Study questions for the lectures 1-4

Transcription

1 Study questins fr the lectures What infrmatin(s) culd yu btain frm a genetic apprach f studying mutants defective in a particular prcess? [L: 1, S: 9] The ptential functin f the prtein f interest and hw imprtant the prtein is fr survival thrugh identificatin/islatin/survival f MUTANTS The number f genes invlved in certain prcesses The rder f these genes (prteins) in the prcess and their relatins by applying a genetic apprach called COMPLEMENTATION ANALYSIS The interactins between different genes (prteins) by using genetic apprach called GENETIC SUPPRESSION 2. Hw wuld yu define permissive cnditins in respect t temperature sensitive mutants? [L: 1, S: 7-8] Permissive cnditins: cnditins under which a cnditinal mutant gene prduct can functin (ex. The permissive temperature fr a temperature-sensitive mutant) Permissive cnditins in respect t temperature-sensitive mutants is the temperature at which a temperature-sensitive mutant gene prduct takes n a nrmal, functinal phentype; the mutant gene prduct behaves nrmally, even thugh there is a mutant allele present 3. Define (r cmpare and cntrast): a) gene expressin; transcriptin; replicatin; translatin; b) gene; allele [textbk/internet] a) Gene expressin: the prcess by which gene prducts (RNA transcripts and prteins) are made frm the instructins encded in DNA Includes bth transcriptin and translatin Transcriptin: the prcess by which an RNA cpy f a gene is made (DNA RNA) Replicatin: the prcess by which the DNA duble helix unwinds and makes an exact cpy f itself (1 DNA 2 DNA) Translatin: the prcess by which ribsmes decde mrna t synthesize a prtein (RNA prtein)

2 b) Gene: the basic unit f heredity; a cmplete chrmsmal segment respnsible fr making a functinal prduct Allele: ne f a number f alternative frms f a given gene (r DNA sequence) that can ccupy a given genetic lcus (lcatin) n a chrmsme Ex. The gene (DNA sequence) cding fr eye clur has several variatins (alleles), such as alleles that cde fr blue eyes and alleles that cde fr brwn eyes 4. Explain by using yur wn wrds the meaning/significance f gene expressin. Gene expressin: the prcess by which gene prducts (RNA transcripts and prteins) are made frm the instructins encded in DNA This is imprtant because many cellular prcesses rely n these gene prducts (ex. prteins); prteins can act as enzymes which catalyze bilgical and chemical reactins 5. What are the rles f mdel rganisms in mlecular bilgy studies? Chse tw mdel rganisms and explain yur reasning. [L: 1, S: 14-17, textbk pg. 229] Mdel rganisms are simpler mdels used t study mre cmplex rganisms Mdel rganisms are useful fr medical research because they have specific characteristics that resemble a human disease r disrder Example 1: Drsphila Shrt life cycle (2 weeks) Ease f culture (easy t maintain) High prlificacy (females can lay up t 100 eggs/day) Example 2: Muse 85% f the DNA in mice is the same as humans Less than 1% f muse genes have n detectable hmlg in humans Physilgy is similar t that f humans (since mice are als mammals) 6. What are three main functins f DNA? Explain the imprtance f each f them. [L: 2, S: 7-17] 1. Stres infrmatin infrmatin (sequence f bases) is used in cding fr prteins and different RNAs, and used as regulatry signals

3 2. Replicates faithfully imprtant in the preservatin f genetic infrmatin a. DNA replicates semi-cnservatively, in which 2 strands f parental DNA separate and each serve as a template fr synthesis f a new daughter strand b. Infrmatin is preserved since ne strand predicts the sequence f the ther strand 3. Has ability t mutate imprtant in the variability f genetic infrmatin a. Mutatins can frm new alleles b. Mutatins in the cding sequence culd result in alteratins in the prtein prduct r cause n prduct frmatin (knckut) c. Mutatins in the regulatry sequence culd alter regulatin f expressin f the prduct 7. What is (are) the rle(s) f phsph-diester bnds in DNA structure? What is (are) the rle(s) f hydrgen bnds in DNA structure? What is (are) the rle(s) f hydrphbic interactins in DNA structure? [textbk pg ] Phsphdiester bnds link adjacent nucletides (f the same DNA strand) t frm a DNA chain (bnd between phsphate n the 5 carbn f sugar and OH grup at the 3 carbn n adjacent sugar) Hydrgen bnds frm between the nitrgenus bases n ppsite strands f the DNA chains; these bnds prvide ne type f frce that hlds the 2 DNA strands tgether Hydrphbic interactins in the DNA structure causes the highly negative (hydrphilic) phsphate backbne t face the utside f the DNA structure and the nn-plar (hydrphbic) bases t face the inside f the DNA structure 8. What nncvalent interactins are invlved in maintaining the duble-helical cnfrmatin f DNA? [L: 3, S: 3-6] Hydrgen bnds sharing f a hydrgen atm between 2 electrnegative atms (between adjacent bases n ppsite DNA strands); in DNA, H-bnds between A & T, and G & C Van der Waals interactins a weak electrical attractin between all types f mlecules (frming temprary diples between adjacent mlecules)

4 Hydrphbic interactins nn-plar grups (ex. Nitrgenus bases) are hydrphbic and thus hide frm water (causing them t aggregate t the inside f the DNA structure) NOTE: H-bnds, Van der Waals, and hydrphbic interactins are individually weak; but if there are lts f them between 2 mlecules, they becme very strng 9. Learn t recgnize nitrgenus bases (A,T,G,C,U) and respective nucletides. [L: 3, S: 9] RULES: Purines: 2 rings Pyrimidines: 1 ring Guanine has a =O n tp, adenine has a NH 2 Base with the O (guanine) will NOT bind t anther base with the same O (uracil/thymine) TIP: shrter wrd = bigger structure (and vice versa) 10. Describe Meselsn-Stahl experiment and explain hw it shwed that DNA replicatin is semicnservative? First, they grew E. cli in a 15 N medium fr several generatins (until the DNA had becme denser due t incrpratin f the 15 N int the nitrgenus bases), then transferred it t a 14 N medium

5 After centrifugatin (t see density f DNA), they fund that the DNA replicated in the 14 N medium was intermediate in density between light ( 14 N) and heavy ( 15 N) 1 st generatin In the 2 nd generatin, nly DNA f intermediate and light density was present These findings were cnsistent with semicnservative replicatin; if replicatin was cnservative, there wuld have been 2 bands in the 1 st generatin f replicatin (ne heavy and ne light), but instead, there was nly 1 band f intermediate density 11. What is meant by saying that a DNA strand has plarity? That tw strands f DNA are antiparallel? That the strands are cmplementary t ne anther? [L: 3, S: 19, 24] DNA strand has plarity the plar phsphate grups face the utside f the DNA structure (interact with water), and the nn-plar bases face the inside (hide frm water) DNA strands are antiparallel ne strand is arranged in a 5-3 directin, and the adjacent strand is arranged in a 3-5 directin DNA strands are cmplementary t ne anther the nitrgenus bases n 1 strand that are H-bnded t the bases n the ther strand are cmplementary t ne anther (adenine always binds with thymine and guanine always binds with cytsine) 12. If a C cntent f a preparatin f duble-stranded DNA is 20%, what is the T cntent? C = 20% G = 20% CG cntent = 40% ttal AT cntent must = 60% ttal A = T = 30% Chargaff s Rules: # purines = # pyrimidines # A = T # G = C 13. What is the difference between nucleside and nucletide? What des dntp stand fr?

6 Nucleside: nitrgenus base + 5-carbn sugar Nucletide: nitrgenus base + 5-carbn sugar + phsphate (= nucleside + phsphate) dntp = dexynucleside 5 -triphsphate d: dexy N: a generic placehlder fr a nitrgenus base TP: triphsphate 14. Describe the cnfrmatinal characteristics f B DNA (r A DNA, Z DNA, triplehelical DNA). When des this (any f the abve) frm f DNA ccur? (Red = imprtant) A-DNA B-DNA Z-DNA Orientatin Right-handed Right-handed Left-handed Majr grve Deep & narrw Mderate depth, wide Minr grve Shallw & brad Mderate depth, narrw Very shallw, single grve Deep & narrw Bases/turn Rise/base pair alng helix axis 2.3 Å (0.23 nm) 3.4 Å (0.34 nm) 3.8 Å (0.38 nm) Pitch/turn f helix 25 Å (2.5 nm) 34 Å (3.4 nm) 45 Å (4.5 nm) Base tilt w/ respect t helix axis 20 Perpendicular (0 ) 7 Diameter 2.6 nm (chubby) 2 nm 1.8 nm (skinny) Cnditins Lw humidity (75%), high salt, DNA-RNA & RNA-RNA helices in vitr r in viv High humidity (95%), lw salt; mst DNA in viv High MgCl 2 (>3M), NaCl, r ethanl, prmter regins Triple-helical DNA (H-DNA): frmed when purines make ne strand and pyrimidines the ther; then the 3 rd strand culd be accmmdated in the majr grve

7 15. Hw des high salt cncentratin influence denaturatin kinetics f DNA? Explain yur reasning. High salt cncentratin prevents the denaturatin f DNA by prviding catins t shield the negative charges n the 2 strands f DNA; thus, preventing the 2 strands frm repelling each ther 16. What are the classes f DNA sequences in genmic DNA (based n renaturatin kinetics)? [L: 5, S: 8] Unique (single cpy) slw renaturatin (1 t few repeated DNA sequences) Mderately repetitive mderate renaturatin (10-1,000 repeated DNA sequences) Highly repetitive fast renaturatin (1, ,000 repeated DNA sequences) 17. What is Ct analysis? C 0t: the prduct f the cncentratin f single-stranded DNA and time. When the cncentratin f 2 cmplementary strands in a slutin is high, then it takes a shrter time fr hybridizatin t ccur than it des when ne r bth f the strands are present at a lw cncentratin C 0t analysis is a technique based n the principles f DNA reassciatin kinetics that measures hw much repetitive DNA is in a particular genme C 0t analysis utilizes a C 0t curve t measure the sequence cmplexity f DNA samples DNA frm rganisms with small genmes have lw sequence cmplexity and reassciate much faster (have a lwer C 0t ½ value) than denatured DNA samples frm mre cmplex rganisms C 0 = initial cncentratin f DNA (nucletides/l) t = reactin time (s) 18. Wh received a Nbel Prize fr 3D DNA structure? [L: 3, S: 19] Watsn, Crick, and Wilkins (1962)

8 19. If yu had tw slutins f DNA, ne single-stranded and ne duble-stranded, with equivalent absrbance at 260 nm, hw wuld the cncentratins f DNA cmpare in these tw slutins? (Yu can use a diagram if it makes it easier fr yu t explain.) Since duble-stranded DNA (dsdna) have stacked bases, it wuld have a lw absrbance f 260 nm light (hypchrmic effect); and since single-standed DNA (ssdna) have unstacked bases, it wuld have a higher absrbance (hyperchrmic effect) Therefre, if yu had 2 slutins f DNA (ne single-stranded and ne dublestranded) with equivalent absrbance, the cncentratin f dsdna must be greater than that f ssdna in rder fr the dsdna t absrb as much 260 nm light as the slutin f ssdna