7.

}lzRecitation

2011'

Summarv of Lectures 1- -3: Origin of life: its unity and diversity: It is thought that earth evolved about 5 billion years ago a1d-wa9 inhibitable for the firsi 1 billion yeari. As earth cooled down, organic molecules were formed and the first evidence of life (prokaryotes) appeared about 3.8 billion years ago in oceans. Photosynthetic organisms likely originated about 2.5 biliion yeais ago. These organisms increased th9 91ygen concentration on earth and infl"uenced the evolution of multi-cellular aerobic organisms about 1 billion years ago. The true multi-cellular organisms evolved about 500 million years ago. Because all living organisms have_originated from a common aniestor they share a genetic makeup-and are comprised of cells as their building blocks. Many of-the Processes
as bacteria, yeast, flies and mice

that occur in the simplest of cells alsb occur in complex organisms. Thus, biologists can use model systems such for their research linowing that they can extend their conclusions to other organisms including the human beings.

Chromosomes and Genes: Each single piece of DNA in a cell is a chromosome. A eukaryotic cell usually contains 5-50 linear chromosomes. A chromoiotr," usually has a few thousand genes on it, all in a row. A gene is a short region of DNA (a few 1000 nucleotides) that encodes the RNA or a protein that has some specific cellular fuiction. Two alleles (or versions) of the same gene may differ at only a single nucleotide, and often affect the

identity of only a single amino acid in the protein product of the gene.-This singleamino acid change may alter a gene pioduct such th"at it works differently or not it all. the changed_ function 9l tryt protein,may_affect the, itl""otyp" of the organism possessing that allele; for instance, a single nucleotide change in the DNA of a plant can result in wrinkled seeds instead of round seeds.

Monohybrid cross: A monohybrid cross is when you cross two (i.e. homozygous) organisms that differ by a single characteristic, such as plants that produce rounded versus wrinkled seeds. The two olganisms you cross are"called the P (parental) generation. Often the first cross is between two homozygous individuals, AA X aa. The offspring will al'l receive"one allele from each parent and will have the genoptypg Aa. They will display whichev"er of the two phenotypes is dominant. If you cross two F1 organisms iogether. (Aa to Aa), you create the F2 generation. The genotype bf utt otgutrism is the alletes possessed by that organism (AA or aa). The phenotype of in organism is hdw thif organism lboks on the outside (round or wrinkled). Dihybrid cross: A dihybrid cross is when you cross two true-breeding organism-s that-differ by jyo characteristics, such as plants that produce rounded green (AABB) veisus wrinkled yellow (aabb) seeds. The F1 will be genotypically aieb and will display the dominant shape and dominant colo-r phenotyPe. If you cross two F1 orgaiisms togethet, you create the F2 generation. Each parent passes on one of their two alleles at each gene to the"new ptog6ry, und th.tr the F2 will ionsist of a phenotypic ratio of 9:3:3:1 (if the genes are independent from
one another).

Cell division: Mitosis consists of four phases. In prophase, the chromosomes become condensed and visible- In metaphase, the chromosomes all alignilong the_ientral axis of the cell. In anaphase,_the sister chromatids sepaiate from each other such that Jne copf of the genom,e goes to each daughter cell. In,telo-phase, the separated -that you 6n no longer see individual DNA molecules. Right after miclei decondense their chromosomes so mitosis, cytokinesis occurs, which is the division of everything else besides the nucleus (i.e.-!he cytoplasm and all of the organelles contained in the cytoplasm). Meiosis has two divisions; meiosis 1 looks different from mitosis, but meiosis II looks similar to mitoiis.ihe result of meiosis is the production of haploid gamete cells such as eggs or sperm, each of which has half the number of chromosomes as a diploid somatic cell.

Ouestions:
1. Below is pictured the nucleus from a skin cell taken from a mouse (named Mouse #1), with the chromosomes shown in bold.
a) Is the cell haploid or diploid? Why did you answer the way that you

Dipl"tl. [.r,lt" did? l.oi

LL,t**r;on

.-,. \

Let's say we blew up a region of the longest chromosome to look at its sequence at one small

Ci /* "rr*, {n *ri gene. '

....

5'

-CCAGTATACGGATTACGTAC-

3'

5'

-CCAGTATACGGATTACGTAC.

3'

b) Is Mouse #1 homozygous or heterozygous at this gene?

'E& u..{,ro.U

ff".{.r'**.!r-5**4 ho* . LH rJ r--J

Now we zoom in on the same exact gene on the long chromosome in a cell from a mouse named Mouse #2 and find that its two homologous chromosomes have the sequences: 5',-CCAGTATACGGAeACGTAC-3',
5'

Sequence I Sequence 2 c) How might we write the genotypes of Mouse #1 and Mouse #2 if Sequence #1 encodes a functional protein but Sequence #2 encodes a protein that no longer works? (Note: Use the upper case or lowercase A while writing the
genotypes).

-CCAGTATACGGA&ACGTAC:3'

6\
2. Consider a hummingbird in which the body color is controlled by the B gene. These humming birds are either

green or yellow, where green color is dominant to yellow color. Give the expected ratio (both phenotypes and genotypes) of the offspring for the following crosses. Genowplc ratlo
(-roSS

l'henotvpic ratio

6b x bl5

uttsPnng:
Offspring: Uttsprmg: Offspring:

Cross 2: Cross 3:
LTOSS 4:

bbxbb
BbXBb
Bb Xbb

Alt

13

AI

tt

(r,.e--,'
q'n.{,to"J

LL

Att

i11{t:t{rL:li'l

\ BLt tbt

5G...-^.

\,n"Jlo.
,r,eft".

IG"-.^:l

3. Tomato plants can be tall or short and have notched or smooth leaves. You cross a tall, smooth leafed plant with a shoit, notched leafed plant. All of the progeny are tall, and notched leafed. -Q'LI n"{c'l'Yol n a) Which traits are dominant and which are

recessive?

1u

I (l^'tr-t; <-'"o*\ Le-O-'

^
-

issociated with the dominant phenotype and the lower case letter for the allele qssociated with the recessiae phenotype.

b) What are the genotypes of the two true-breeding parents? Ilse the letters H or h to represent the alleles. of the height gene and the lette/s S oi i to represent the alleles of the leaf gene. In each case, use the uppercase letter for the allele

{-rH
4.

,,
:4).

Lt, 55
S[..r {

tal\,Lr',rootx
chromosomes (2n

A schematic of a diploid nucleus prior to DNA replication is drawn below. It contains two pairs of
The genotype of the cell above is DdEeFf, where the D and E loci are on the large chromosome and the F locus is on the small chromosohe. This cell was taken from an F1 organism that resulted from the cross of two true-breeding parents such that Parent 1 is DDeeFF and Parent 2 is ddEEff. Draw and align the chromosomes as they could be when in metaphase of meiosis I. Include the D, d,E, e, F, and f alleles on the drawing and assume no recombination. Given your drawin S, draw the chromosomes in each of the two_products resulting froir meiosis I. Include the D, d,E, e, F, and f alleles on the

^otclv1,t

drawing.

F.

t{t:
F--tf r

Ou
X
I

J
u-l- J- ,r

I
l
1

' 1: f*"'."'

F{

"+l

l?*-11'r"{'-"t
E

*-t
l.Aa&,rglre

rt

7.012 Recitation 2 - 2011.

Summarv of Lecture 4: Recombinition and crossing over: F{omologous recombination is the reciprocal exchange of DNA between two homologous chromosomes that occurs only during metaphase of,meiosis I. During homologous recombination, two horiologs (e.g. the chromosorne #7 inherited from dad and the chromosome #7 inherited from mom) break at the same plaie and switch ends with each other. Homologous recombination results in a change in which alleles are on the same chromosomes together (e.g. a cell with " AB" on the maternal homolog and " ab" on the paternal homolog can give rise to gametes containing " Ab" or " aB" chromosomes). The two recombinants ("Ab" and "aB") will always be created in equal frequencies because one of each kind is created by a single recombination
event.

Linked and Unlinked genes: Two genes can either be unlinked, completely linked or somewhat linked. Genes that are unlinked are uzually located on different chromosomes from each other or are located far apart from each other on the same chromosome. Genes that are completely linked are very close to each other on the same chromosome. Genes that are somewhat linked are located on the same chromosome with a map distance of <50cM. Unlinked genes assort independently from each other; this means that an AaBb parent would create their four possible fametes (AB, ab, Ab, and aB) each 25% of the time. Genes that are completely linked always segregate with eacfi other. This means that an AaBb parent would create only two of the four possible gametes. GeneJ that are somewhat linked to each other show a pattern that is in between what is seen for unlinked genes and completely linked genes.
Complementation and Epistasis tests: Yeast is capable of growing in a very simple growth medium that only contalins minerals, salts, ahd sugar. From these components, the yeast can make all of the molecules it needs. But if the yeast is fed amino acids and nucleotides in the growth medium, then it would much rather use them than makeihem itself. For example, if arginine is provided to the yeast, then the yeast will import and use it. If arginine is not provided, the yeast will make arginine using a biochemical pathway that takes a precursor and coiverts it into^arginine through a series of steps. The genes that encode the enzymes that catalyze the reactions in this pathway ai6 caled arginine synthesis genes. A yeast tha! is mutant in one of its arginine synthesis genes is called in Arg mutang wild-type yeast can grow on medium lacking arginine, but Arg mutants cannot. If you were interested in determining which genes are necessary in a yeast to synthesize arginine, then you could perform a genetic screen. In this screer,, you *ould mutagenize thousands_oty"?tt cells and plate all the mutant yeast cells 6n medium containing arginine. You would then "replic a plate" all of the colonies that arose on the medium containing arginine onto medlum lacking arginine. An Arg mutant could grow on the medium containing arginin6 bul not on the medium lacking arginine. You would screen through the thousands of yeast cells for the ones showing this Arg mutant phenotype.

After isolating your mutants, you would use complementation tests to determine how many arginine synthesis "discorrered. Todo this, you would mate each Arg mutant to every other Arg rnrrtant, and ask genes you haX ioh"ttt"r the resulting diploid could or could not grow on medium lacking arginine. You would assign your mutants into comple"m"trtutiott groups., where the simplest assumption is that each mutant in the same complementation group has a riutation in the same gene. Once you knew the number of genes you,had discivered, you w-ould use epistasis tests to determine the order in which these-genes act-in the pathway leading to arginine synthesis. To do ihis, you would create double mutant yeast that lack two of the genes you identified and determine which single mutant phenotype was displayed by the double mutant.

linked. In one specific fly whose,Senotype is GgBb, "8" material chromosome iz,And "b" is linked to "G" on the paternal chromosome #2. linked to "g" on the

f- I" ft"it fti"s, the "B f b" gene and

Ouestions:

the "G I g" gene are

. is Lf b
!

a) List all parental type (non-Lecombinant) gametes by genotype that could be produced by this GgBb fly.

b) List all reiombinant gamttes by genotype that could be produced by this GgBb fly.

Gb q^ol
G F."

F:

c)Say that40% of allgametdsproducedbytheGgBbfly are"Bg." Whatpercentageof allgametesproducedby

tu^tl ,l i

thisflyarerecombinants?

d) As the recombination frequencyNet*een two genes on the same chromosome rises, does the physical distance between those two genes lower or rise?

% gGr % !G;. t1)or- *)

o1u p^rc,*ltu(

a to)1o--) % /t<_to^o a ?O%

n*"*r,^a-Lse-

cl.l{t.^.q-

2. Tomato plants can be tall or short and have notched or smooth leaves. You cross a tall, smooth leafed plant with a short, notched leafed plant. All of the progeny are tall, and notched leafed.

a) Which traits are dominant and

which are recessive?

j li

. -

lr
{

Jtlri'.

'l

',,:..';i

b) What are the genotypes of the two true-breeding parents? Llsc thc lctters H or h to represent the qlleles of the height gene and the letters S or s to represent the alleles of the leaf gene. In each case, use the uppercase letter for the allele associated zuith the dominsnt phenotype and the lower case letter for the allele associated with the recessiae phenoty1te.

,:ri",.
l-t \", ss
3. Name an

i'l

ri a(

c) Two tall, notched F1 plants were crossed. If these two traits are

unlinked, what ratio of phenotype do you

lr:
i :.' : ,r .. I i
,.1,,

expect in the F2 generation?

^ organism that may

l(\ s,

j''n' 'l
exist

{i l.i ii
lit',.

,t

a) In a haploid state:

b) In a diploid state:

c) Can adopt either

state:

d) If you were mutagenizing the last organism and looking for a resulting recessive mutant phenotype, would you mutagenize the haploid or diploid form?
t'

Yi'r:.'.'i

4. You discover a series of

fruit flies that all display a recessive "lri.ini-f7y" phenotype. Each mutant fly is homozygotic for the mutation causing the mini fly phenotype. You cross each mutant fly with the other flies in the series and score the phenotype of the resulting F1 progeny as follows.

ala

blb
mlnl
X

clc
normal normal
X

dld
normal normal normal
X

ele
normal normal

fl
mn

ala

blb
clc

dld
e/e
f

lf

mlnl normal normal normal mini

normal normal normal

mlnl

normal mlnI normal

mlnl normal
X

normal normal

mln normal normal normal

X

normal

a) Place the six mutations into complementation

groups.

I {

[ f

il-i
r: 1.i ;' ;'

b) Are mutations b and c in the same gene or in different

c) You cross a

genes? [r;

homozygous"glg" mutant animal to af a andblb animals. Both crosses yield mini-flies. What is vour

conclusion?
5. You are studyin$ a biochemical pathway results

in the synthesis of comfound F in yeasts. Compounds A through E are intermediates in this biochemical pathway. You have isolated 5 different haploid-mutantyeast straini, each of which are defective in a single gene that encodes an enzyme that acts in this pathway. The table shows whether each mutant can (+) or cannot (-) synthesize compound F (and thus can grow on minimal medium) when provided with each of the compounds shown along the top of the chart in their growth medium.
Compound A
B

D
+

Mutant
1

2
.) 4

+ +
+ + + +

+
+ + + +

Given the chag draw the biochemical pathway that produces compound F. Indicate the correct order of the steps, and which enzyme (out of enzymes #7-5) catalyzes each reaction.

2 E-; -{

f.,az

l.v1

t-1

-.-

r(

[^L {
t,/

L.

rt

rr

.-

l)

't)

i\

,f

't

7.012 Recitation 3 - 2011

Summarltof Lectures 5 & 6:

Pedigree Analysis: A pedigree shows how a trait runs through a family. A person displaying the trait is indicated by a filled in circle (female) or square (male). Simple human traits that are determined by a single gene display one of four modes of inheritance: autosomal dominant, autosomal recessive, X-linked dominant or X-linked recessive. Autosomal traits are due to genes that lie on chromosomes #1 - #22. X-linked traits are due to genes that lie on the X chromosome. Females are diploid for X-linked genes, but males are haploid. For a male to show an X-linked recessive trait, he only needs to inherit the X-linked allele from his mother, making X-linked recessive traits more common in males. The females are usually the carriers. X linked dominant inheritance is extremely rare.

Bonding: Biological molecules are assembled from a small subset of atoms i.e. H, O, S, N, C and P. There are many types of bonds that hold molecules together. Covalent bonds result when two atoms share a pair of electrons between them, such as the bonds between C and H in methane. lonic bonds occur between atoms of the opposite charge, such as Na+ and Cl-. Hydrogen bonds occur between polar molecules, such as molecules of water, because of the partial negative charge on the O and the partial positive charge on the Hs' Van der Waals forces are very weak forces that occur between all atoms because of random unequal distributions of electrons. Hydrophobic effects promote the association of hydrophobic molecules together in order for them to avoid water and thereby increase entropy.

Questions:
Extra Linkage Problem While working with a type of beetle that is normally smooth, large, and grey, you discover three mutations that lead to the recessive phenotypes bumpy, small, and white. You cross true-breeding smooth white beetles to truebreeding bumpy grey beefles and get all smooth grey F1 beetles. Then you cross the F1 beetles to true-breeding bumpy white beefles and, after analyzing 800 F2s, calculate a map distance of 5 cM between the smoothness and color loci. What are the four phenotypic classes you got in the F2, and about how many of each did you get?

gb \ + At, , ,{.'.-.-r1\ l, F; K qR
fi

c'b &L, i. (r\ur I- \-,. +'A \, .T ) ,, rtr5 ,

_

,(l ou\ I j,' f:J

I,i

i=:ltb
n\, !l; a3(C

At

i:
i

Al5 * \. JL
''. i,

\rL

,'

1,.,[ i,. j

'J /

z-y'

you cross two true-breeding parents to get all F1 beetles that are large and smooth. You cross the Fl to truebreeding small, bumpy beetles and get: 228 large and smooth, 19 small and smooth, l6large and bumpy,237 small and bumpy. What were the phenotypes of the two parental beetles (P generation)?

it :,'lfr :-:: p*,LL*--/,41il11"1,.g,

IN

.'l - \'

ic' " tlt\

)r 1.,".,r
,t
t.

{-"r\
('tt(

F"i +

'j

What is the map distance between the smoothness and size loci?

il

"#.: lcl ?-?f \ , rrrI J 1'.*(Nr/L d, ,rL +)

cr(i

I
tC)U

,.-,.'^r

i ts : rTD x AD J

-q-.!

orl

i'{ ,
C. e)l

AD

IqJ
lD

(L

^'l
11( 'u "r 1,'

!--<_-

"li, ;\-L' ^

lriu /f l

,r

a.

tu

=)

c fi\

1.

ln the box below, write the most likely mode of inheritance of the following pedigree?

^,^'
At\
5

.d.o

AA

'c\ 4
Ar.,

4r,{rgo*".,

W.<e- s s,t*,-

Given each consistent mode of inheritance, if the couple in question decides to have a child, what is the probability of that child being affecte d? (Note: Use the appercase or lowercase A to represent the a//e/es for lhe domnant and recessive traits).

lfz^,r, (, 't A-)

>/l

to"

A,..),4.^

P(o.,.)

i'lt(

P(ui,t

,ro-)i llc''/r,

'/e

2. Consider the pedigree below showing the inheritance of two X-linked diseases, hemophilia A and hemophilia B. Hemophilia A is due to a lack of one clotting factor, and hemophilia B is due to a lack of a different clotting factor' Each clotting factor is a protein that is encoded by a specific gene located on the X chromosome. Note that no A and hemophila B. individualshown in this pedigree is affected
I

lA

Rrrenotypically normal

ffi

Rtre"t"o with Hemophilia A

Affected with hemophilia B

a) Write the genotypes for the following individuals at both the hemophilia A and hemophilia B disease loci. Clearly define your genotype symbols. lj.*.,rh,l;o

Xo ,, lrq,*1i
Xc'

lndividual
1

Genotvpe

y^y

*'

A
{1

ai{r,{t
^tLe
{.1
{q

yrsy
V'J_kb

.D,r..r,,g;{ l-tr"'*,:L,t,r. A

2 3

VAyA

4
5

y*k*

XAY YIY

B X = tV,,^,,i f-k. J,[,i.c

r, ifu

vAV v

yu X b
sY

b.' X

i)r$1,,t;.-{

{"irtrl",l

lj

ft{i,{.Lr

b) How do you account for individual 5 not being affected with either hemophilia A or hemophilia B?

JytrLv J

KAY xl'Y

J-.^Ji,, t'{ :

xAx* x Bx b
(N
o,

to^ " B)

^".1)

0
(7-.

-r

nlo

u.

f I \'fY
(,fu n,.*r*

{}

.012 Recitation

4'

201'l'

Summary of Lectures 7 &

8:

Biological macromolecules: All cells, both eukaryotic and prokaryotic, are composed of the same m{qr classes of macrd'molecules: lipids, carbohydrates, nucleic aiids and pioteins. These macromolecules are formed through condensation /dehydration reaction which results in covalent bonding of specific monomers to form polymers. Conversely, the hyitrolysis of the polymers results in the formation of monomers. Lipids or fats are predominately hydrocarbon chains that are used.as_energy storage and insulation. Lipids are hydrophobic in niture. Modifibd lipids form phospholipids, steroid hormones, cholesterol and some vitamins. Gtl *"-branes are composed of tipia bilayers, wliich separate the aqueous inside of thecell (the cytoplasm) from the aqueous outside of the cell (the extra cellular environment). 9"" specific lYpe of hpid, called_ phospholipids, has polar phosphate groups as heads and two long hydrocarb,on tails that are very hydrophobic. irnorirt'tottiridsform lipid'bltayers iniquebus solutions that expos-e the phosphate_head gg"qs o1"1+ side of the layeiand hide the hydrophobic tails in between the two rows of head groups. Only small hydrophobic molecules .un pus through .eil -"-braoes. All large or polar or charged molecules must cross the membrane through proteins that act as pores or channels.
Carbohydrates include monosaccharides and disaccharides like glucose and lactose. A monosaccharide is .o-po#d of carbons flanked by H or OH groups and cante linear or-in a ring structu1e. Many monosaccharides hnk6d together form polysaccharides like starch and cellulose. Carbohydrates are used as an energy source/ as a source foi carborU ut d can be attached to other molecules to serve as recognition markers for the cell. Proteins are formed as linear chains of amino acids of which there are 20 distinct $pes. The order of the amino acids in the chain dictates the shape that the protein will take and therefore the funition of the protein in the cell. There are 4 levels of protein struciure: primaiy, secondary, tertiary and quaternary. The precise sequence of amino acids in a polypeptide chain constitutei the primary stru,cture of proteins. A pr_otein's secondary structure (cr- helix and 13- pi"ui6a iheets) is comprised of regular repeated spatial patterns in different region of polypeptide chains and is predominantly stabilizei Uy hyatog"n bonds. _The different interactions between the side chain groups of the amino acids determine theieriiaryitructure (the shape in threedimensions) of proteins.. . ftuaternary structure results when two or more polypeptide chaini in a protein bind to and interact with each other. proieins perform all possible functions within a cell except storage.of hereditary information. Proteins can be structural, cin be used to transport materials, can be used for locomotion of cells and be used as enzymes or biological catalysts. It is worth mentioning that the RNA may also have enzymatic activity.

Ouestions:
Oil is fluid at room temperature but butter is solid. Which tlpes of fat do you think predominates in oil and butter (saturated / unsaturated)?
1.

4 ;)
'1t

ur,

t(t{t^rr'{i.,n

-J t ftuid'{5
vngahu'*heu{ &4
c,r,{ cka;^ 4

C;

ln a< r"'ort-

<

tha o

Lu({,c

2. Each cell in an organism has a plasma membrane that defines its

boundariet however the plasma membranes What factors determine the fluidity of the plasma membrane? of different cell typJs show different fluidity.
utn saf

wa4J* s '{- {"'l+\

t f-a'&ft a"c',cj le^(1*1,\ -5 \, (t;;A;'t, ( I*tqr{rro Ie*f *,a{*4.

I

"

t {b; c('|4y

3. When

biomedical researchers design drugs that must enter cells to be,effective, they often add methyl (-CHJ groups, which make the drugs morelitety to enter the cells. Why does this work?

CHI it L br{{t: l)a tN

I,p,ri 6,dnoo,.

rronp

oLw

**t

r;h i ch
hg d"*p t"
b

{f,,o,1pi^,

{l*
ee {d

e\t^ ^oy k"tg i c v14 6'{ t'lr* ',o

I
n

+L

fr

-{

#l*

F-

lipid bilayers, but instead have lipid monolayers. The structure of one lipid they can use to make up their monolayers is shown below. Why would you not need a bilayer (i.e. but instead could use a lipid monolayer) if you used this specific kind of lipid to make up your cell
4. There are some rare organisms that do not have

membrane?

Lu'Q

/ "4

f?\

SoL'&

i{1r

A V
f--

-\
R1

Nofe: R1

R2 represent charged groups and " A" represents bonds between C and H atoms.

4', &'rr.pis$ic ev,J d'gdiiop},1},c S16cvcJ* 5. A certain protein has this structure, where a line indiiates a hydrophilic region and a box represents a

[,t\4^^grarw c^x

st:ll {tt* th" l.

-

"$*^g

f*46 5

hydrophobic

region.

ydrophilic

ydrophobic
Why do you think the protein might fokl-f,ris way when dissolved in salt water?

l,l -> Coo ,^4,*rr{ *l wa4.e{ d*ul l{1a*p h*lo, r, ls.r Fa,w 5 6u6,tscf -+ 9\',!}daa fi*^ uoLn G,^r{ Sc'l{t

/tra^yhJio Swh.c+1 Lxpos,J

grL

ii.

How do you think the protein might fold differently if it were dissolved in cooking oil?

\v,

Tnva-tuio.

*)

ftgl^F k,bic Svr{a'cs,s Lrf

oftJ, hy/^*p L',lio

jw-{c.

ca.5

6. You are studying a protein that functions in

your gut. If you boil this proteirg you will disrupt hydrogen bonds and ionic bonds, Uut nbt covalent bonds. If this protein is most active (100% active) in the cytoplasm of the gut cells, what percentage of activity do you predict it will have:

b*ln d

"

i.
ii. iii.

At

1oo'C ( Loooto (ptrI agl b"/o)

treatment 0'
/O

After protease

If it is suddenly exposed to the acidic environment of the lysosomes.

Llooolo (p., kop s Oo/, )

sorution

to-"o,o H'
_. Substrate
I

:iii*::: rr

\^s.-HH
.:::ii::ii::+

FH- \r lIH t+7 . /\ --trl

tt-c=c/ H Jl J- t..^rt Sa"-gzC-H t*

"+

::::i':i::i::i:.",.:'_..:_..:

-- -E# li:iliiiliri':-'ii:i'i'! a:i:i1:i:i:..:i:::i:i:i:i:i:i"::i..;i::i;ili;i.'l+ili::.-ijil:.r:.1i1ii:.i:.:i:...i'..':.:i::...::.11:l:-di1;1i'71fi i:.i;'{!i; """i;ilii:rlililil::..:1.. i+ijiiiiiiiiii#, P.._"t9i' ..-jiiiiiiiiiiii.iiij,,:

a) For each side chairU state the strongest type of interaction it could have with the substrate in the configuration ,i,o*r, below. Your choices are: Coaalint, Hydrophobic, lonic, Hydrogen, snd Van der Waals. Also classify each R group as hy drophobic, polar or char ged.
R

Crouo

Name of Amino acid

Classification ot lt Group

Interaction(s) of R Group

with Substrate

(i)
\2)

Asaon{;. n.rJ
Cu^ s"{.oih-e,

Ckcrore4
S{ia6-lt1

lLo{

0s(r.r

l{qcl,*.as-.

1^r,

ik,

uta

tJ

(3) (4)

V a, l.t'rue, *srran'^q,l*,

F{,nr[i-nL6icPo

i-{",rr.*ufot- i r. f v,^

,Lu V, U

[.,'-

i-ir"

rj*,"- ^ ,\

b) Describe the effect of the following substitutions on the enzyme substrate interaction.

i.
tl.

Amino acid # 1 substituted to methionine. Amino acid # 1 substituted to lysine. Amino acid # 3 substituted to alanine

I_^hrr-trr^

tu<a_ke^ gil

( **y"t *t
n+r1c,{.,w

no,'yt

ulur)

-C^4-rn

[.n

rnl,{n*ol

*"

/t,

u r'f

,L I r*rg
)

lll.

Zr.{, , ' , { .-.r 1i,^,to,r

L^,^ f\oGr -t

l.on ,, *l ,"

jI
c) One way to study the interactions betweeri substrate and protein is to syrrthesize molecules similar to the substrate ind see if they bind to the protein. Shown below are 3 "substrate analogs" along with the normal

substrate. Explain, in terms of the inieractions you described above, why each of the analogs binds or fails to bind to the protein.

ooato

t) Nt.-,
*T

neaJa{u,u

f ^rp
St-n

(rLwK-

^:1;,t*L

,,frd
H3C\zP

lt:ffii:$:ffi.i

t) t,;,lrn i,,l.r.i{U in Shanyr,f. {^$ Mry,fe,

vtswira"l (hury4

ot'oe

Y, I

(b

",RJ9J
Analog
(binds)
2

".*dr
Analog 3
(does not bind)

l\

AaAJ,o^.1 por;-l,w .^^d (av seg

Analog 1 (does not bind)

^-p

f'qPr,r"I S''on

STRUCTURES OF AMINO ACIDS

GENERIC AMINO ACID:
frotein slnrhesis
utrique

t".*",*".

"*-Sside.ba,n, ;fiil;;il';,"*\---y' ffi:',:,{**'

srouDs to form

"",* ,---*31' are lidedltuoush lhese,

the \

>

oooo -\,
rtl "-f--a", NH, ALANINE (ala)

\, NH,
ARGININE

o-."ao
H

o"co

H-{4I?GI,CII--N\C,M?
Mu
II

"i,*o<l
Ihr. NH, ASPARAGINE (asN) oo 9o aJ.+.G.J! ' '

"i*,oll M,
ASPARTICACID
(asp)

(ae)

oo
I I

v
-SH
NH3

"" !o
s--b-an.cn,-C' ' ' \o'

ao \Y

H-{-{lI?
+

^!t,
GLUTAMIC ACID
(glu)

,h.,

t*,

H--{-H
NHI +
I

CYSTEINE (cys)

oJuro**
(elr0

GLYCINE (glv)

Y }-'* "i,l'\i*
t r''ori,
(his)

oo Y,g lt H--{--{-{l,cH, /t M, G,
ISOLEUCINE
(ile)

oo

tl H--4-CH, --{--C{, tt NH3 CXt3

LEUCINE
(1eu)

\YH

...C.

r-t--or"o,o,o,-*,'
N{3
I

LYSINE
(lys)

o".ro

ov,o

"-i--c,,o^-r--o,. "i-.",Ja)\*, METHIONINE .

&,,

"\J"

""a,

-\Y
H-{<ft,--oH
I

{"

,,>< 1J*;*'
i#3"-l

"J-*i,

NH3

."*-" &'
TIIREOMNE
^6,

"i*t: *S+
t, .HH t,

ffi"I^^,,^"

SERINE
(ser)

'7-l*.
M]
H

"" r

cH,

Hl
TRYPTOPHAN

(ttr)

(trp)

H
il:jlosn'rE

VALINE
(va1)

7.012 Recitation 5 -2011 Summarv of Lectures 9 & 10:

Biological macromolecules- Nucleic acids: Nucleic acids [Deoxyribonucleic acids (DNA) / Ribonucleic acids (RNA)I are polymers specialized for the storage, transfer and use of genetic information. They are comprised of nucleotides (A, T, G, C and U) each of which has phosphate group, a pentose sugar and a nitrogenous base. Polymers of nucleic acid are formed by linking the 5' phosphate group of one nucleotide to the 3' hydroxyl group on the pentose sugar of another nucleotide. DNA is the hereditary material in the cell and is usually doublestranded. The RNA is usually single-stranded and there are three major types: ribosomal (rRNA), transfer (tRNA) and messenger (mRNA). The mRNA gets translated to proteins, ln comparison, the IRNA and rRNA are involved in the synthesis of proteins. Nucleotides also serve as energy sources (ATP and GTP) and second messengers
(cAMP).

Enzymes: Enzymes are biological catalysts. They act by lowering the activation energy of a reaction. Each enzyme has a specific three-dimensional conformation and an active site to which the substrate molecules can bind. The complex then gives rise to product (P) and the enzyme is released in its original form to catalyze the reaction once again. Each enzyme is specific for a particular reaction. Enzyme functioning may be regulated by various factors. These factors may include i.e. prosthetic groups, metal ions / cofactors, coenzyme, substrate concentration, pH, temperature, inhibitors, allosteric modulators and the feedback inhibition by the end product. They are usually proteins. However RNA may also have enzymatic activity. Reaction Kinetics: An endergonic reaction (one with a positive AG) cannot occur spontaneously, although it may be coupled to an exergonic reaction. An exergonic reaction (one with a negative AG) can proceed spontaneously. Exergonic reactions do not necessarily proceed quickly; this highlights the difference between thermodynamics and kinetics. The thermodynamics of the reaction are dictated by the difference in free energy between the substrate and the products. The kinetics of the reaction is determined by the transition stage and how much energy must be added to form the intermediate. Enzymes lower the activation energy needed for a reaction to proceed, but do not change the free energy of either the reactants or the products, ATP-The energy curency of the cell: Nucleic acids [Deoxyribonucleic acids (DNA) / Ribonucleic acids (RNA)] are polymers specialized for the storage, transfer and use of genetic information. They are comprised of nucleotides (A, T, G, C and U) each of which has phosphate group, a pentose sugar and a nitrogenous base. Polymers of nucleic acid are formed by linking the 5' phosphate group of one nucleotide to the 3' hydroxyl group on the pentose sugar of another nucleotide. DNA is the hereditary material in the cell and is usually double-stranded. The RNA is usually single-stranded and there are three major types: ribosomal (rRNA), transfer (IRNA) and messenger (mRNA). The mRNA gets translated to proteins. ln comparison, the IRNA and rRNA are involved in the synthesis of proteins. Nucleotides also serve as energy sources (ATP and GTP) and second messengers
(cAMP).

Respiration: Glycolysis is the process of breaking down the sugar glucose into two pyruvates, with the goal of creating energy in the form of ATP and NADH. lf oxygen is available, pyruvate will be fed into the Krebs cycle, which produces more NADH; NADH is then reoxidized into NAD+ by the electron transport chain. lf oxygen is not available, pyruvate is fed into the fermentation pathway, which reoxidizes NADH to NAD+ and converts pyruvate to either ethanol (e.g. in yeast) or lactic acid (e'g' in humans).

Que.stions:
1. Nucleic acids are of two major types: double helical DNA and single stranded RNA.

(Y
44a) Draw the chemical str$ture. of a DNA r66leful

"nor.

*?\
-L

J y

.!i

<'>.

"{--\_l-o-q)
,

F-

Label the 3' and 5'

b) How is a nucleotide different from a nucleoside?

f-'t tbogc f rnX

fr.i.rcb-{iUr, {tot 3 pLl-r"-L {*-frt

I

a&{c'cfr,.,t{o

r / -ot+

at

+L

a+ 1+-

rtwrkos,ulq,

c) Why the two strands of DNA are anti-parallel to each other?

A(t"*rr
in DNA?

Co^plcn<',-[r,"0

M-6"^.lta

g+
G1

carrp<,4,t,V

(rr<- p6':y

d) RNA is single stranded. However, it cdn have a very complex three-dimensional structure. Certain regions of RNA may show intra-molecular base pairing. ls the base pairing observed in RNA similar to or different from that

-f ) A pa,r< , may be definedr,tr^, V lo, gppo ut 4o uleach enzyme as biological catalysts. The active site of 2. Some proteins act as enzymes that
Ois<,-".ra-'.

(i*;l *',

A^-t;pa"*lto[

C pavt

.;l

Tn

funrA

binds to the substrate molecules. ln each case, the active site is comprised of only 10-1 5 amino acids. Would you expect that these 10-15 amino acids that form the active site are immediately adjacent to each other in the primary structure? Explain your answer.

lttot'[ &t n lLtu

f

,aChtv'q"

{i!<,

fr-si.'!.*c.t r_r\-

fi*L br

d-,F.n^

{.

(h*.^ a.*o[ 6-*e- 6*-7\.,,/ f,or.*hL, 6,0 &U;*, ,? +L prutc"r, -rz qil*-( fL (u<a(ie;* *d *,\,re
pvly1up'l:

t,u, o{- {'l*

ak,

a[

r,..o,".- .

<-

( &1, dar *(g p- c{<e'{i

/ a.$,* s;,Jrp

3. Enzymes, which are usually proteins, act as biological catalysts. Of the properties mentioned in the followihg
table, state which do enzymes NOT change. Properties A G of reaction Rate of reaction Does enanme chanoe this orooertv? Yes or No.

r)o
\F-

4. Working in a research lab you happen to identify a novel protein. This protein serves as an enzyme, which catalyses the following reaction.

B+C

AG = negative

a) You observe that if you increase the concentration of substrate A, there occurs a simultaneous increase in the rate of reaction, but only up to a certain point. Explain why.

A+ 9o*-r. Lom.cr,ui rat'.c- 4 , & C**d 4k r6tL l,t,(,

b) Draw the energy diagram for this reaction. Label the axes, products, reactants, DG, and energy of activation.

Vou sa(.w-o'fa tlt a((r",,t . S fta, i ( t F\^r,ry11{'^ v-*bc,il g V^o, ,

,

Srls{*Q

A(,,

tl*. *..,y1\

,v
!Lr..,".
("ord,,..4*{g.

(
5. This is a diagrarn of ATP
)1"

t'*t

"-[]

)-\) -lr^N -$-o-f -o--

o o

/=11 'ti

'\_-/

66H

OHOH

a) Draw a line to show which bond that is broken when this molecule is used as an energy source.
b) Star * the atom(s) that can form a hydrogen bond with the complementary base.

6. A representation of the mitochondrion is shown below'

H'

a) Why is the mernbrane important in chemiosmotic

pathway?

M,t-wbrar.r

rra,tq"{ cu,,45

+l-

chovXa_

b) Wher\ $lucose is metabolized, some of the energy is captured in the formation of reduced NAD (NADH + H+).

s.para-L.'"l --+ 4' E'varq.a oVol lo

e"n,t

nal',* 4ff

1"&^I.al t'

-:

What is the fate of NADH in mitochondria?

U"*4.t crt P/-;N o( i{s g,(ech**, 4o +k

gLsr{*n *ru*sg,-4

cJno,n

,t";r(

bG,t,,It
,

{L

chv.n-, o

(-vl's (ra,,U*l t\a-4-s<w,4 G r {l*

{o,

*,.{'it

n( ATP
(g.

c) lf aerobic respiratioh is occurring in this cell, 02 is consumed. What is the role of 02 in this cell? What is produced from the 02?

^c(<

dr +L {r,*i^rvl sfus4*n a(C.*p{,r,

b*& Ttl, *+ 1 Ho o

d) lf aerobic respiration is occurring in this cell, COz is released. What is the source of this C Oz? What is the ATP that is generated used for?

L0.r4

(ou'+

< {*n'

"''4u**"1'^l+l
i

ir.. +L
4.s[l'11'.,J

**kLlo,*^ o(
f rocers9s

GLu cos rc ,

AIP I
.

rtt

{u ,

in *L

I . "*.X V _- r.,c (*,,n,t 1

a,-[l

v'

7.012 Recitatio n

1l - 201L

p.lymorphism (SNP) is a DNA sequence variation occurring when -a single nucleotide members of a species or between paired chromosomes in an individual. SNPs are "throughout the genome. A SNP can result in different alleles of a gene, where one of those alleles may be found associated w'it} a disea"se or trait' SNPs are also lound in non-coding regi'ons of genes or intergenic regions between genes. SNP mapping is used in forensics and a genetic tool / marker for a trait or disease.

Summarv of Lecture 19:

"".t".ttd" in the geno;e differs between

SNpJ;figt"

Micro?xayglNot all cancers respond equally well to all treatrnents, so knowing the specific $pe or sub$p-e-of ."r,cer is ifii-o'iiairtasgccessfui treatment. 3ome cancers can be identified by histological toolg-but oth6i are best ipt&*technology that expression profile. A DN.+"microarraf|g character ized by determinifr ists A DNA microarray consis of an ion of manv dif allows comparison of cells based upon representing a gene- When a DNA spots arrayed series of thousands of microscopic fft ill hybrid les will hvbridize to the 6lls, individual microarray is probed with mRNA iso , so more mRNA i-shlv exoressed. more mRNA will be made fi6 appropri?lg DN4,tgIJ L _o__/ ? -" -1 from that spot will be greater. do-tp:onding dNA spbt, and the signal will hvbridi

jl-yo.r id"ntify

u hypothetical Gene R in humans that encodes for protein,"R" which is involved in maintaining low blood ch6lesterbl level. This gene shows anguto,samaklqmillnt mode of inheritance and the affected SNP^(SinBle nucleotide individrrals are at a hisher risk of"developins a cf,ffiidisorder@)'.-You come across a SNP (Single nucl higher rist of-de"eloping c;4fi;i;;&;feDTYou come to GenJR. Y-ou decide to use this SNP as a marker for CD. The two alleles o{ poiy-o.phirt"; Aat i;tightly linked bNi' (C u'"a C) u.e showi for each individqa] in the following pedigree. Indiaiduals fficted by CD are shadcd in

Ouestions:

;Jt"td;;il;;;i
black.

0i

l.A
2

E,*f*, +t" C u. G sv! 6lldL e n 0/t0 '^u+l l*

l'.;nlt66,'L

Auto sspv/;-

Aona

MfrN1

-f" {
f,

ltg.

d'qro*,{,

GIC
di,{

CIC $t
d
0,,

GIC
Dd''

CIC
Dd
3

D,t

IA

Nu-t

Lci^ra

."* G (/vp et$lL ,A D/,o *"r-4 L

^b

d.,ss"c,1y

pL*-t31<,
f,*lq4

94c 9,lc @c ds'i dd. Dd

dd

a) Assuming no iecombination between this SNP and Gene R, which allele of the SNP is I associated R- allele in individual #L?

+"

ctc d4
linked to the disease

ol:<eo"lo,{.1,1

TI'q- C* a{{x"la

b) Assuming no recombination between this SNP and Gene R, what is the probability that the offspring of individual #g and individual #4 has the disease?

CIC

Uc' G <AJp 6vlt&- l,^la,r-to d,,w,rJ,

showing.
(!1

2. Describe what the image below is

0olo I

Summary of Lecture 20: Protein localization: The proteins undergo different types of post-translational modification i.e. cleavage of signal peptide, glycosylation (addition of carbohydrate moiety that occurs in golgi bodies), phosphorylation (addition of phosphate group) and lipid addition. Each protein contains an inherent iignal sequence that serves as a zip code_for the translocation of a protein,to its sPecific destination. Pioteins foing to the specific orgahelles have a short stretch of amino acids that allows them to bind to docking proteln receptbrs present on the membranes of appropriate organelles (i.e. nuclear localization signal foi nuclear proteins). Theprotein_s in the cytosol have no signal sequence. The transmembrane pioteins have a stietch of L5- 20 hydrophobic amino acids as thejr signal sgqu-enge. Thi-s signal sequence is bound by a signal recognition particle (SBP), the SRP aids in the docking of the nascent poiypeptide to the ER rec6ptor. The signal sequence then enters the ER lumen by passing through a ihahhei in the receptor. Th-e signal sequence is then removed and the protein synthesis once again resumes. The protein is transported to the membrane via E& golgi bo-dy and vesicles that fuse with membrane. Piotein localization can be determined by techniques such as fluorescence imaging or by creating a fluorescent protein.
L. You are interested in four different proteins in a yeast cell: protein L is a cytosolic protein, protein 2 is a secreted proteiry protein 3 is a nuclear protein and protein 4 is a transmembrane-protein. You plan to

Ouestions:

study howthe prot-eins are localized to their specific destination by creating the following mutations in the genes encoding proteins L-4.

a) Mutation A inactivates the SRP (signal recognition particle). Indicate how the localization of each protein (1-4) will be affected by this mutation. In additional state whether each protein will function as st.hr,u"{rCe. t*., .ft-r,yi:,r; fir,l} it does in a wild-type cell. il.v{r"*{ Cc"*!. "'.,^i Y*11'.,,a{- 5i$rrr,rl

----TroGin ll-U,j e {tf.{. ('t*"roli. p,b{L;..t lr"ny.-. ur3 Jrrln6rl pe 9t.6i4. Nc"a.r. I tv t {*.n({.ir, I'foteln I: fuc ee{ac{" CS;;].'. Protein2: Pn:t,^ ca,.\{ [{-.,*c^o{r',| ( I fr,5., ix c,5{r-ltl) :: V.tt aa+ !ro.^g \VT f*,'({,';,1 Profein 3: Vu off, -t , L,,r(r,r-r' r:r\id ra( hoy{- 1...r s,5rwr{ pep*.r,Lu Llgj'^rnl \^z,y C, ,.,,t.[ Protein3: Mr. e&-c-{ c S,(rrvr{ .:ep*.rrLl- fuo:'^ei1 \^/'Y G",,,"C{,;," l"r-rrkr-r' f,.r*i"."^J Protein 4: Pi.;d,"- C.,"iJ 6{- {r6:.n5ri:. k <i t* ce.ri r-1t.t1tl-ej,.t- r'{q,* * i., (,,r{,.1.,, {J ';.f *{ (
w

{.,

r-

f,*rJ,g.

C}",it \err,...

b) Mutation B prevents the fusion of vesicles to the golgi bq4y membrane. Indicate how the localizationof each protein (L-4) wlll be affected by this mutation. In additional state whether each protein will function as'it does in a wild-tyPe cell. .'ofo.g f !ra"s-,-- te77."s- p"s-(e,^l ca"l

]:',g.".l'+r'i;'t ffi{rrir:[_q6K+;;*"t.;tr|",i,,,c*.l.{.*..llr.tJ;lL}.6"tit'-:rf,''ln.({,*.'' ^r"^., protein 2: p,.r {.,.. .'.,..-J tr' f-r-j6.i[;'i,;', ix r ,i'v,e ,r, ..;J'l t:jn
protein3: Protein4:

#S*+-tl':,.j:l;'*;fiY
:,: ;1 ft*-""t

*"8
'

;t

rrr"r-*- i,. , .]{, { t_ rr,.r{(*r. u-.r. p^i,,Tli...1;,i.,:-r;.?i*-',,r**tl,xfl"'"'5'J',''i,,:-r-rr..-,<t.o(- rett "c.*r.n-< (<.,5",ti rrr ws3...,.-J-, v;rr {y-r Ar.rq*
;

'ol

tl,.-/tdJ:_f_

'
Summary of Lecture 2L:

7.012

Recitationl2 - 2O1l

Cell receptors and signaling: A cell responds to signals from its surrounding environment. In general, the signal molecule binds to a receptor, and conveys the message to the inside of the cell and then the cell changes its activity in response to the signal. These signals can be autocrine r (chemicals act on cells that produce them), paracrine (diffuse to and out of a nearby cell) or endocrine (hormones that travel through blood to reach the target cells).
Signal receptors can either be membrane bound or cytosolic. G protein-linked receptors are examples of membrane-bound receptors. On binding to their ligand, these receptors are activated and in the active form, they interact with a GTP binding protein (G protein) to activate it. Once in the active GTP bound state, the G protein activates additional components of the signaling pathway. Once the G protein hydrolyzes the GTP into GDP, the signal is stopped. The same signaling molecule can elicit different responses in different cell types. These responses may either be direct i.e. rt is the function of the receptor itself and occurs at the plasma membrane or indirect which are more common and involve second messenger like cAMP etc. In either case a cascade of signals gets initiated, each step adding towards amplification of the signal. The result is a change in the activities of the cell.

Ouestions:
1. Following is the schematic of a signal transduction pathway that is activated by the binding of Epidermal growth factor (EGF), produced by one cell type, to its specific membrane receptor on a target cell. The major steps involved in this pathway are outlined below:

. . . . .

EGF ligand binds to the EGF receptor. Ligand bound EGF receptors become actiae through phosphorylation and homodimerization. Actiae EGF receptor causes Ras to exchange its bound GDP for GTP and become actiae. Actiae Ras actiaates the kinase cascade (RAF, MEK and MAPK) through phosphorylation. This increases the expression of c-myc gene which results in cell proliferation.

EGF Receptor

Cytosol
ma membrane

'ltr*"]-T
c-myc transcription

Ve'1.",,*.ll,Ht)
a) What

would be the effect of each of the following treatments/mutations on cell proliferation?

Explain why you see this effect.

i. ii.

iii.

A mutation that results in the constitutive homodimerization of EGF receptor. :, Co,.sJi{r.dr.t' CLtt 0rol;{t t.n{it,.. Pak*(v,\ At wAvS c';.,-. Treatment of cells with a Rai specific phosphatase. ' :' Cr,i -0." tf*. r.fl"^ crn,.,w;,*f h-'.'cJ,v^fur{ . (iq ""u.,f ,^k,{il't,t Treatmenf of cells with PD9809, an inhibitor of MAPK.

A{,

tt/l{"

'

;:i (ull*F.of,{q-rrr'['u*'- fc'r.'''*.f' bt' f,ur"{,tr*{re{ (':'\"* { ;'J't,t1iri;f^-f &TAPru

I

v
\

\.i
2. You decide to engineer mammalian cell lines, each expressing a specific mutant variant of either the EGF ligand or the EGF receptor (EGFR). Nofe; A cell line is a single type of cell which continuously grows in culture.

Cell line-lhas a mutation that results in the deletion of only the signal sequence of EGF ligand. Cell line-2 has a mutation that results in the deletion of only the transmembrane domain of
EGFR.

Cell line-3 has a mutation that results in the deletion of both the signal sequence and transmembrane domain of EGFR.
You incubate each of these mutant cell lines with fluorescent antibodies that specifically bind either to EGF or the EGFR. You then observe these cell lines under the fluorescent microscope to study the localization of EGF ligand or EGFR.
a)

In cell line-l

i.

Where do you expect to find the EGF ligand (c\ll membranelcytosoU ceII culture mediumlV

Explain your choice.

{fu p^t";-,, is w,+ -h-sns l' cale I +" -t-h*- kn- a^ul, r\.w\o,vtJ .;q1ht. *l,uo it is ix +[L cgtorol
Cg+er.f Nr"
si qno,ul Je[urf-n C{- ww:e\,v-t

11.

3'k(' v
I

5'

j,

eoirdsffids to Sg"tl

@where

do you expect to see the base sequence that EGF (close to the 5' end or the 3' end)? sequence of

s/ffi;ilJ
rt
$' 1nc'r,[

.
.

CGs* -g* {"rre-

t *^r{,

In the mRNA transcript of EGF, where do you expect to seethe base sequence that corresponds to signal sequence of EGF (close to the 5' end or the 3' end)?

C(,rcJo +i". El
C (o

(-v,J,
(close to the N-

In the EGF ligand, where do you expect to see the signal sequence

r< -{ o +}.'-L U- +g r ,-rr r^w J

pi.p{,'e{+.

b) If cell line-2 is incubated with EGF ligand, do you expect these cells to proliferate? Answer as Yes/No and explain your choice. +L -("o,-,S*<r (t^,rr- t}o *^;. $J- ECFrt- wt-td ,^L.['J Dt,tr-{.,'""-.

If cell line-3 is incubate'a with EGF ligand, do you expect these cells to proliferate? Answer Yes/No and explain your choice.
c)

Nu I "C "ft"c p l^s,o. lg calr*{; ^ r{. f"G*'la. J J {u^c-{r.* ,lnt -e (lnttu- 'n -' E-4F-.

rn

(r-"src,

,rt- &*,t'

-thc

"e r**r'l

d 6c
as

JrtqI

fr,"",.C c(

plv-t

t)

exce-p{
cg{*<*1,

+L"'',{r*'{ tqpn

--ou,Lolo*v's{

lik*.t", t$" {i,,.=* rt ix

+|a

The wnt signaling pathway is one of the most important in biology. It is required.for cell proliferation and its inhibition leads to programmed cell death. As diagrammed below.
a a a a

(Practice Problem)

Wnt ligands bind

to

These receptors bind to disheoeled (Dsh) and actiuate its function. The Dsh inhibits GSK313, a kinase.

frizzled receptors.

GSK\B phosphorylates the transcription factor ftcatenin (Bcat). The phosphorylated ft catenin is unstable and gets degraded. Thus wnt signalinginhibits GSK3B, promotes ftcatenin stability and translocation to the nucleus. GSK3P can also be inhibited by addition of Lithium, acting through Pl3 kinasel Akt patfuaay. Cyclin D gene transcription is qctiaated by ftcatenin.

@ Wntw
ligand

receptor

Li

nucleus

. Gyclin

anscriPtio

gen6

.t

Cg

c\i'

-)

(etl
,or
o

h6

i c{

,',, u,

Compare the expression of ryclin D protein levels in the pairs of cells de3cribed below and explain vour reasoning. In eidl case both cells of the pail ale treated with wnt ligand, For your aniwers you should con{ider only those components that are shown in the schematic abooe or listed as bultet points in the exptanation ind explain only in the space prooided. State the changes that utill be elicitetl by the follouing mutations.
a)

t* ^.i;".tJ7'tu Mutant cells that express a constitutively active Dsh p"rotein' b)

-lrc..,s(',pf*.. (d,'",,{ [.e
C..., s-{,-1, c)

Mutant cells having a Frizzled recePtor that lacks its ligand binding domain. \.,Jn-l l;",c^J cc^-l (,;.1 . Si1no,l1",1 pc'l(-a.^ ,..[,{,{.1 .

(q t l,; I [,..I cr.. ,'{., ltJL" 1cl,.oIt"r

t't

Li {

\,iutant cells in which p-catenin lactt its CSXap phosphoryla'tion site'

{,r.t b.f'.' *r (o,^.-.{,i"'"'c- nprus:!1^ 'I' G<klg*> {o^r{,'i'i.'l <c'{'''i3 "[ P. t.'l -t (.^<{.i,-{.i* ('rcl'., D {2."5q.,p'f ...-', of $-c"{ -1
C^s{,'{*{;"E
wild type ce l tleated with Chiron
92060, a GSK3p

d) Untreated wild type cells with

F-<a.{ ca^r"'{ 6.c i^h,r,,{rr{ [.., 6<r3f .-> f"...r{,.{,,ji"r 6c{',.,,1) (o.' l{,+-.1,w *r a.., s..,p'1,;.'' o4' (q rl;x D,

inhibitor.

Y( Q.,t'!6 ir ,xk.(,tcl -.) (<L:p c.,,.'.-l ,v-.r:.rlr.,.r-lc [?'c"] ' ) P'(/,{ c-diu'.{)--4 ("^,1,ju.1.,;., CqrJ;^ | -f.n^<.r'p{i;-,',

7.0L2 Recitation 1.3 - 20 0

Summary of Lecture,22 & 23:

Viruses: Viruses are particles that consist of a protein coat surrounding a genome. This genome encodes the few proteins that a virus needs, such as the coat proteins and any other proteins necessary to get inside the host cell ind make copies of its genome. Viruses that have no lipid bilayer surrounding their coats typically dock onto some protein on the suiface of cells and inject theh genomes into the host. Viruses that have a lipid bilayer surrounding their coats typically fuse their own membranes with the host membrane, such that the entire viral particle is a6sorbed into ihe host cell. Ornce a virus is inside its hos! it can create many new viral particles. To this ind, a virus takes over the host machinery and uses it to make lots of coat proteins and lots of viral genomes. The genomes are then packaged into the coats and the new viral particles escape from the host cell. The viruses escape either by lysing the cell or budding off from the cell. Viral genomes can be single-stranded RNA" single-stranded DNA, double-stranded RNA, or double-stranded DNA. Rekoviruses: These are viruses whose genomes are RNA strands that are converted to DNA upon entry into the cell. Retroviral genomes contain a gene that encodes the enzyme Reverse Transcriptase (RT). Reverse transcriptase is a DNA pol1'merase that reads a strand of RNA as a template, and synthesizes the complementary strand of DNA. Retroviruses use RT to convert their RNA genomes into DNA such that these pieces of DNA can now randomly integrate into the host cell's genome. In this way, the virus hides out in a chromosome and gets replicated and passed on to all daughter cells of the original cell it infected.

Tumor suppressors genes and proto- oncogenes: These are normal genes that work in a regrrlated fashion in a normal ceilio properly control the cell rycle. The wild-type function of a tumor suppressor gene is to inhibit the cell rycle in any c-ell that is not supposed to be actively growing and dlviding. Both homologous versions of a tumor suppresior gene must loose iheir function to transform a no_rmal cell tb a cancerous type. The wild-typg function oi an oncogene is to promote the cell cycle in any cell that is supposed to be actively growing.and divid ing. One of thdtwo homologous versions of an oncogene must gain a function or increase its function for a cell to bicome cancerous. Normaliellular counterparts of the oncogenes are called the proto- oncogenes. Some of these genes are carried by oncogmic viruses and are designated as v-oncogenes. The v- oncogenes can be linked to poient promoters thit lead-to their inappropriate and high level expressionn leading_ to deregulated. cell division. Oire example is the Rous sarcoma virus (nSV). fhis_retrovirus infects the chickens, thereby causing Em -Here the viral genome contains a gene that it has stolen at some point from a host cell. This to acquire tumors. g"r," i" u. oncogene called src thai is involved in cell signaling. Th9 vlrgs ggrrigs a mutant v.ersion of src that produces an overactive form of the normal cellular kinase src. When RSV infects a celf the mutant src is iranscribed and translated" creating an overactive cell signaling protein that promotes growth and division in dricken cells to form tumors. Otheiexamples indude the avian leukemia viruslthat causes leukemia and human papilloma virus responsible for cervical cancer.
Cancer Viruses: Viruses can also cause cancer. RSV is a type of retrovirus that infects chickens, thereby causing them to acquire tumors. The way that RSV causes tumors is that the viral genome contains a gene that it has stolen at soime point in history fiom a host cell. This,gene is an oncogene called src and-it is involved in cell simaling. The i'irus carries a mutant version of src tliat produces an overactive form of the normal cellular kinase rrl. Wh"L nSV int"cts a cell, this mutant src is transcrib6d and translated, creating an overactive cell signaling protein that tells the drickenls cells to grow and divide. Ihus the chickenis cells form tumors.

Retinoblastoma: This is a cancer of the retina. In Familial retinoblastoma, multiple tumors in the retinas of both eyes occur in the fust weeks of infancy when the fetus inherits from one of its parents a chromosome that has its dB locus deleted or otherwise mutated. So in this form of the disease, a gerrnline mutation Plus a somatic mutation of t}e second allele leads to the disease. ln sporadic retinoblastoma a single tuinor appears in ore eye sometime in early drildhood before the retina is fully developed and mitosis in it ceases. In this form, both in}erited RB genes are normal and a single celJ musibe so unlucky as to suffer a somatic mutation (often a deletion) in b;th in ord.er to develop inti a tumor. Such a double hit is an exceedingly improbable event, and so only rarely will sudt a tumor occur.
Cancer therapy: Most cancer patients are treated with some combination of surgery,-radiatior! dremother-apy or immunotherify. Radiation aid chemotherapy have the disadvantage of destroying healthy as we! as malignant cells and thus'c-an cause severe side- effects. Drug desigrr is a very eipensive process. Drugs must be specific (i'e. they can't inhibit other proteins in addition to their targets or else they will cause side effects), must work at a low concentration (so that the amount that needs to be taken by the patient is feasible), and must not be metabolized by the patient either too quickly (so that taking the drug is ineffuctual), too slowly, or into toxic byproducts'

Questions:
1.

Why do some people think that viruses are alive and some people donlt?

r c^^ f.a l;fulr[t , organitld, L*c. No--lrv,n3 f ?<.f+ieJ', (a,a,{ rcf li csJt ea i4r oe,6, tte c,,LL[5, ne rvloholis,n, zlc.
Lirr*X popcl'lc-r', Co.n
Qat

(wl

adayt

2..The following sequence is a short viral gene from a double-straaded DNA virus that actually encodes three different proteiis. the sequence sl,rown is-from the kanscription start site to the transcriptionil end site. The upper strand of this gene is used as a template in transcription. C.+ai,, Ui(urr Stdrl bt6pa

-\

ca* t.< arrl lc,c c G, 1l+ rcxl" o* 5'| -TACTCTATCGCTTTAGCGGTATGCTATCAGAGCCATGCATGCATC- 3' ? 3' .ATGAGATAGC G UV\TC GC CATAC GATAG -5 ' **
l,

(ect,*t
l"&"

Te,xp

,tf|"{;$. -,f
a

,fu

a) How do you know that it is true that these three s

proteins are

ilced from alternative splicing of

single transcript?'\ica"t

s"{"r'\,I g r* a1/t q.n"""*.S 6cu E^c^ll crar{ (a}^f a.rS " T* $ 0 hc^tog rn-[rons clo.I{ hauw- 1+.4- rpoeu +i: ehr $*(f. fin r rp liara* pr-tr'$oi*s. ^n,]

b) How many amino acids long would ead:r of the three proteins be that are produced from this gene?
Pn

{'i^ | i

{ oots, pr.{ai.r 0;

ll

ac"'r

r..'(r-.* 3-'

ao,'s

c) What is a major advantage for the virus to using this strategy?

d) What is a major disadvantage for the virus to using this strategy?

3 d:{&** pM6;- p"o.t..rll f-" 6,n! ct^.\ k*p t-* <n*ll

t,

a s,^glg *,rwA- tro^eaa,-p'l , E4;ai^l

virus is an RNA virus that does not replicate via a DNA intermediate. The virus typically infects vertebrate epithelial cells. The following is a schematic of the influenza virus'
3. Influenza
Matrix Protean

[5i"g(<- $a1g -Jal.i,' cAn *le4 up atl (lx<. p*biu a4 on*!

Lipid Bilayer

Nucleoprotein

RNA

a) Influenza

i.

virus is unable to make more viral RNA within the host cells using exclusively the host cell proteins' Explain why this is so.

NeLnl*

a^

tUJk'd.t4e+'l+^4 tuak.gohtr,.,+r+a. Hest

doetil

Aor^e

#,'rl

ii.
b)
stranded RNA

Explain how the virus overcomes this issue and rdplicates its genome in the host.

Bric'ns

t\ or e-(\hd49 olh
in

lLuk-fup,
i$

Based on your answei to question above, would you classily

AruA po[4'"vr{'c In{luenza virus as a plis stranded/ minus

# f {"uf4ffM
(]

virus? (.o..*^

#t{*g.4-s6n uir"s

f;

{'*'

$rs+#*

rd

/t,.$,.d4

\irrs5

, 6r

e".{"rr \rir&l

p tr.+r'c.ac ,^.u

t.t 6^t iA a
2

{r+rrcl,o-*l Lllb

lq

A^AtolJ

4. Viruses can also cause cancer. One such example is the Rous sarcoma virus (RSV) that causes sarcoma/ a cancer of connective tissues. The virus does so by inserting itself near the cellular c-Src gene, a non-receptor tyrosine
kinase.

How does RSV convert the c-src to its mutated form? rC +c-...in\j r lr,le*ro.' s( c's.<- -*) v-src C- src-r conversion of c-src to its mutaGd form help the virus? How does the

g-t.q-

gr.k', I

r.{r c'o

<{,

{u{rW src

c.r.(',,,,e{,

I wkcl leulr {-

Con g{,

{o{,\rt- &;ll

d,ui

s..-, ao|

experiment Ras was the first oncogene to be discovered. Ras is part of a cell signaling V ir;, I Fe1t,c^{1.0,. pathway. ihe input for ihis pathway is an extracellular protein growth factor, and the output is to_induce. , iranscriftion of genes necessary for ihe cell rycle to occur. Ras is a GTPase that is active in the GTP-bound form but inaciive in tlie GDP-bound'form. Ras was discovered in the Weinberg lab via the following experiment. Human tumor DNA was cut into pieces, and each different piece was put into a different mouse cel1. The mouse cells were then grown in Petri phles. Only the mouse cell that took up the mutant allele of the oncogene could grow and divide enough to form a colony of cells'
5. Weinberg's famous a) Do you

think thdt the mouse cells had their own versions of Ras before the experiment began? If yes, do you

b) In this experiment, it seems that there was only one mutation necessary to make the mouse cells overproliferate. We know, however, that cancer results from an accumulation of mutations. Why then did this

.il :l;":'"";l:
work?

:","";i

ff T' !:!;;rH", [:' L? ;:,-#*

h'4fu I

experiment

-f\g- lsag. '*g c,at c*) 6rtr a ^v{a{,; ; ,. a p rv{: - t;4t o gt^Z. / wLitl 4hD{.ars a1 t do*.!ro,t'7 Cl,Se ^cul c) If a paiient hhd a tumor that was caused in part by mutations in Ras, do you think it would be a good
therapeutic decision to treat the cancer patient with a drug that targets and inhibits Ras?

d) Do you ihtnk it would be a good therapeutit deci3ion to provide this cancer patient wrlh a wild-type copy of
the Ras

Nop. A; 41alt J in pw| a, lyut i, 6^ TJuol{1 c." e s<e *1,'o.l peo(o- gwayre {"r- 11e. ^*l L4ll d,*r'ii.-. 'Tot q!;"4 V)T 'Qct vq/,ld Le- vtt91 lcu,l ,
gene?

think that this experimentalrtethnique would work to identify tumor suppressbr genes? 14ft{y or why not? l*Jo . ar^ o. dt" Jo k*nr(o,^ o c+{l , boJt', rclil.r a( 1L+ {u.m<r cu4pNJtcr 4t^g F',, C Lt ',, ^<1,,t ^LtJ. Zn lL; r rr9gr,^brJ, n.'ftr;n 4 ha1pc,', {o 'fltt. m *-.ss copter 6{ 6. hetinoblastoma is caused by a mutation in the retinoblastoma lumor sufpresi6r gene. There are several medranisms, which can cause a cell to lose its normal gene and thus be predisposed to develoP into a tumor. These may result in a "lgss of heterozygosity" or "LOH".
e) Do you a) What do you mean by LOH?

1+,+

A{ w"*tCt^V t.elp, nrt"A /Las grk,l'.{./ condi4v{'ia, lLat ^ c.(,'",45, Frui4i., c*tl ,^r / o,'lk. UY .oci^ w,J.l ( or."{"c r *l;r @n dt (uln.r'- ac4;";+i
+|.-:rcG.

b) Many clinicfar{s and scientists ari curr'ently tryinf to develop cancer treatrnents that are more specific and targeted than chemotherapy. If a patient had a tumor that was caused in Part by mutations in Rb do you think would be a good therapeutic decision to provide this cancer patient with a wild-type copy of the Rb gene?

lZb he*e.t.olg r vr''l -tnl".ri !.rI qc.r'(1pe i< +l-, qo*, 1l1+ c'tt i, bC (u*rg i,. 1\'+ {,'nz!,o- wt *pg ..'g +lc -Lbo^{ lv-4"*1qqau s becau x if ,s ,..--r -lta^ctt\ru.l,
Rb co*r*n i1 u^.+e,l t.o & qg ,,e , F* vt t1,w4 +k Fn[,cnJ * I
ur,al{,wal,sa

,G-

'L{ a gc'"'a-/.Ju ""'fa'{io"' n" lo*qcr

it

V.r.

,tL

6,1N

L*ty ,,,6" '{lw A6 fi**' w**lt/ {tu*l *,*f (nncr,r e$tk#ou*tL, 0-u'.,t,n,4*li< W{d.efrd, Lor/r.r-f, t'{ ^ I,:{ rhc F.e at. {dir * l.l *l** i { fq{,* r | ( ,, 3

^ vI

repi*' "( 4k

p6

7'012 section 14 - 2011

Main concepts from Lectute 25:

Cancer therapy: Most cancer patients are treated with some combination of surgery radiatioo chemotherapy or immunotherapy. Radiation and chemotherapy have the disadvantage of destroying healthy as well'as malignant cells and thus can cause seve_re side- effects. Drug design is a veiy expensive process. Drugi must be specific (i.e. they can't inhibit other Proteins in additioil to their targetg or else they will cause side effects), must work at a low concentration (so that the amount that needs to be taken by the patient is feasible), and must not be metabolized by the patient either too quickly (so that taking the drug is ineffectual), too slowly, or into toxic

byproducts.

Drug Designing: Drug design is a very expensive piocess that is the focus of many biotechnology companies. Drugs can be compounds that ifibit the overactive mutated forms of proteins that cause dominant diiorders. Drugs can also treat recessive disorders (generally iaused by the loss of function in genes) if the drugs are compounds that provide the patient with whatevei protein they are missing. Successful drugs must be specific (i.e. they can't ifibit other proteins in addition to their targels or else they will cause side effects), must work at a low ioncentration (so that the amount that needs to be taken by the patient is feasible), and must not be metabolized by the patient either too quickly (so that taking the drug is ineffectual), too slowly, or into toxic byproducts.
Chronic myelogenous leukemia (CML): This is a myeloproliferative disorder characterized by increased prolifiration of the granulocytic cell Iine without the loss of thet capacity to differentiaie. Consequently, tfre peripheral blood cell profile shows an increas-ed number of granulocytes and their immature precursors. CML is an acquired abnormality that involves the hematopoietic stem cell. It is characterized by a cytogenetic aberration consisting of a reciprocal translocation between the long arms of cluoirosomes 22 and 9 tt(9;22)1.-The translocation results in a shortened chromosome 22, an observation first described by Nowell and Hungerford and subsequently termed the Philadelphia (Ph1) chromosome after the city of discovery. This translocation relocates an oncogene called ABL frorn the long arm of chromosome 9 to a specific breakpoint cluster region (BCR) in the long arm of chromosoire 22. The ABL oncogene encbdes a tyrosine protein kinase. The resulting BCIi/ABL fusion gene encodes a chimeric protein with strong tyrosine kinase activity. The expression of this protein leads to the development of the CML phenotype. Gleevec: is a drug that works against the CML. The drug Gleevec specifically inhibits t}re kinase activity of Abl, thdreby ceasing ihe aberrant phosphorylation in CML patients and sending their cancer into remission.

Questions:
1.

Consider

patient who has CML, and answer the following questions.

a)

body? 11o.

Would the Philadelphia chromosomal translocation be present in all of the cells in the patient's Tlprcnlg .1++ *ronsl$c^liza etft,,l i< r r-N- antt QxcL*t,V *o euhtL blo"l
be present in all or the cells in the patien/s

blood system?

,,*:lL1;Sfr[l$1*'.*.-*.mal translocation all

c)

How maiy independent tiines did the-Philadelphia chroinosomal translocation occur in the patient? -;-1,,, fr"r3le64'{,o^ i haApt'. ,t cli w^a rarc,

t/hil4 t^)h;u, blonl e{s I y rrr,,locg-te I ax, cir a^la4d rn 1[r<- bl*,I, no( 1+& c.dls ,n 4+L LG.,l LrcuLto",l qtis,e+) r^,':',I/ erftlt-l *l:1.P|'l'&tp{':a (A"o*5'^",

hap

d) Could the patient pass CML onto his/hei kids?

W l..i-{r,

penel o^tL u^l in a srngb (+tl ,

5e rF '{

p rc|"

U,,

f 4r-'^l"rq-lz r c",*- (e-,*4ig c+Llt, a! opltn*el 1o G"nott u co"'yo i'"t'e^ o( s,*oke cert! 6t]' $or.^ a1,ls [up.-' / "g6) .
6L-.t
czll5

pa*<+o{

c.,,

{*

p^ge,g1

2. For each of the

following medicines / treatments, answer these three questions: work for?

a) Which disease / condition does this treatment

b) What is the target of this'treahnent?

c) Why does this treabnent

work?

Herceptin (an antibody directed against the Her2 growth factor receptor expressed
breast cells)

in

Gleevec

fle,c;g*in

A) T,e*|r,yga 't2- bru7,4 5.a^ct-r b) Tary'tt -Hu- HF-1L1 i^-*h {*o4a *tcyLor o) T"^h166fu.6..,c(;". o( HEn-2 6s lLat tf c.e,\nct prv,oe- cctt d,ujstin,
Gter^lc,c

ct) Tre-{-+r-J q.
b

Cor,U

To"611 '{J,a A$L-Sq- f,m;^ pro{r.a Ore-lat [, *r",ct.cn'ta.

L["ro,-so,*s Q ay,l X7,
Lnh;br-lt

st

C)

4\- *lros,:w k,nae2 6-cl,v,t, '? ,+AL-B|JL wi.n 1,-&..,. 5" *:Lel it Ct a-,,l p **L ccrl divislon u.^toa(rwtl6ltlq
,

/-

7.012 Section L5

'20LL

Ions can move across membranes through pumps and channels. These channels or pumps are integral membrane proteins that cross the membrane via transmembrane domains. Pumps are ATPases that set up the conc-entration gradients of ions across cell membranes, such that K+ is high inside cells and other ions (such as Cl-, Nai, and Ca++) are high outside cells. A membrane potential is only set up by ions that move freely across the membrane through open channels, creating one side of the membrane that is more positive. This movement of ions does not dissipate the concentration gradient because the number of ioni that move to generate a membrane potential is very small compared to the number of ions that need to be pumped to create a concentration gradient. Ions are free to move across the membrane through open ion channels. Two forces act to dictate this movement - the concentration gradient and the membrane potential. Ions move down their concentration gradients through channels, and ions move towards the side of the membrane that harbors the opposite charge.

Main concepts from Lecturc 26:

Neurons are the cells of your nervous system that make connections with each other in order to allow you to think and feel. Each neuron has a cell body (where the-nucleus is), dendrites (multiple small m.embrane projections that receive signals from o-ther cells or from the environment), and an axon (one large membrane projection that sends signals to other cells). The place where the dendrites of one neuron communicate with the axon of another neuron is called a synaPse.

Action potentials are the characteristic changes in membrane potential that-propagate down the length of axons, unidirectionally at each point on the membrane from the hillock to the terminus. An action potential begins when a threshold membrane potential is reached at the axon hillock, and voltage-g-ut"9 irla+ channe"ls are induced to open. Once Narrushes iry the inside of the cell becomes positive, and this induces the voltage-gated K+ channels to open. Thus K+ rushes out, restoring the membrane potential back to normal (roughty -60mV). Action potentials do not vary by amplitude; the maximal membrane potential is always +SOinV. Instead, action potentials vary in frequency. The- axons of motor neurons are ioated in a myelin sheath that ailows action potentials to travel down axons faster by allowing them to jump from node to node between patches of insulation.

Questions
1. Shown below is a plot of axonal membrane potential as a function of time, measured at a single position on the axon.

r{+r

+50mV

? a

* t

-70mV

{B

t
Time

For each of the three time points indicated, fill in the following table. Name the phase of membrane potential that describes the time point, the channels/pumps that are involved, and explain how these channels/pumps maintain that phase.

Time

Phase

All the ion

channel/ Pump involved
lvo"1l

Explanation

point
A

k+

/\T?o<<-

R<-s{,^
B

(a,x rtTPact

k* (1,".,o I

Es4atLEIvs h,'{h taq**Jrsr#,^r tt6 No-j & (o'74 ov4<,ahn a^1J d,1i' /(.* /'nL(clralioa inlflLQ", t*to trl*^hli <lu.t nC,s*i na n(,,'|tbr//V-. na&nfi; al

VolS^**-\o&4
hfotariza.{to,.'

U*r

c{nanrncl

kll.^t < Na+ {o rra<( tnlo 4ia. ^wv{ ir. Cr.erarrvrq 4'4. &) C@T* i^(tcly
,*t[o^,,

Cull t

Vuf{aX6,*Xakd
Pepo\orir,o**i*.,

K*

cna*rvl

lC* 4r ru(( A1,f o* *A-e ngl.ry"o5 I b.,^X,-^g *l'l,,t, rv"<icLr brclc J.z i't s tv,r*+ul (- ) d',eg+

2. For each of the following cases where neurons are stimulated to the normal threshold level, draw on the axes below how an action potential would look relative to a normal action potential.

i. ii. iii.

Voltage gated Na* channels fail to close. Voltage gated K* charurels fail to open. Voltage gated Na* channels never open.

()

Th{*, ne-s{,hj rns.n(rar-r- prtt^{,'^t CI{ -?o^V c}opendt or ^ <4"*i N"{ g^J'L*l i,.cid<-a*al ovlstck 0{- W c.eXl, 5,h.& 'll,E- fl6( Nf che*ry,tr ${,J, clox-, +i.e Not

q^ol +M n{ uv 'e(ailibriu^ po-6"{"iul gral,+..l wr\\ e1l.[,'r,a{r, 6q l.J.r 5{ecP , *?0-'V (b* *"cl' LiXher is u^Lu'-^.} 6C k,gl*- -FL^^

o'il ^t('alty

-fL"e ;t s+ttt -f t'{ Dp*n fot 6t) \Jh;t4 VG K+ cLa^^^"1t Cr*j of"o,
pl^.,c' Lqr,

c{u,ana'} "'---/

g,{ilt le4 b4 r'onr Sl";[4 o^( o$*bV tltpotw'teA n-e'.onJ i{i''(-{ *{o q*f 6^dc +" +I<- *lp*r/ ne t{t^6 p'{ot*'!"1' &ol:c,s c^ nucrr [o^3.- -t,^s,
Euw..

i;; )

;(

no{

pob^I,'al tw^ct"ts An'i"'ltl1 '&cL'!i''po'[aurf'c"l U po<tibi Le1uc'v dap'ta-iYe"lJon &'pa^'I( a^ nCL IJ*l c}unw(s
w,**^1rv,,,4,-

2
l,

7.012 Section 1.6 - 2011 Main concepts from Lectures 25 & 26 A neuron receives signals at its dendrites and sends signals down its axon. A synapse occurs wherever the axon terminus ofone neuron meets the dendrite of another neuron (or a muscle cell). At a synapse, the electrical signal of an action potential is converted to the chemical signal of a neurotransmitter, and then this is conirerted back into in electrical signal in the post-synaptic cell. This occurs because, when an action potential reaches the axon terminus of a pre-synaptic cell, voltage-gated Ca++ channels opery and intraceilular calcium induces the exocytosis of neurotransmitters into the synaptic cleft. The exocytosed neurotransmitters then bind to receptors in the dendrites of the post-synaptic cell. These ligand-gated ion channels are then opened, leading tb changes in membrane potential that are summed at the axon hillock. If the sum of all these changes is greater than threshold, the post-synaptic neuron will fire an action potential, first at the hillock and then at each subsequent location down the axon to the axon terminus.

Neuromuscular junctions are synapses where a nerve cell contacts a muscle cell. The neurotransmitter that is released from the neurons al neuromuscular junctions is Ach (acetylcholine). The release of enough Ach will trigger the muscle cell to contract. Ach is cleared from the synapse by an enzyme that cleaves Ach called Ach esterase. Nerve-nerve synapses use mdny different neurotransmitters. Some neurotransmitters are excitatory; their receptors allow the flow of ions that causes the inside of the post-synaptic cell_to become more positive, making the cell closer to the threshold needed to fire an action-potential. pther i'reurotransmitteirs are inhibitory; their receptors allow the flow of ions that causes the inside of the postsynaptic cell to become more negative, making the cell farther from the threshold needed to fire an action potential.
There can be many, many inputs to a postsynaptic cell, and the summation of all of these ilputs oc:YJS across the cell body. If the excitatory inputs are sufficient to depolarize the membrane at the axon hillock, the voltage-gated Na+ channels at the axon hillock will open and an action potential will be generated.

In the adult, the cells of most organs and tissues are differentiated and do not divide, but have a finite lifespan. Examples of such tissues are the skin, blood and intestinal lining. Cell replacement is acc&nptshed 6y specialized stem cells, which are able to self-renew throughout the life of the organism. Stem c'ells are pirtiy determined, while progenitors are more determined. Stem cells reside in a specific cellular enviro^nment, called a niche. Cell-cell signaling between the niche and stem cell regulates when the stem cell will divide and whether it will divided symmetrically, to create two stem cells, or asymmetrically, to create a new stem cell and a progenitor cell Theprogenitor goes.on to divide further to create *ore piogenitors which later differentiate into cells needed for replacement in a specific organ. Progenitors hurrE h-it"d division capability. Each stem cell type has specific potency. The zygote is the onlf naturally totipotent cell (that can form all cell types). Embryonic cells are multiPotent (pluripotent, many fates), while adult stem cells can be pluripotent.but are oftenbipotent or unipotent. A set of cell typei that arise from the same stem cell (oi progenitor) is a lineage. Progenitors,mayhave less potency thin the stem cell from which they arise. Stem Cells may be able to repair or replace the damaged tissues in d.iseases i.e. stroke, Parkinon's diabetes, muscular dystrophy and heart disorders. However, stem cells are rare, and difficult to isolate. Recent work has shown that stem cells can be generated from adult cells by expression of a small set of genes. Dopamine is one of major neurotransmitters in the mammalian brain that regulatg.t.-"o4t cognition and locomotion. Dopamine acts on two types of receptors: the D1 receptor is an inhibitory ligand-gate.d channel, the D2 te."ptot activates the G pioteins, and is excitatory. The released neurotransmitter is taken back into the presynaptic cell, for re-use.
1.

Questions

a) On what part of the neuron are the dopamine receptors localized?

Arrrn (, eco-;u*. eL Siq^c"l b) The D1 receptor is inhibitory and transportsk+ ibns. Would K+ be moved into or out of the postsynaptic c6ll? Explain the mechanism underlying this inhibitory effect.

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2. GABA is a major

inhibitory neurotransmitter in central nervous system (CNS). It a_cjs by binding to ^ GABA-A receptbrs that are ihloride channels and GABA-B receptors that activate K+ channels via G proteins.
a) In response to GABA, would you expect both types of receptori to activate ion flow with the same time course?

Explain.

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b) K+ concentration is high inside the neuron, while Ca2t, Na" and Cl- ion concentrations are high outside. Passage of Na* into the neuron is responsible for an action potential.

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in or out of the neuron?

i.

In what direction will ions flow when the GABA-A recePtor is activated

,f o*^ ;{ r r*ntu-n{rca.{r*in etatfis. ,l , P ^.gc^"$an ii. How does this flow alter the likelihood of an action potential in the post-synaptic neuron? Explain' Ct- ;rn0u x ,^o.ke-s *L +l'e hanon u^sre ne{a*te, G^4 '",<,rjgo6 clZcrcoffr#rc p1"ra"$re,tt 1x{:uo't,:r^1, Ftk{ '{ ftL.'*'il {;-^ +t f^**'-tn[ iev4f. c) Strychnine is a toxin thit competitively iirhibits the'etfects of GXBA, often_cafising lethal muscle contrlctions. The graphs below iho* a t-efer"nce "normal" action potential. Draw the approximat tracing of membrane potential changes you would observe in the post- synaptic cell, after treatment with GABA alone or GABA plus strychnine. fq.f.re ,.5 qr^.es-t;^_. .

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d) Give two possible mechanisms by which

neurotransmitter is removed from the synapse?

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e) You make a synapse between two neurons.

(en,lo cetr*o e i'* J Neuron awh^en stimulated secretes epinephrine, which can bind to and activate ligand gated Na* channels. This neuron expresses the GABA-A and GABA-B receptors. Neuron 2 when stimulated secretes GABA, which can bind to GABA-A and GABA-B receptors on a post-synaptic neuron. This neuron expresses the epinephrine receptors that act as ligand gated
Nan channels.
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3. Classify the

following as totipotent, pluripotent, bipotent, unipotent or differentiated:

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Cells that can differentiate into many cell type*s. :7 Plr^ri po"f+"1 *G*i ' pc *e rr{ A zygote that is about to start cleavage. =) Celis isolated from developing brainlortex that ploduce motor neurons or interneurons when 15, po*+al provided with specific growth factors. :) cells that form the hematopoetic, bone, cartilages, muscle and fat cell Human *"r".r.hy-al

=? Intesiinal stem."tis-*iuiteptenish the differentiated intestinal crypt cells.

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lineages.

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A patient has a rare recessive genetic disorder due to the loss of a specific gene function. This disease causes a decrease in immune furiction. The patient receives a bone mirrow transplant which is successful in alleviating the symptoms of the disease, such that patient can lead a normal life.
a) Why does the bone marrow transplant relieve the patient's symptoms?

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b) your patient marries a man who happens to be carrier for the same genetic disorder. What is the probability that their offspring will inherit the disease if ^ i). the couple had a ihild before the patient had any bone marrow transplant no longer showed any {\ the couple had a child after the pitient had. a bone marrow transplanf and / symptoms of the disorder?

Explain your answers.

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7.012 Section L8 - 201L

Main concepts from Lectures 28 &,29

In the adult, tlie cells of most organs and tissues are differentiated and do not divide, but have a finite lifespan. Examples of such tissues are the skin, blood and intestinal lining. CelJ replacement is accompiished by specialized stem cells, which are able to self-renew throughout the life of the organism. Stem cells are partly determined, while progenitors are more determined. Stem cells reslide in a specific cellular environment, called a niche. Cell-cell signaling between the niche and stem cell regulates when the stem cell will divide and whether it will divided symmetrically, to create two s-tem cells, or asymmetrically, to create a new stem cell and a progenitor cell The progenitor goes on to divide further to create more progenitors which later differentiate into cells needed for ieplacement in a specific organ. Progenitors have limited division capability. Each stem cell type has specific potency. The zygote is the only naturally totipotent cell (that can form all cell typ-ei). Embryonic cells are multipotent (pluripotent, many fates), while adult stem cells can be pluripotent, but are often bipotent or unipotent- A set of cell types-that arise from the same stem cell (or progenitor) is a lineage. Progenitors may have less potency than the stem cell from which they aiise. Stem cells may be able to repair or replace the damaged tissues in diseases i.e. stroke, Parkinon's diabetes, muscular dystrophy and heart disorders. However, stem cells are rare, and difficult to isolate. Recent work has shown that stem cells can be generated from adult cells by expression of a small set of genes.

A chimera is a single organism (usually an animal) that is composed of two or more different populations of genetically distinct cells that originated from different zygotes involved in sexual ieproduction. Iithe diffeient cells have emerged from the same zygote, the organism is called a m-osaic. Chimeras are formed from four parent cells (two fertilized eggs or early embryos fused together). Each population of cells keeps its own character and the resulting organism is a mixture of tissues.

Questions
1. Classify the

following as totipotent, pluripotent, bipotent, unipotent or differentiated:

a) Cells that can differentiate into many cell types. p[u.ip ok,a{ b) A zygote that is about to start cleavage. T"*i po**n{ c) Celli-isolated from developing 'd;

provided with specific growlh fictors. B, p "*evr{ Hu*un mesenchymil cells that form the hematopoetic, bone, cartilages, muscle and fat cell Iineages. ?lvr;p"**q4 e) Int-estinal stem'cells that replenish the differentiated intestinal crypt cells. Un; po-le,n{ f) A neuron. Di t{b...e#,''.""k4

brainioltex

thad

produte motor neurons or interneurons when

b) Using intestinal cells, you do a pulse chase experiment by first putting the cells in a culture mediuri containing radioactive dttp for a short time and then allowing them to grow and divide in medium containing a lot of non-radioactive dTTP.

for performing this pulse chase experiment? a) -' What is the rationalei+n": iL"q']r Jde ;,1' U+ 4h{- rf,rl s .:tkt re"{,t;^ 4lc b) What is thd relationship of the rapid turnover of a cell with cell division?

16{*r6(4lv'{, lo,tfra

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a disease causes a decrease in immune which is successful in alleviating the symptoms of the disease, such that patient can lead normal life. a

a) Why does the bone

marrow transplant relieve the patient's symptoms?

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i.

The couple had a child before the patient had any bone marrow transplant?

5 E{
ii.
Explain your answers.

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The couple had a child after the patient had a bone marrow transplant, and no longer showed any symptoms of the disorder?

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Outline the steps required to make a mouse that is chimeric (or mosaic) for the two coat colors.

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7.012 Recitation L9 - 20lL of Lectures 30-32 Summary The immune system protects the body from foreign entities that have invaded it, such as bacteria and viruses. It does so by making proteins called antibodies, which recognize and direct an attack against foreign entities, which are often called antigens. A person's body produces billions of different antibodies. Many of these randomly generated antibodies have the potential to recognize proteins that are made in one's own body. The immune system however has a way of distinguishing antibodies that act against "self" antigens from that against "non-self I foreign" antigens. It dbes so by destroying or preventing the proliferation of any antibody-producing cell that recognizes a self-made antigen.

Our bodies make billions of different antibodies. These are proteins and thus are encoded by genes. However, our genomes contain less than 30,000 gene9. So it is not possible that we would have a different gene to encode for each different antibody that we generate. The explanation for this is that there is a cluster of segments of genes (called V, D, and ] segments) inthe section of the genome that encodes antibodies. Every antibody-producing cell rearranges these DNA segments to join one V segment to one D segment to one j segment, thereby creating one gene thit makes one antibody.-Every different antibody-producing cell rearranges tfis cluster of DNA segments differently. Thus all antibody-producing cells contain only one gene that encodes an antibody, but every cell contains a different arrangement of that gene thus leading to antibody diversity. Other than cells of immune system, every cell in your body contains the exact same DNA as every other cell because no such rearrangement occurs.
_

The immune system provides humoral and innate immunity. There are three important cell types that play critical roles in the humoral arm of the immune system - phagocytic cells, B cells and helper T cells (T,r). When a phagocytic cell encounters a foreign particle in thebody, it eats that particle and displays pieces of the particle on its surface using the cell-surfacetisplal protein MHC class IL A T; cell has a protein on its cell surface called T cell receptor (TCR). Every Tn cell has a different TCR that recoghizes a different antigen (because every T" cell rearranges the TCR gene in the genome differently, in a mechanism similar to VDj joining). If u T11 cell encounters a phagocytic iell displaying the antigen recognized by its own TC& then that Ts cell undergoes . itonit expansion. fhe -i"y T' cells of this kind thi:n go searching fo-r a B cell that recognizes thesame antigen as that Ts cell. If the Ts cells find the right B celf then that Bcell undergoes a clonal expansiori These B cells make secreted antibody that recognizes and attack the foreign antigen. The immune system displays memory; we know this because the second time the immune system encounters an antig-en, the response of B cells and T cells is-faster and stronger. The principle behind vaccination is to expose an organism to some of the antigens of a harmful ioreign particle that will spark the immune system's memory in case the actual entire foreign particle ever invades that organism.

Another type of immunity is the innate immune response. This responseis however not antigen specific and does not generate immune memory. Thi.g cgmqences immediately upon pathogen entry and involves phigocytes and cytotoxic T cells-(Tc). If-the phaSocytes cannot rapidly elirninate pathogen, infi-ammation isinduced with the synthesii of rytokines and acute phase proteins.

HIV is a retrovirus that infects the T' cells of our immune system. HIV gets into our Ttt cells by docking onto a protein called CD4 that our T" cells have on tl9ir surface. Our Tg cells have CD4 on theii surfacei because CD4 helps T' cells recognize the MHC class II molecules on the surface of macrophages, which is the job of a T,-, cell. However the HIV virus has evolved to have a glycoproiein-on its surface thit binds to CD4, thus targeting HIV to T,, cells. T_is glycoprotein Itio hus the ability to fuse the lipid bilayer of HIV to the cell membrane of our Tg cells, thus dumping the conlents of the HIV virus into our T' cells. The HIV virus harms our T" cells, thereby lepleting our immune systern and therefore our ability to fight t-he virus. HIV also q.tiit ty due to it hiving a reverse transcriptase that is highly mutagenic. This mutates "ury allows the fitV to be constantly changing the amino acid makeup and the shape of its viral proteins so that our immune system iannot gain immunity to the HIV.

1. Viral infections can be treated using anti-viral drugs, or they can be prevented in the first place through the use of vaccines. Some vaccines are just ilrjections of viral particles that have been inactivated in some way (such as extreme heat). Other vaccines are injections of a single viral protein that has been purified and produced using recombinant DNA techniques. How does a

Questions:

."

vaccine

work? / y) *txcc,hg, iE *n i,xa-c(,,)a4.(J I a#e^"tr*hJ px'$k"1*" *$tl4,{ w)'tl el;ci-6- a.r* s4.g{np*nlg ttr*,rAaw6.* th{.1tew'rc, #*"ttn "fdl*# **.0^aorf t4'[.{ J &A{- *ot"r'{t" ++" rhaC{rt e pa,4k qaa t r e-*sil1 C(eara/ , ^utd.
Pof*r

2. Antibodies are very useful reagents in molecular biology. To make an antibody, one injects the protein of interest into an animaf (e.g. mouse or rabbit) and then extracts the antibody later. r

rl (lo^a.l at4i$61'lr7r tro.- ;s' oLqlrrl ,r.en ,etgttt! F'-c+tts a^" *ha 4tryl tl',t{tt1*^' ipi*op"et e]a +ld, P'oki.',.'p(,v'(e*,*, ")Fir;U;"Trrv.n""il"liuoaydiffeiiromamonoclonalantibodyi r+r.qtti2lg cr sirtqlg Lp;{"pe, {n^ On B-att r c'^{ A ^o^. c(an4,, 6.+; b ,.tl,a cr.nt r b) Do you think that immune reJponsd would be specific for only that one s'pecific kind of rYbbit '
antibody? Why or why not? \lo , +|^*- pr.q-tl ,a of

3. The cellular arm of the immune syst6in employ-s cytotoxic T lymphocytes (T") and natural

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cells. The T. cells can recognize the viral infected body cells. a) What proteins are involved in the presentation of the antigen to the Tc lymphocytes?

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b) Explain why the Tc lymphocytes d6 not reiognize an infected cell if the virus is latent i.e. viral capsid protein does not occur.

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4. Shown below is a schematic of the

production of a heavy chain polypepti4e io, un antibody' At the top is the chromosomal arrang-ement found in an immature B cell, at the bottom is shown the heavy chain polypeptide. pro.bsindicatea by each arrow. Choose the one best option for each from: - Label ih" homologous recombination J transcri Ption ,./ translation J translocation d ligation V t DNA rearrEngement V
,

splicing

a) Indicate on the diagram below where you would expect to find each of the following

components: ^

Promoter
start codon

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.

Transcriptional termina tor

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b) Indicate on the diagram below the variable and the constant region of the heavy chain polypeptide and the N and C terminus of this polypeptide.

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5.

Why do the HIV patients succumb to simplistic infections?

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symptoms. Explain ho* an unrelated infection that ictivates the humoral response pathway may lead-to the development of a full - blown HIV infection.
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