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CHAPTER 12 The Reproduction of Cells CELL DIVISION: terminology genome: total genetic information in a cell prokaryotic genome = often a single DNA molecule eukaryotic genome = usually includes many DNA molecules (each DNA molecule contains 100-1000s of genes) CELL DIVISION: terminology replication and distribution of DNA is manageable becase it is packed into chromosomes chromo- = color some- = body CELL DIVISION: terminology every eukaryotic species has characteristic chromosome number in nucleus SOMATIC CELLS = body cells (2n) GAMETES = sperm, egg (1n) CELL DIVISION: terminology DNA is associated with various proteins that maintain structure CHROMATIN = DNA-protein complex --normally, a long thin strand --however, it condenses after duplication to prepare for division CELL DIVISION: terminology each duplicated chromosome has 2 sister CHROMATIDS each chromatid goes to separate cell during division CELL DIVISION: terminology MITOSIS-division of nucleus CYTOKINESIS-division of cytoplasm MEIOSIS—production of gametes (occurs only in ovaries & testes) FERTILIZATION-fusion of gametes CELL CYCLE MITOTIC (M) PHASE usually shortest part of cell cycle INTERPHASE much longer (90% of cell cycle) CELL CYCLE G1 = Gap 1…growth S = synthesis of DNA G2 = Gap 2…growth, preparation for cell division M = mitosis (or meiosis) ***cell grows by producing protein and forming organelles*** CELL CYCLE: interphase 1. 2 centrosomes present, each with a pair of centrioles 2. microtubules extend from centrosomes, forming radial array (asters) CELL CYCLE: prophase 1. chromatin more tightly coiled discrete chromosomes 2. nucleoli disappear 3. mitotic spindle forms 4. centrosomes migrate away INTERPHASE CELL CYCLE: prometaphase 1. nuclear envelope fragments 2. microtubules “invade” to interact with chromosomes 3. each chromatid has a kinetochore 4. some microtubules attach to kinetochores CELL CYCLE: metaphase 1. centrosomes now at opposite poles 2. chromosomes align at metaphase plate 3. all kinetochores attached to microtubules CELL CYCLE: anaphase 1. paired centromeres of chromosomes separate 2. chromosomes move toward opposite poles 3. poles of cell move further apart CELL CYCLE: telophase 1. nonkinetochore microtubules lengthen cell further 2. daughter nuclei form at poles 3. nuclear envelope arises from existing fragments 4. chromatin becomes less coiled 5. cleavage furrow develops CELL CYCLE: finer points each of the 2 joined chromatids has a kinetochore kinetochore = structure of proteins and chromosomal DNA at centromere CELL CYCLE: finer points What is the function of nonkinetochore microtubules? in animal cell, elongate cell during anaphase (motor proteins drive microtubules past each other, using ATP) CYTOKINESIS in animal cells, occurs by process known as cleavage first sign is appearance of cleavage furrow (begins near old metaphase plate) PLANT CELL DIVISION no cleavage furrow during telophase, vesicles derived from the Golgi apparatus move along microtubules to the middle of the cell where they coalesce, producing a cell plate cell plate enlarges until its surrounding membrane fuses with the plasma membrane along the perimeter of the cell 2 daughter cells result…each with its own plasma membrane checkpoints a checkpoint in the cell cycle is a critical control point where stop and go-ahead signals can regulate the cycle generally built-in stop signals halt the cycle until overridden by go-ahead signals major checkpoints are found in G1, G2, and M phases for many cells, G1 checkpoint seems to be most important (if no G1 signal is received, cell exits cycle into nondividing state known as G0) cancer cells escape cell cycle control tumor: mass of abnormal cells within otherwise normal tissue benign tumor: abnormal cells remain at original site malignant tumor: becomes invasive enough to to impair the functions of one or more organs metastasis: spread of cancer cells to locations distant from their original site Chapter 13 Meiosis and Sexual Life Cycles heredity transmission of traits from one generation to the next --offspring resemble parents --however, there is variation genome whole complement of genes gene passed as hereditary units coded information segments of DNA --polymer of 4 different kinds of monomer (nucleotides) --information is passed in form of sequences of nucleotides --most genes are programs for the synthesis of enzymes or other proteins one chromosome = 100s – 1000s of genes LOCUS = specific location on chromosome ASEXUAL REPRODUCTION single individual is sole parent all genes are passed to offspring (changes can occur due to mutation) FORMS: mitosis, budding budding SEXUAL REPRODUCTION two parents give rise to offspring unique combination of genes in offspring inherited from parents SOMATIC CELL any cell other than sperm or ovum in humans, each somatic cell has 46 chromosomes chromosomes can be separated based on: (1.) length (2.) position of centromere (3.) banding patterns KARYOTYPE micrograph of organized chromosomes homologous paired chromosomes are GAMETES in humans, each has single set of 22 autosomes + 1 sex chromosome union of gametes called fertilization or syngamy zygote as zygote develops, genes are passed on precisely to all somatic cells thanks to mitosis MEIOSIS I-INTERPHASE chromosomes replicate sister chromatids (b.) centrosomes replicate (a.) MEIOSIS— Overview MEIOSIS I— PROPHASE (a.) chromosomes condense (b.) homologous chromosomes pair (c.) SYNAPSIS = homologous chromosomes attached to one another --appears as tetrad (cluster of 4 chromatids) --crossing-over occurs (chiasmata) MEIOSIS I— PROPHASE (d.) centrosomes migrate to poles (e.) nuclear membrane & nucleolus disperse (f.) spindle fibers capture kinetochores (g.) chromosomes move toward metaphase plate MEIOSIS I— METAPHASE chromosomes line up at metaphase plate (in homologous pairs) MEIOSIS I— ANAPHASE (a.) sister chromatids remain connected and move toward poles (b.) homologous chromosomes move toward opposite poles MEIOSIS I— TELOPHASE cytokinesis and telophase occur simultaneously MEIOSIS I MEIOSIS II THREE EVENTS UNIQUE TO MEIOSIS 1. SYNAPSIS 2. homologous chromosomes (not sister chromatids) pair at metaphase plate 3. sister chromatids do not separate at anaphase I. SOURCES OF GENETIC VARIATION A. independent assortment --alignment of chromosomes at metaphase plate is random --# of possible combinations at metaphase = 2n (n = haploid #) --223 (~8 million) possible combinations in humans independent assortment SOURCES OF GENETIC VARIATION B. crossing over --produces recombinant chromosomes, which combine genes inherited from mother and father --begins early in prophase I --in humans, 2-3 crossovers occur per chromosome pair --at metaphase II, chromosomes can be oriented in nonequivalent ways crossing over SOURCES OF GENETIC VARIATION C. random --ovum (~8 million) + fusion of gametes sperm (~8 million) zygote (1 of ~64 million) CHAPTER 14 Mendel and the Gene Idea Mendel brought experimental and quantitative approach CHARACTER: heritable feature (i.e. flower color, height, etc.) TRAIT: variant of color (i.e. purple vs. white flower, tall vs. short plant) Mendel and the pea probably chose garden peas because they come in many varieties use of peas gave Mendel strict control (they usually self-fertilize) --each flower has male and female organs Mendel’s process only tracked “either-or” traits (no traits with a continuum) started experiments with true-breeding (pure) plants pure = all offspring are the same when the plants self-pollinate Mendel’s process HYBRIDIZATION = mating (crossing) of two true-breeding varieties P generation F1 generation F2 generation P = parental F = filial (son) Mendel’s process Mendel used very large sample sizes he also kept very accurate records observed patterns in flower color, along with 6 other characters Ex. P: F1: F2: purple flower x white flower purple 3 purple : 1 white Modern explanation of F2 ratio (1.) alternative versions of genes (alleles) account for variations in inherited characters --DNA within locus can vary in sequence of nucleotides (2.) for each character, an organism inherits 2 alleles, one from each parent (Mendel didn’t know about chromosomes) (3.) if 2 alleles differ, the dominant allele is fully expressed; the recessive allele has no noticeable effect (4.) the two alleles for each character segregate during gamete production = LAW OF SEGREGATION Terms homozygous: pair of identical alleles heterozygous: 2 different alleles (not true-breeding) phenotype: organism’s traits (appearance) genotype: genetic makeup testcross: recessive homozygote x dominant phenotype w/ unknown genotype Terms Mendel developed Law of Segregation by following a single character monohybrid: F1 organism produced by cross dihybrid cross: looks at 2 traits (are traits independent, or are they inherited as a package?) --results in 9:3:3:1 ratio Terms Law of Independent Assortment independent segregation of each pair of alleles during gamete formation Probability Scale from 0 to 1 (1 = certain to occur, 0 = certain not to occur) Ex. coin… ½ heads; ½ tails probabilities of all possible outcomes must add up to 1 each coin toss is an independent event (compare coin toss to gamete formation) Rule of Multiplication probability x each = of each probability independent event probability of series of events Ex. probability of 2 heads = ½ x ½ = ¼ Ex. Probability of a white flower Rule of Addition probability of an event that can occur in 2 or more different ways is the sum of the separate probabilities Ex. probability of having a heterozygous individual in the F2 Mendel’s impact since Mendel’s study of peas, his principles have been extended to diverse organisms (most with patterns of inheritance far more complex than peas) for Mendel, each character was determined by one gene (with complete dominance) the relationship between genotype and phenotype is rarely simple Using probability PpYyRr x Ppyyrr Incomplete dominance F1 hybrids have intermediate phenotype Ex. CRCR x CWCW (red) (white) Incomplete dominance Codominance two alleles affect the phenotype in separate, distinguishable ways Ex. human blood groups (surface proteins on RBCs) M = one type N = another type MN = produces both proteins Codominance Codominance Tay-Sachs brain cells of baby are unable to metabolize gangliosides because crucial enzyme doesn’t work properly as lipids accumulate in brain, brain cells gradually cease to function, leading to death Tay-Sachs organismal level: allele is recessive (need 2 copies to have disease) biochemical level: intermediate phenotype with incomplete dominance (heterozygotes lack symptoms) molecular level: produces equal amounts of normal and dysfunctional enzyme molecules (illustrates codominance) Dominant alleles do not subdue recessive alleles (they don’t interact at all) Ex. round vs. wrinkled pea seeds R = codes for synthesis of enzyme that helps convert sugar to starch in seed r = codes for defective form of enzyme ***in recessive homozygote, sugar accumulates in the seed; as seed develops, high sugar concentration causes osmotic uptake of H2O Multiple alleles most genes in populations have more than 2 allele types Ex. ABO blood groups (A, B, AB, O) --letters refer to carbohydrates on surface AB A B O Other patterns pleiotrophy—ability of a gene to effect an organism in many ways Ex. gene for sickle cell anemia multiple symptoms epistasis—gene at one locus alters the phenotypic expression of a gene at a second locus polygenic inheritance --quantitative characters; usually vary along continuum --2 or more genes have additive effect --Ex. skin pigment, height epistasis polygenic inheritance Nature vs. Nurture plants—sun, wind, and water can affect the expression of genes humans—nutrition height exercise body shape tanning skin color experience intelligence twins have phenotypic differences due to different experiences product of genotype is not a rigidly defined phenotype most traits are multifactorial (both genetic and environmental influences) Human disorders humans are not convenient subjects for genetic research --long generation times --few offspring --breeding experiment unacceptable to study humans, the results of previous matings must be studied --family histories are used to assemble pedigrees --Ex. widow’s peak, attached earlobes pedigrees not only help us understand the past, they help us predict the future Human disorders 1000s with simple recessive traits --Ex. albinism, CF genes code for proteins of a specific function --allele that codes for genetic disorder codes for malfunctional form of protein, or no protein at all disorder only in homozygous recessive individuals --heterozygotes are carriers Human disorders most genetic disorders are unevenly distributed in populations Ex. cystic fibrosis --1/2500 of European white descent have disease --1/25 whites are carriers normal allele codes for membrane protein that functions in Cl- transport between cells and ECF defective allele results in defective protein which allows abnormally high extracellular [Cl-] thick coating of mucus Human disorders most genetic disorders are unevenly distributed in populations Ex. Tay-Sachs --1/3500 births of Ashkenazic Jewish population dysfunctional enzyme fails to breakdown class of brain lipid symptoms: seizure, blindness, decreased motor and mental performance…death within years Human disorders most genetic disorders are unevenly distributed in populations Ex. sickle-cell disease --1/400 births among African-Americans caused by single amino acid substitution in Hb protein of RBCs If O2 content is low, sickle-cell HB crystallizes into long rods (crystals deform RBCs into sickle shape) example of pleiotrophy treated with regular transfusions Human disorders DOMINANT ALLELES Ex. Achondroplasia (Dd) --rest of population (dd) lethal dominants less common than lethal recessives --arise by mutation in sperm or egg --if kills offspring before reproduction, won’t be passed on lethal recessives can escape elimination if they are late-acting --Ex. Huntington’s disease --no phenotypic evidence until age 35-45 genetic testing Multifactorial disorders genetic + environmental components heart disease diabetes cancer alcoholism manic-depression CHAPTER 15 The Chromosomal Basis of Inheritance SEX-LINKED GENES genes located on sex chromosome for males, single copy of mutant allele yields mutant phenotype sex-linked inheritance LINKED GENES genes located on the same chromosome seem to be inherited together the number of genes in a cell are far greater than the number of chromosomes linked genes in Drosophila GENETIC RECOMBINATION general term for production of offspring with new combinations of traits Ex. YyRr x yyrr Ex. if 50% of all offspring are recombinants, said to be 50% frequency of recombination 50% frequency of recombination is observed for genes that are located on different chromosomes LINKED GENES do not assort independently because they are located on the same chromosome (they move together through meiosis and fertilization) if genes are linked, should see 1:1:0:0 ratio if ratio varies, indicates recombination occurred crossing over = mechanism for breaking linkage (non-sister chromatids break at corresponding points and swap fragments) production of recombinant gametes production of recombinant offspring Linked genes led Alfred Sturtevant, one of Mendel’s students, to construct a genetic map. map = ordered list of loci along a particular chromosome hypothesis: calculated recombination frequencies from experiments reflects actual distances between genes on chromosomes (farther apart increased chance of crossing over) 1 map unit = 1% recombination frequency max % = 50% (cannot distinguish from independent assortment) genetic map genetic map Linkage maps, etc. a linkage map cannot be used to put together a scaled model of the chromosome (provides the order of the genes, but not precise locations) CYTOLOGICAL MAPS =locate genes with reference to markers on chromosomes Sex chromosomes although they behave as homologs, there is very little crossing over In humans, anatomical signs of sex begin at ~2 months for embryo (before that point, gonads are generic) 1990—SRY gene discovered --if expressed testes --if no SRY ovaries --codes for protein that regulates many other genes sex determination Sex chromosomes also carry genes unrelated to sex (mostly applies to X chromosome) Fathers daughters Mothers sons and daughters if sex-linked trait is recessive, female will only express it if she is homozygous since males only have one X, homo/heterozygous does not apply Sex-linked disorders color blindness Duchenne muscular dystrophy --1/3500 males in U.S. --progressive weakening of muscles and loss of coordination (rarely survive early 20s) --lack key muscle protein = dystrophin hemophilia --absence of one or more proteins required for blood clotting --history… X-INACTIVATION in females, one X chromosome in each cell becomes inactivated early in embryonic development inactive X compacts into a small object known as a Barr body --a few genes remain active; most do not selection of which X becomes inactive is totally random --thus, females have a mosaic of two types of cells (½ with active X from mother, other ½ from X-inactivation ERRORS AND EXCEPTIONS IN CHROMOSOMAL INHERITANCE NONDISJUNCTION members of a pair of homologous chromosomes (a.) do not move apart properly during meiosis I, or (b.) sister chromatids fail to separate during meiosis II as a result, one gamete receives 2 copies of the same chromosome, while the other gamete receives no copy (other chromosomes usually distribute normally) nondisjunction ERRORS AND EXCEPTIONS IN CHROMOSOMAL INHERITANCE NONDISJUNCTION if one of these gametes combines with another gamete, result is ANEUPLOIDY (an abnormal chromosome number) 3x (2n + 1) = trisomic 1x (2n – 1) = monosomic (mitosis then transmits the anomaly to all embryonic cells) if organism survives, usually has a set of symptoms due to an abnormal dose of genes ERRORS AND EXCEPTIONS IN CHROMOSOMAL INHERITANCE POLYPLOIDY organism has more than 2 complete chromosome sets 3n = triploidy --due to nondisjunction of all chromosomes + normal gamete 4n = tetraploidy --2n zygote fails to divide after replicating its chromosomes; mitosis 4n polyploidy is fairly common in plants…extremely rare in animals ALTERATIONS OF CHROMOSOME STRUCTURE breakage of chromosome can lead to 4 possible changes: 1. deletion = chromosomal fragment lacking a centromere is lost (chromosome will be missing genes) 2. duplication = chromosome fragment may become attached as an extra segment to a sister chromatid 3. inversion = chromosome fragments reattach in reverse orientation 4. translocation = fragment joins nonhomologous chromosome alterations of chromosome structure ALTERATIONS OF CHROMOSOME STRUCTURE a 2n embryo that is homozygous for a large deletion (or a single X with a large deletion in males) is usually missing many essential genes and is therefore lethal HUMAN DISORDERS aneuploidy—results are usually so devastating that spontaneous early abortion occurs ALTERATIONS OF CHROMOSOME STRUCTURE HUMAN DISORDERS (cont.) Down syndrome—1/700 children born in U.S. --extra chromosome 21 (trisomy 21) --severely alters phenotype (characteristic facial features, short stature, heart defects, mental retardation) --prone to leukemia and Alzheimer’s --most are sexually undeveloped and sterile --frequency correlates with age of mother Down syndrome ALTERATIONS OF CHROMOSOME STRUCTURE HUMAN DISORDERS (cont.) nondisjunction of sex chromosomes seems to upset genetic balance less (Y has relatively few genes, females can deactivate faulty X) ALTERATIONS OF CHROMOSOME STRUCTURE XXY (male) – Klinefelter’s Syndrome (1/2000 births) --male sex organs --breast enlargement --normal intelligence --abnormally small testes, sterile XYY (male) – no well defined syndrome (usually taller than average) ALTERATIONS OF CHROMOSOME STRUCTURE XXX (female) – (1/1000 births) --healthy --cannot be distinguished from other females except by karyotype XO (female) – Turner Syndrome (1/5000 births) --only known viable monosomy in humans --sex organs do not mature; sterile --hormones can produce normal secondary sexual characteristics --normal intelligence ALTERATIONS OF CHROMOSOME STRUCTURE HUMAN DISORDERS (cont.) chromosome 5 deletion = “cri du chat” --mentally retarded --small head --unusual facial features --unusual mewing --die in infancy or early childhood ALTERATIONS OF CHROMOSOME STRUCTURE GENOMIC IMPRINTING expression of trait depends on which parent passed along the allele --Prader-Willi: mental retardation, obesity, short, small hands and feet (chromosome from father) --Angelman: laughter, jerky movements, motor / mental symptoms (chromosome from mother) genomic imprinting Chapter 16 The Molecular Basis of Inheritance DNA structure: history Griffiths—studying Strep. pneumo. in 1928 --2 strains: (a) pathogenic (b) harmless variant --process: (1.) heat-killed pathogenic strain (2.) mixed remains with living harmless variant (3.) some of the cells were converted to the pathogenic strain (4.) pathogenicity was inherited by all offspring of transformed bacteria TRANSFORMATION: change in genotype and phenotype due to assimilation of external DNA Griffith’s experiment DNA structure: history Avery—sought to identify pathogenic substance --process: (1.) purified chemicals from heatkilled pathogenic bacteria (2.)tested each to see if it was the “transforming” substance (3.) only DNA worked still much doubt in scientific community: --bacterial DNA --little was known about DNA --most still believed that protein was the source DNA structure: history viruses that could infect bacteria were studied these viruses are called bacteriophages, or just phages --to infect, a virus must take over the cell’s reproductive machinery --viruses that infect bacteria (bacteriophages) are widely used in molecular genetics DNA structure: history bacteriophages protein coat DNA DNA structure: history Hershey & Chase = process: (1.) used different radioactive isotopes to tag phage DNA and protein (2.) T2 with E. coli in radioactive S (radioactive atoms incorporated into protein) (3.) T2 with E. coli in radioactive P (radioactive atoms incorporated into DNA) (4.) each set of labeled samples allowed to infect cells (5.) after infection, samples whirled in a blender to shake off loose phage parts (6.) centrifuged Hershey & Chase experiment DNA structure: history other circumstantial evidence: --before mitosis, cell doubles DNA --during mitosis, amount of DNA divided equally Chargaff = DNA is polymer of nucleotides --composition: (1) nitrogenous base (A,T,G,C) (2) pentose sugar (3) phosphate group --DNA composition varies between species, but bases are always present in a characteristic ratio DNA structure DNA structure: history Watson --helical --width of helix = 2 strands (double helix) --spacing of N-bases vision of rope ladder with rigid rings helix makes full turn every 3.4 nm (10 layers of base pairs / turn of helix) at first, Watson thought “liked paired with like” DNA structure: history purines = adenine, guanine (2 organic rings) pyrimidines = cytosine, thymine (1 organic ring) Since width of helix is always uniform, must be formed by PURINE + PYRIMIDINE A = T 2 H-bonds C = G 3 H-bonds DNA structure DNA structure: history REPLICATION MODELS conservative— parental double helix remains intact and an all-new copy is made semi-conservative—two strands of parental molecule separate; each functions as a template for synthesis of a new complimentary strand dispersive—each strand of both daughter molecules contains a mixture of old and newly synthesized parts DNA structure: history REPLICATION MODELS DNA structure: history REPLICATION MODELS Meselson & Stahl (late 1950s) DNA replication: basics takes just a few hours to copy human DNA 6 billion bases in one cell ~ compares to letters in 900 books as thick as the AP Biology text extremely low error rate ~ 1 per billion nucleotides DNA replication: step 1 begins at origins of replication (specific nucleotide sequence) --circular bacterial chromosome has 1 origin --eukaryotic cells have 1000s once sequence is recognized, replication “bubble” opens DNA replication: step 1 replication proceeds in both directions replication fork at each end of “bubble” DNA replication: step 2 DNA polymerases catalyze elongation of new DNA strand --rate: 500 bases / sec in bacteria 50 bases / sec in humans energy for process derived from nucleotide triphosphates --hydrolysis of pyrophosphate drives polymerization --structure of nucleotide triphosphates nearly identical to ATP (use deoxyribose instead of ribose) DNA replication: step 3 2 DNA strands are anti-parallel --sugar-phosphate backbones run in opposite directions 3’ OH– o o o o o o o o o o o o o o o o—P 5’ --new DNA strands elongate only in the 5’ 3’ direction (nucleotides are only added to the 3’ end) DNA replication: step 3 DNA replication: step 3 DNA replication: step 3 (cont.) 2 approaches: strand 1: LEADING STRAND synthesizes 5’ 3’ (continuous) strand 2: LAGGING STRAND synthesizes short fragments of DNA --known as Okazaki fragments --100-200 nucleotides long --joined by DNA ligase DNA replication: step 3 (cont.) DNA replication: step 4 DNA polymerases cannot initiate synthesis (can only add nucleotides) start of a new chain (primer) is a short stretch of RNA --primase joins RNA nucleotides to make primer (~10 nucleotides long) --DNA polymerase eventually replaces the RNA --leading strand only needs one primer; each Okazaki fragment needs its own primer (primers are converted to DNA before ligase joins the fragments) DNA replication: step 4 DNA replication: step 5 other proteins involved in replication: helicase—untwists double helix at replication fork single-strand binding protein—lines up unpaired strands, holding them apart while they serve as templates (most models suggest that replication proteins are stationary and “reel in” DNA DNA replication: step 5 DNA replication: summary DNA replication: step 6 proofreading enzymes initial error rate is 1 in 10,000 base pairs (a.) DNA polymerase proofreads as it adds each base --if an incorrect nucleotide is found, it is immediately replaced (b.) mismatch repair—used if error evades DNA polymerase or occurs after replication is complete --special proteins used to make corrections --error in correction protein leads to one form of colon cancer DNA replication: step 6 (cont.) proofreading enzymes (c.) maintenance requires frequent repair due to environmental damage nucleotide excision repair --usually, damaged segment is cut out by nuclease --resulting gap is filled with proper bases DNA replication: step 6 (cont.) DNA replication: step 7 ends of DNA molecules --usual replication machinery provides no way to complete the 5’ ends of daughter DNA strands --as a result, each round of replication produces shorter and shorter DNA molecules --eventually, essential genes would be deleted telomeres = multiple short, repeated sequences DNA replication: step 7 Chapter 17 From Gene to Protein Genes provide the instructions for making specific proteins. nucleic acids and proteins can be seen as two different languages TRANSCRIPTION = synthesis of RNA under direction of DNA (use same language) --just as DNA provides template for replication, it provides a template for transcription --messenger RNA carries genetic message from DNA to protein-synthesizing machinery of the cell Genes provide the instructions for making specific proteins. nucleic acids and proteins can be seen as two different languages TRANSLATION = synthesis of protein under direction of mRNA --change in language: nucleic acid sequence must be converted to amino acid sequence of polypeptide --ribosomes are sites of translation (they facilitate linking of amino acids) transcription / translation in prokaryotes in eukaryotes Potential problem: only 4 nucleotides (A, C, G, T) available to code for 20 amino acids 1 base code 4 amino acids 2 base code (4)2 16 amino acids 3 base code (4)3 64 amino acids triplet code = genetic instructions for a polypeptide chain are written in the DNA as a series of 3nucleotide words triplet code TRANSCRIPTION DNA mRNA transcription is an intermediate step: --only one of the 2 DNA strands is transcribed = TEMPLATE STRAND --mRNA molecule is complimentary to its DNA template (not identical) TRANSLATION mRNA tRNA protein CODON = mRNA base triplets --Ex. UGG tryptophan --codons are read in 5’ 3’ direction along mRNA --takes 300 nucleotides along an RNA strand to code for polypeptide that is 100 amino acids long 1960s: Nirenberg deciphered first codon (UUU phenylalanine)…AAA, CCC, GGG followed --all codons deciphered by mid 1960s --AUG = start --redundancy (but no ambiguity) table of mRNA codons READING FRAME Ex. 5’--the red dog ate the cat—3’ Ex. 5’—t her edd oga tet hec at—3’ The genetic code is nearly universal shared by range of organisms from simple bacteria to complex plants and animals --genes can be transplanted between species and still successfully undergo transcription and translation --a few species, such as Paramecium, have a few codons that differ near universality provides evidence that the common language has been present since very early in the history of life TRANSCRIPTION SYNTHESIS & PROCESSING OF RNA --RNA polymerase pries apart DNA strands --combines RNA nucleotides as they are added along the DNA template --just like DNA, RNA elongates in a 5’ 3’ direction TRANSCRIPTION—overview TRANSCRIPTION 1. INITIATION promoter (upstream) --DNA sequence where RNA polymerase attaches and initiates transcription --determines (a) actual start point and (b) which strand will serve as template terminator (downstream) --sequence that signals the end of transcription TRANSCRIPTION 1. INITIATION TRANSCRIPTION prokaryotes = RNA polymerase recognizes and binds to promoter eukaryotes = require RNA polymerase + transcription factors (called transcription initiation complex) 2. ELONGATION --as RNA polymerase moves along DNA template, ~10-20 bases are exposed --enzyme adds nucleotides to the growing 3’ end of the RNA strand --~60 nucleotides are added per sec. in eukaryotes --many RNA polymerases can work simultaneously TRANSCRIPTION 3. TERMINATION --transcription proceeds until after RNA polymerase transcribes a terminator sequence --prokaryotes = transcription stops right at end of termination signal --eukaryotes = transcription carries on 100s of nucleotides past terminator TRANSCRIPTION 4. MODIFICATION OF RNA AFTER TRANSCRIPTION --enzymes in the nucleus modify pre-mRNA before releasing it 5’ = immediately capped with a modified form of a guanine nucleotide --protects mRNA from degradation by hydrolytic enzymes --once in cytoplasm, acts as attachment sign for ribosome 3’ = enzyme makes poly (A) tail with 50-250 adenine nucleotides --inhibits degradation; helps ribosome attach TRANSCRIPTION 4. MODIFICATION OF RNA AFTER TRANSCRIPTION TRANSCRIPTION 5. RNA SPLICING --average length of transcription unit in eukaryotic DNA = 8000 nucleotides --takes only ~1200 nucleotides to code for average protein (means that there are long non-coding sequences) --non-coding sequences (INTRONS) are mostly interspersed between coding sequences (EXONS) Signals for RNA splicing are short nucleotide sequences at the end of introns --particles called small nuclear nibonucleoproteins (snRNPs or snurps) recognize these sequences --several snRNPs join with additional proteins to form a spliceosome TRANSCRIPTION 5. RNA SPLICING TRANSCRIPTION snRNPs TRANSCRIPTION WHY HAVE INTRONS? --introns may play a regulatory role in cell --alternative RNA splicing + depending on which segments are spliced, several different proteins can result + may be reason why humans get by on relatively few genes (only 2x more than flies) --may also facilitate evolution of new proteins + proteins often have discrete regions = domains + introns increase the probability of useful crossover exons (expressed genes) proteins TRANSLATION the message in codons along mRNA is interpreted by tRNA --tRNA functions to transfer amino acids to a ribosome cell keeps cytoplasm stocked with all 20 amino acids by (a) synthesizing them (b) taking them up from the surrounding solution molecules of tRNA are not identical --each type of tRNA links a particular mRNA codon with a particular amino acid TRANSLATION TRANSLATION as a tRNA molecule arrives at a ribosome, it bears a specific amino acid at one end at other end is a nucleotide triplet called ANTICODON --anticodon base pairs with complementary codon on mRNA codon by codon, the genetic message is translated as tRNAs deposit amino acids in the order prescribed ribosome joins the amino acids into a chain TRANSLATION PROKARYOTE = tRNA transcribed from DNA template EUKARYOTE = tRNA produced in nucleus, transported to cytoplasm --in both types of cells, tRNA molecules are used repeatedly (pick up amino acid…deliver to ribosome…repeat) TRANSLATION tRNA molecle is a single RNA strand, only ~80 nucleotides long (most mRNA are 100s of nucleotides long) molecule folds to form clover-shaped structure (when flattened…it’s actually more L-shaped in 3D) --held together by H-bonds between complimentary bases TRANSLATION TRANSLATION TRANSLATION if there was one tRNA for each codon, there would be ~61 tRNA (in reality there are about 45) some tRNA have anticodons that can recognize 2 or more different codons versatility is possible because rules for base-pairing are not as strict as those for DNA and mRNA codons (called wobble) TRANSLATION Ex. U on tRNA anticodon can pair with A or G in 3rd position of mRNA codon most versatile tRNA are those with inosine (I) in 3rd position --I can associate with U, C, or A --CCI can bind to GGU, GGC, or GGA wobble explains why codons for the same base can differ in the 3rd base, but not in another position TRANSLATION aminoacyl-tRNA synthetases --correct match between tRNA and amino acid must occur before codon and anticodon can match up --each amino acid is joined to correct tRNA by aminoacyl-tRNA synthetase --20 different enzymes…one for each amino acid --enzyme active site fits only a specific combination of tRNA and amino acid --enzyme catalyzes covalent bonding of amino acid to tRNA (driven by hydrolysis of ATP) TRANSLATION aminoacyl-tRNA synthetases RIBOSOMES facilitate coupling of mRNA and tRNA have 2 subunits: large and small constructed of protein and rRNA (in eukaryotes, subunits are made in nucleolus) rRNA gene on chromosomal DNA transcribed --RNA processed, then assembled with proteins imported from cytoplasm --ribosomal subunits are exported via nuclear pores RIBOSOMES large and small subunits combine to form functional ribosomes only when they attach to mRNA prokaryote and eukaryote subunits differ in size and molecular compositions --useful in medicine…drugs like tetracycline and streptomycin can paralyze prokaryotic ribosomes without effecting eukaryotic ribosomes rRNA is most abundant type of RNA RIBOSOMES P-site = holds tRNA carrying growing polypeptide chain A-site = holds tRNA carrying next amino acid to be added to chain E-site = discharged tRNAs leave ribosome RIBOSOMES BUILDING A POLYPEPTIDE 3 stages analogous to transcription: (1) initiation (2) elongation (3) termination all 3 require protein “factors” to aid process energy required to elongate chain provided by hydrolysis of GTP BUILDING A POLYPEPTIDE INITIATION --brings together mRNA, tRNA with first amino acid, and 2 ribosomal subunits 1. small subunit binds to mRNA + special initiators tRNA 2. downstream from leader is initiation codon, AUG, which signals start of translation 3. large subunit joins mRNA, initiator tRNA, small ribosomal subunit 4. initiator tRNA sits in P site; vacant A site is ready for next aminoacyl-tRNA BUILDING A POLYPEPTIDE INITIATION BUILDING A POLYPEPTIDE ELONGATION --amino acids are added one by one to the preceding amino acid --each addition involves several elongation factors (a.) codon recognition (b.) peptide bond formation (c.) translocation BUILDING A POLYPEPTIDE ELONGATION BUILDING A POLYPEPTIDE TERMINATION --translation continues until mRNA stop codon reaches A site --release factor binds to codon in A site --causes addition of water instead of amino acid --hydrolyzes completed polypeptide from tRNA BUILDING A POLYPEPTIDE TERMINATION POINT MUTATIONS mutation—change in genetic material of a cell (or virus) point mutation—chemical change in one base pair if a point mutation occurs in a gamete or in a cell that gives rise to gametes, it may be transmitted to offspring and to a succession of future generations – – if the mutation has an adverse effect on the phenotype of a human or other animal, the mutant condition is referred to as a genetic disorder Ex. sickle-cell anemia POINT MUTATIONS TYPES OF POINT MUTATIONS SUBSTITUTION – – – – replacement of one nucleotide and its partner in the complimentary DNA strand with another pair of nucleotides silent mutations—due to redundancy of genetic code, have no effect on the encoded protein Ex. CCG (mRNA GGC) changes to CCA (mRNA GGU)…glycine results in both cases substitutions of interest are those that cause a detectible change in the protein occasionally leads to an improved product or one with new, unique capabilities usually fit into category of missense mutations—the altered codon still codes for an amino acid, but not the right one nonsense mutations can also result—where the alteration changes the codon to a stop codon TYPES OF POINT MUTATIONS INSERTION and DELETION – – – additions or loses of nucleotide pairs in a gene effects are usually more significant than substitutions frameshift mutation results—reading frame is altered, causing a number of incorrect amino acids to be produced TYPES OF POINT MUTATIONS MUTAGENS interact with DNA to cause mutations Ex. X-rays, radiation, chemicals summary— transcription and translation