INSUFFICIENT NaCl) --> not enough wtr moves by osmosis into secretions so become very viscous --> sticky mucus build-up in lungs (--> infections, pancreatic duct is usually blocked so digestive enzymes secreted by pancreas don't reach small intestine so has poor digestion). In some parts of Europe 1/20 have allele for cystic fibrosis (--> chance of both parents having it = 1/20 x 1/20 = 1/400 --> 1/400 of developing disease)

ABO blood group system

ex. of co-dominance, of great med. impt.ance nd vital to find patient's blood group nd match it correctly (othrwise blood will coagulate --> death), 1 gene decides ABO group (gene I or i). I^A, I^B = co-dominant, i = recessive: all 3 alleles cause production of glycoprotein in mbr. of RBC; I^A alters it by adding ANTIGEN ACETYLGALACTOSAMINE (if this is given to B they will produce anti-A antibodies) to glycoprotein as antigen in RBC's mbr.; I^B has GALACTOSE added to glycoprotein; I^A I^B has acetylgalactosamine + galactose (--> produces neither anti-A nor anti-B antibodies if given either blood type); i = recessive allele bc. produces basic glycoprotein w.o. additional galactose/ acetylgalactoseamine (e.g. are 6 diff. genotypes: I^A I^A, I^B I^B, ii, I^A I^B, I^Ai, I^Bi). Antigen A in blood group A produces anti-B antibodies in blood plasma to defend against B blood; B had anti-A antibodies

Eukaryotic chromosomes

comprises DNA + proteins (+other substances, like pos. a. acid lysine on histone tails, binding to DNA, coiling it, methyl coiling it, acetyl uncoiling it etc.), DNA = single, immensely long linear molecule, associated w. globular histone proteins wider than DNA. Are many histones in chrom. w. DNA wound around them. Adjacent histones in chrom.s are sep. by short DNA stretches not in contact w. histones, linker DNA, (=euk. chrom. rather looks like string of beads during interphase) In euk.s are diff. chrom.s carrying diff. genes.. Euk. chrom.s = too narrow to be seen w. light microscope in interphase, but during mitosis/meiosis chrom.s become shorter, fatter by supercoiling (prophase 1) so are visible if stains binding to DNA/proteins are used. First stage of mitosis = chrom.s can be seen to be double, are 2 chrom.s w. identical DNA produced by rep. When chrom.s are examined during mitosis diff. types can be seen, differing in length, centromere position (where 2 chromatids join tgthr; centromere can be positioned close to end/ near middle), are at least 2 diff. types of chrom.s of every euk. but in most species are more than that (in hum.s are 23 types of chrom.). Each chrom. types carries specific seq. of genes of certain loci arranged along linear DNA molecule, many chrom.s contain over 1k genes in seq. Crossing expt.s were done in past to discover seq.s of genes on chrom.s in D. melanogaster + othr species. Base seq. of whole chrom.s can now be found, allowing more accurate, complete gene seq.s to be deduced. Having genes arranged in standard seq, along type of chrom. allows parts of chrom.s to be swapped during crossing-over of meiosis

Sex chromosomes

determine sex, in hum.s dep. on rel.ly large X chrom. w. centromere near middle + small Y chrom. w. centromere near end. X = has many genes (over 2k), crucial to both sexes (=all hum.s have X) Y = only small no. of genes (78 GENES), small part of which has same seq. as parts on X, but remainder is only found on Y, not req. for fem. development SRY/TDF gene on Y causes fetus to develop into male, initiates male feature development like testes, high testosterone production, bc. of this gene fetus w. XY = male. As XX lacks SRY/TDF ovaries develop inst. of testes, fem. sex hormones are produced. As fem.s have XX pass on one X to each egg cell, but each sperm either has X or Y (=sex of offspring determined in moment of fertilisation) Autosomes = all chrom.s NOT determining sex

Discovery of meiosis

discovered by microscope examination of dividing germ-line cells, when imprv.d microscopes had been developed 19th cent. giving detailed images of cell structures was discovered some dyes specifically stained nucleus of cells, dyes revealed thread-like structures in dividing cells (=chrom.s), 1880s onwards group of German biologists carried out careful/detailed observations of dividing nuclei gradually revealing how mitosis/meiosis occur - prep. of microscope slides showing meiosis = very challenging, suitable tissue can be obtained from developing anthers (ståndarknappar) inside lily bud/testis of a dissected locust (vandringsgrähoppor), stained and then squashed on microscope slides. Often no cells in meiosis are visible/images aren't clear enough to show details of process (although, even if prep.d slides are made by xperts it's hard to decipher images). Key observation was in horse threadworm Parascaris equorum that are 2 chrom.s in nuclei of egg, whereas fertilised egg contains 4 chrom.s (=chrom. no. doubled by fertilisation) --> theory of special nuclear division in sex cells; seq. of events in meiosis = wrkd out by careful observations in rabbit cells from ovaries between 0-28 days old (advantage of this species = female meiosis starts at birth of rabbit cells and occurs slowly over many days) Mosses' sporophytes grow on main moss plan and consist of stalk and capsule in which spores are produced to be carried by wind

Huntington's disease

due to dominant (rare) allele HTT gene on CHROM. 4, gene product = protein HUNTINGTIN (function still unknown, researched). Dominant HTT allele --> degenerative changes in brain w. symptoms starting at 30-50 yrs age, changes to behaviour, thinking, emotions become incr.ly severe. Life expectancy after start of symptoms = ca 20 yrs. Pers. w. disease = eventually needs full nursing care, typically succumbs to heart failure/pneumonia/some othr infectious disease. Bc. late onset of symptoms many ppl w. Huntington's already have had children so children will prob.ly develop disease; gen. test can show predisposition to illness (if have dominant allele as runs in fam.) but most ppl at risk choose not to take test. Ca 1/10k have copy of allele (=very unlikely both parents have copy, which would ensure developing disease).

Independent assortment

due to random orientation of chrom.s, this recombination + random mating incr.s gen. variation. 2^n = no. of possible chrom. sorting combos due to random orientation. Random fertilisation: 1 spermatozoa (=1/ 8 million combos of chrom.s from parent) x 1 oocyte (=1/ 8 million combos of chrom.s from parent, simply judging from random orientation, indep. assortment) = 64 trillion diploid combo.s possibilities (=you'll probably never see anyone identical to you as genes also differ - children would differ if 2 twin couples would mate) Indep. assortment = observation that alleles of 1 gene seg. indep.ly of alleles of othr genes. Genes on diff. chrom.s = UNlinked --> seg. indep.ly in meiosis. Hwvr, genes on same chrom. = linked --> DON'T seg. indep.ly, except for linked genes far apart on chrom. as crossing over occurs more freq.ly further genes are from each othr on chrom., can make it appear genes are unlinked; LINKAGE GROUPS = sev. genes are close tgthr so often inherited tgthr as in parental gametes (likely to be inherited tgthr bc. is smaller chance crossing over will occur in between these genes; ex. freckles + red hair in hum.s); linkage map = diagrams describing rel. gene locations using recombination freq.s - shorter distance between 2 genes = less likely crossover will happen between them (is proportional to how often recombination occurs between certain traits; is measured in centiMorgans, 1 cM = 1% recombination between 2 genes - ex. in sample of 100 ppl only once were trait x and y not inherited tgthr = 1cM)

How to sequence a genome

genome is first broken up into small lengths of DNA. To find base seq. of small DNA fragment single-stranded copies of it are made using DNA polym., process is stopped before whole base seq. has been copied completely by sep.ly putting mix. of small quantities of non-standard nucleotides carrying each of 4 DNA bases (so are added as primers, but DNA polym. can't wrk from them) in reaction --> 4 samples of DNA copies of varying lengths are produced, each w. one of 4 DNA bases at end of each copy. These 4 samples are sep.d by length using gel electrophoresis. For each no. of nucleotides in copy is band in just 1/4 tracks in gel from which seq. of bases in DNA can be deduced. Major advances in technology speeding up project of base seq.ing: - development of coloured fluorescent markers to mark DNA copies (diff. colour used for copies ending in each of 4 bases) - sample being mixed tgthr, all DNA copies are sep. in one lane of gel acc. to no. of nucleotides (due to gel electrophoresis) - laser scan along lane to make fluorescent markers fluoresce - optical detector used to detect colours of fluorescence corresponding to each no. of nucleotides - computer deduces base seq. from seq. of colours of fluorescence detected

Clones

groups of gen.ly identical org.s derived from single original parent cell. Bc. zygotes, org.'s first cell = gen.ly diff. grows, develops into adult org., if reproduces sex.ly offspring = gen.ly diff., but some species can also reproduce asex.ly (then --> gen. identical offspring) Production of gen.ly identical org.s = process called cloning; pair of identical twins = smallest clone to exist (bc. are 2), result of hum. zygote dividing into 2 cells (so develops sep.ly but acc. to same principles)/ embryo splitting into 2 sep., but identical twins = NOT identical in every way w. ex. diff. fingerprints so better term for them = monozygotic; more rarely identical triplets/quadruplets/ even quintets have been produced. Sometimes clone can comprise large no. of org.s, ex. commercially grown potato varieties = huge clones; large clones = occur when cloning happen again nd again but even so all clones can be traced back to one original org. Many plant/animal species = have nat.l cloning methods; "clone" wrd first used early 20th cent. for plant produced asex.ly (comes from Greek word for "twig") - plant cloning methods vary, can incl. stems, roots, leaves, bulbs (ex. single garlic bulb when planted uses its food stores to grow leaves, which produce enough food by photosynthesis to grow group of bulbs, all gen.ly identical) = clone; /strawberry plant grows long horizontal stems w. plantlets (young/small plants) at end which grow roots into soil, photosynthesise using leaves so can become indep. of parent plant, healthy strawberry can produce new/more gen.ly identical new plants in this way during growing season Nat.l cloning methods in animals = less common, but some species canle, ex. Hydra (cursive) clones itself by process called budding; female aphids (bladlöss) can give birth to offspring entirely produced from diploid egg w.o. male, produced by mitosis inst. of meiosis (--> offspring = clone of mothr) Stem cuttings (short stem lengths) can clone some plants artificially (if root develops from stem --> indep. new plant), e.g. Ocivaum basilicum root are espec. easy; w. most species stem's cut below node (positions on stem where leaves are attached), then leaves are removed from lower half of stem, if are many large leaves in upper half can also be reduced, lowest third of cutting = inserted into compost (sterile w. plenty of air nd wtr)/wtr (clear plastic bag w. holes prevents excessive wtr loss from cuttings in compost), rooting normally takes few weeks nd growth of new leaves typically indicate cutting has developed roots; sev. factors may affect if successful rooting

Prokaryotic DNA structure

have one chrom. of circular DNA molecule (= has no ends) (bc. have same DNA structure, UNIVERSAL CODE, GMO is possible), at least in most prok.s, w. all genes req.d for basic life processes of cell; as bacterial DNA isN'T associated w. proteins (= no histones/nucleosomes) is often described as naked. Bc. only one chrom. present is usually only one copy of each gene, but 2 gen.ly identical chrom.s are briefly present after replication in prep. for cell division (through binary fission), are moved to opp. poles, cell undergoes cytokinesis. Chrom. is attached to cell mbr. through ATTACHMENT SITE. Some prok.s have plasmids, small xtra circular DNA commonly found in prok.s but rare in euk.s, are small, circular, naked DNA containing a few useful genes perhaps useful to cell but not needed for basic life processes (ex. antibiotic resistance often in plasmids as those only are beneficial when antibiotics are present but not othrwise), not always rep.d as chrom. is nor at same rate (=may be sev. copies of plasmids present in cell, plasmid may/not/ be passed over to both daughter cells from cell division). Plasmid copies can be passed over from 1 cell to anothr using pili, allowing spread through pop.; is possible for plasmids to cross species barriers (happens if plasmid that is released when prok. cell dies is absorbed by diff. species cell), is nat.l method of gene transfer between species, also used by biologists to transfer genes between species artificially. Viruses (NOT PROK.S; but still) can have genomes coded for in RNA, as some viruses only have RNA, then are retroviruses (slightly more dangerous), nd use host cells' DNA to replicate own RNA)

Gene

heritable factor consisting of length of DNA, influences specific characteristic: genetics = branch of bio. concerned w. info. storage in living org.s and how this info. is passed on to progeny, are 100s/1000s of bases of DNA in gene. Expt.s in 19th century = showed were factors in living org.s influencing specific characteristics and that these were heritable --> intense genetics research from early 20th century onwards, word "gene" invented for heritable factors. One big q. = chem. composition of genes? Mid. 20th century strong evidence genes comprised DNA (bc. by Hershey-Chase expt.). As are only a few DNA molecules in cell (max. 46, normally, in hum.s) yet have ~ 23k genes must comprise shorter DNA chunks --> each chrom. carries sev. genes (so must be linked genes as well and some must be inherited tghtr).

Gene no. comparison between species

hum.s might be expected to have most genes due to our intricacy yet e.g. large tree Populus trichocarpa, black cottonwood, has ~46k genes (estimated, bc. difficult to count all genes) - but w. our microbiota we have ca 4.4 million genes, inst. of just 23k. Most complex species doesn't necessarily have most no. genes (although complexity and genome size are correlated but aren't directly proportional) bc. of proportion of genes being transcribed varies and amount of gene duplication does so as well

Haemophilia

more severe sex-linked disease (life-threatening). Although are some rare milder forms most cases = due to inability to make COAGULATION FACTOR VIII; if untreated life expectancy ~ 10 yrs; treatment = infusing Factor VIII, purified from blood donors. Factor VIII gene = located on X chrom. by recessive allele. Freq. = 1/10k in boys. Fem.s = can be carriers of recessive allele. Freq. in girls = theoretically (1/10k)^2 (if fathr has haemophilia daughter must become carrier). In practice, have been fewer cases of girls w. haemophilia due to lack of Factor VIII than this bc. then father must haemophiliac nd decide risk passing condition to children

Sickle cell anaemia

most common genetic disease (are ca 4k genetic disorders) in wrld, due to mutation in gene (symbol: Hb) coding for BETA-GLOBIN polypep. in 1 of haemoglobin subunits (each has 4 alpha, 4 beta); healthy hum.s have gene Hb^A. Is point mutation, base substitution mutation converts 6th CODON on SENSE STRAND at tip of CHROM. 11 of gene from GAG to GTG resulting in new allele Hb^S (mutation only inherited to offspring if occurs in cell of ovary/testes to develop into egg/ sperm) When Hb^S is transcribed mRNA produced has GUG as 6th codon inst. of GAG --> VALINE becomes 6th amino acid inst. of GLUTAMIC ACID --> bc. VALINE is NON-POLAR and GLUTAMIC ACID is POLAR produced haemoglobin will have valine forming London disp. Fs w. othr a. acids --> haemoglobin will stick tghtr in tissues w. low O2 conc. (in tissues w. high O2 conc. are still disc shaped); bundles of haemoglobin molecules formed are rigid enough to distort red blood disc cells into sickle cells (sickle = skäran in Commie sign) Sickle cells can cause damage to tissues by becoming trapped in blood capillaries, blocking them, reducing blood flow. When RBC return to high O2 conc. in lungs haemoglobin bundles break up, RBC return to normal shapes. These changes occur time after time as RBC circulate body. Both haemoglobin nd plasma mbr. are damaged by sickle shapes; life of RBC can be as low as 4 day, bc. body can't replace these rapidly enough anaemia (blodbrist) develops Thus, small changes to gene can have detrimental effects on individuals inheriting gene. Is unknown how often has occurred but in some parts of world Hb^S allele is very common - parts of East Africa 5% newborns have 2 copies of allele, develop severe anaemia (usually die within few days), 35% have 1 copy of allele + 1 healthy --> suffer from mild anaemia as have both both sickle nd disc RBCs, (--> alleles are co-dominant). Why detrimental allele has survived (othrwise by nat.l selection these ppl would have died long ago) = provides protection against malaria (= why disease almost exclusively occurs in Africa, bc. that's where malaria mosquito Anopheles mosquitoes, carrying Plasmodium falciparum viruses; they invade normal RBC, causing them to lyse, burst), so is favourable to be HETEROzygous w. allele Hb^S. Malaria symptoms: fever, shivering, severe anaemia etc. However, malarial virus can't infect sickle cells (even heterozygous ones) --> heterozygous sickle cell anaemia is "good", in regions w. high malaria rates are often high sickle cell rates

Radiation and mutation

radiation + mutagenic chem.s incr. mutation rate --> gen. diseases + cancer New alleles are formed (ex. 1/small no. of bases differ, as SNPs/VNTR/hyper-variable reg.s/STR) by gen. mutations. Mutation = RANDOM change to base seq., can incr. mutation rate: radiation if has enough E to cause chem. DNA changes, ex. gamma rays, alpha particles from radioactive isotopes, short-wave UV rad., X-rays = all mutagenic; some chem. substances cause chem. changes in DNA so are mutagenic, ex. benzo[a]pyrene nd nitrosamines found in tobacco smoke nd mustard gas in WWI. Gene mutations controlling cell division = can cause cell to divide endlessly --> tumour --> mutations can cause cancer. Mutations in somatic cells (incl. cancer-causing) = eliminated when individual dies, but germ line cells' mutations = can be inherited to offspring = origin of gen. diseases --> impt. to minimise no. of mutations in gamete-producing cells in ovaries/testes. Current estimates: 1/ 2 new mutations occur each generation in hum.s (--> risk of gen. disease in children) Hiroshima/Nagasaki bombings: 150k-200k died directly/in few months, surviving 100k ppl have been followed by Radiation Effects Research in Japan + 2k ppl not exposed as control group. By 2011 = survivors had developed 17 448 tumours, but only 853 could be attributed to rad. effects. Apart from cancer othr main factor of rad. predicted = mutations (--> stillbirths/malformations/death). Health of 10k children being fetuses when bombs detonated + 77k children born later in Hiroshima/Nagasaki = monitored w. NO EVIDENCE found of mutations caused by rad. Likely were some mutations but no. = too small to be statistically sig. even w. large no. of children in study; despite lack of evidence of mutations due to atomic bombs, survivors = often stigmatised, some found potential spouses unwilling to marry due to fear of children inheriting gen. disease Accident at Chernobyl, Ukraine, 1986 = involved explosions + fire in nuclear reactor core --> wrkrs quickly received fatal rad. doses as radioactive Xe, Kr, I2, Cs, Te(tellurium) isotopes spread over large parts of Europe - ca 6 tonnes of uranium + othr rad. metals in fuels from reactor = broken up into small particles by explosions, escaped. Estimated 5,2k million GBq, gigabecquerel, unit of radioactivity, of rad. mat. released into atmosphere in tot w. severe/ widespread effects: 4km^2 pine forest downwind of reactor turned ginger brown, died; horses, cattle near plant died from damage to thyroid gland; lynx, eagle owl, wild boar, othr wildlife subsequently started thriving in zone closeby as hum.s left; bioaccummulation --> high levels of rad. fish as far away as Scandinavia, Germany, lamb contaminated w. radioactive Cs banned for time as far away as in Wales; conc.s of rad. I2 in environment rose --> ppl drinking wtr./ milk w. unacceptably high levels; +10k cases of thyroid cancer have been reported that can be attributed to rad. I2 released in accident; acc. to Chernobyl's Legacy Health, Environmental and Socio-Economic Impacts by The Chernobyl Forum = is no clearly demonstrated incr. in cancer/ leukemia due to rad. in most affected groups

Mutations

on average occur 1 mutation/1,2 x 10^8 bases New alleles are formed from othrs by gene mutations Mutation = random change, is no spec. mechanism for particular mutation happening Most sig. type of mutation = BASE SUBSTITUTION when 1 base seq. is replaced by anthr (ex. A being substituted by T/G/C) as entire structure of resulting polypep. can be changed, espec. if resulting a. acids have diff. prop.s of polarity( polypep. can become much shorter nd be cut off at point mutation Random changes to an allele that has developed over millions of yrs by evolution = unlikely to be beneficial (othrwise mutation would have occurred earlier nd survived through nat.l selection) (=almost all modern mutations = harmful/ neutral). Some mutations = lethal (cause death of cell in which has occurred). Mutations in body cells are eliminated when individual dies, but those in cells developing into gametes can be passed on to offspring nd cause genetic disease FRAMESHIFT MUTATION = caused by deletion/insertion of one/ more nucleotides that is not divisible by 3 (bc. then codons would be diff., not frameshift) in DNA seq. so shifts way seq. is read, so DNA seq. is moved forward/backward a few steps Some mutations are silent, some you get similar a. acid from so protein will fold basically same way, some you get completely diff a. acid from so will fold vastly diff.ly/end earlier In rare cases chrom.s can fuse tgthr/split apart to change chrom. no. of species (--> during hum. evolution 2 ancestral ape chrom.s fused to form modern-day chrom. 2, which has inactive region where old centromeres were, is abt. twice as large as chimps' chrom. 2 --> hum.s have 23 chrom. pairs, chimps have 24. Mules are sterile bc. have odd no. of chrom.s as donkeys and horses have diff. chrom. no.

Sex-linkage

pattern of inheritance = diff. w. sex-linked genes due to location Plants like peas = hermaphrodites (can produce both fe-/male gametes). Thomas Andrew Knight's crossing expt.s w. pea plants late 18th cent. --> discovered results were same regardless if character was in male or female gamete, e.g. cross w. pollen from plants w. green stems on stigma placed on plant w. purple stems and pollen from plant w. purple stems placed on stigma of plant w. green stems = always gave same results when reciprocal crosses like where are carried out (in animals: results are sometimes diff.). Inheritance pattern w. ratios diff. in fe-/males = sex linkage True breeding = pure-bred, org. always passes down phenotype to offspring; wild type = commonest phenotype in nat. One of first ex.s of sex linkage = discovered in fruit fly Drosophila (small insect ca 4 mm long nd completes its life cycle in 2 weeks, allowing crossing expt.s to be done quickly w. large no.s of flies. Most crosses w. Drosophila = don't show sex linkage) Geneticists = observed gen. nd chrom. inheritance = showed clear parallels (--> genes = likely to be located on chrom.s). As female Drosophila = have XX nd male = XY --> deduced sex linkage of eye colour could be bound to sex chrom.s (bc. white-eyed male + red-eyed female --> red-eyed female and white-eyed male; red-eyed male + white-eyed female --> only red-eyed offspring) As are very few genes on Y (hum.s, only 78 - most Y-linked mutations lead to sterility so can't be inherited) almost all sex-linked diseases are on X; e.g. red-green colour blindness Variation in traits = discrete or continuous (CONTINUOUS shown by phenotypes of POLYGENIC traits, when 2/ more genes have additive effects on expression of traits). Continuous = gives unexpected phenotypes due to ex. that are 2 UNlinked genes w. CO-DOMinant alleles - in that case no. nd freq. of variants can be predicted using alternate rows in Pascal's triangle (e.g. if are 2 unlinked traits w. CO-dominant alleles in DIHYBRID cross affecting trait phenotypic ratio will be for 5th row in Pascal's triangle bc. every-other row is used, giving ratio 1:4:6:4:1; for these co-dom. traits if only 2 diff. gametes are crossed, not dihybrid as in prev. one, phenotypic ratio would be 3rd row in Pascal's triangle 1:2:1). POLYGENIC TRAITS --> CONTINUOUS VARIATION, as no. of genes incr.s distribution incr.ly becomes close to normal distribution (many hum./non-hum. characteristics = close to normal distribution, e.g. mass, height, intelligence etc.). Closeness to normal distribution = suggests sev. genes are involved = polygenic inheritance = also influenced by environment. Polygenic trait variation = typically continuous (=is range of variation) bc. ENVIRONMENT BLURS DIFF.s IN PHENOTYPES, e.g. hum. skin colour = sev. genes + environment William Bateson, Edith Saunders, Reginald Punnett = discovered 1st exception to law of ind. assortment 1903, always w. higher freq. of parental combos than predicted from Mendelian ratios, but new allele combos = sometimes formed due to crossing over (=results due do linked genes)

Karyograms

show org.'s chrom.s in homologous pairs of decr.ing lenghts. Chrom.s of org. = visible in mitosis, w. metaphase giving clearest view. Stains have to be used to make chrom.s visible, some of which give each chrom. type distinctive banding pattern (called G-bands/Giemsa bands due to Giemsa dye darkening regions rich in A (purine tgthr w. G; 2 rings)-T (pyrimidine tghr w. C; 1 ring) pairs (electrically compatible w. A = neg., T = pos.), even thinnest band contains over 1 million base pairs w. potentially 100s of genes). If dividing cells are stained nd placed on microscope slide nd burst by pressing on cover slip, chrom.s scatter, often overlapping each othr, but w. careful searching cell can usually be found w.o. overlapping chrom.s, micrograph can be taken of it. Originally analysis involved cutting out all chrom.s, arranging them manually, but this process can now be done digitally, arranging chrom.s acc. to size, structure; position of centromere nd pattern of Giemsa banding allow chrom.s of diff. type but similar size to be distinguished. As most cells are diploid, chrom.s are usually in homologous pairs, are arranged by size starting w. longest pair, ending w. shortest, then pair 23 (XX/XY). Karyotype = prop. of an org. (=no. + appearance of chrom.s in nuclei), studied by looking at karyograms. Can be used in 2 ways: to deduce if individual is fe-/male (if XX/XY are present) or to diagnose Down syndrome/ othr chrom. abnormality, usually done by using fetal cells taken from uterus during pregnancy, if are 3 chrom.s 21 in karyotype child has Down syndrome (trisomy 21) Karyotyping by extracting fetal cells by AMNIOCENTIS (fostervattensprov, take out amniotic fluid by passing needle through mothr's abdomen wall under ultrasound guidance, then needle withdraws sample of amniotic fluid containing fetal cells from amniotic sac, has miscarriage rate 1%) or CHORIONIC VILLUS SAMPLING (take sample from placenta, sampling tool enters through vagina, obtains cells from CHORION (one of mbr.s from where placenta develops); can be done earlier in pregnancy but has miscarriage risk 2%), cult. cells to stimulate mitosis, take photograph under light microscope/scan w. computer, arrange chrom.s in homologous pairs based on size, banding pattern, centromere positions --> fetus's karyotype. Normal karyotype = 44XX or 44XY (autosomes, sex chrom.s) Trisomy = having 3 chrom.s of one type Monosomy = having 1 chrom. of one type (rare, but e.g. Turner's syndrome has only an X)

Chromatin

uncoiled DNA, used for macromolecule synthesis, transcription, in G1/0 interphase (then is transcription in chrom.s); chromatin has structure of SOLENOID FIBRE (=wrapped around sev. nucleosomes to pack it into nucleus, takes shape of helix -"wavy")

Autoradiography

used to establish length of DNA molecules in chrom.s. Even though quantitative data typically is strongest supp. for/ against hypotheses images can also be good. Microscopy developments have allowed images to be produced of diminutive structures. Autoradiography = used 1940s onwards to discover where specific substances were located in cells/tissues. John Cairns used technique diff.ly in 1960s when obtained images of whole DNA molecules from E. coli bacteria. At time was unclear if bacterial chrom. was single/sev., but Cairns's images showed was single and showed DNA rep. forks for 1st time; technique then used to investigate euk. chrom. structure. Only made thymine radioactive so that only DNA would be radioactive (bc. are no Ts in RNA) He grew E. coli for 2 generations in cult. medium w. TRITIATED THYMIDINE (thymidine = thymine + deoxyribose linked tghthr, is used by E. coli to make nucleotides for DNA rep.). Tritiated thymidine contains radioactive H isotope tritium so radioactively labelled DNA was produced during E. coli rep.; placed cells onto dialysis mbr., digested cells using lysozymes nd cells gently burst to release DNA onto surface of dialysis mbr.; thin photographic emulsion film placed on surface of mbr., left in darkness for 2 months (during time some tritium atoms in DNA decayed, emitted high E e- reacting w. film); at end of 2-month per. film was developed, examined w. microscope, at each point of tritium atom decay was dark grain to indicate DNA's position. Images showed E. coli chrom. was single circular DNA molecule 11k micrometres long (=remarkably long as E. coli is only 2 micrometres) Autoradiography then used to produce images of euk. chrom.s by othr researchers. Image of chrom. in fruit fly Drosophila melanogaster produced, chrom. = 12k micrometres long (corresponded to tot. amount of DNA known in D. melanogaster chrom., so at least one chrom. contains very long DNA molecule, which was linear rather than circular)

Gel electrophoresis

used to sep. proteins/DNA fragments (neg.ly charged bc. PO4 group! - why pos. lysine binds to it, which acetyl can reduce) acc. to size, involves sep.ing charged molecules in electric field acc. to size/ charge. Contain samples placed in wells cast in gel immersed in conducting fluid, electric field is applied --> charged molecules in sample will move through gel, neg.ly/ pos.ly charged molecules will move in opp. directions. Proteins = may be pos./neg. charged so can be sep.d acc. to charge. Gel used = consists of mesh of filaments resisting mov. of molecules in sample. Euk. DNA molecules = too long to move through gel so has to be broken up into smaller fragments (using ex. endonucleases + DNA polym.). All DNA molecules = carry neg. charges --> move in same direction during gel electrophoresis, but not at same rate (small fragments move faster so move further in given time = method can be used to sep. DNA fragments acc. to size) --> banding pattern characteristic of pers.

Pedigree charts

way to visualise monohybrid crosses as knowing phenotypes in this case can reveal genotypes, can be used to deduce inheritance patterns w. MALES shown as SQUARES, FEM.S as CIRCLES, shaded/ cross-hatched to indicate if individual is affected by disease, parents-children = linked by T (top-bar between parents), roman numerals indicate generation, Arabic fig.s for each individual in gen.; enables gen. counselling for risk for disease in offspring, can be used to determine sex-linkage and dominance/ recessiveness in genes w.o. gene-testing.

Genome

whole of gen. info. of org. Gen. info. = contained in DNA, so genome = entire base seq. of each of living org.'s DNA molecules In hum. genome = 46 molecules forming chrom.s in nucleus + DNA molecules in mitochondria, is same pattern in othr animals but no. of chrom.s may differ. Plant species' genomes = DNA molecules of chrom.s in nucleus + chloroplast DNA + mitochondrion DNA Prok.s = genome is much smaller, consists of DNA in nucleoid region + any plasmids present