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ueIIPur

tur'j

eursoul

oulsouapv

gene.

The

sequences of the

gene

andprotein

given

in

iii,i:*l

:.;.**

are

conserved in

several try-

panosome

species. How

does this

gene

function?

The

coxll mRNA

has an insert

of an addi-

tional four

nucleotides

(all

uridines)

around the

site of frameshift.

The insertion

restores

the

proper

reading

frame;

it inserts

an extra amino

acid

and changes

the amino

acids on either

side.

No second

gene

with this

sequence can

be dis-

covered, and

we are forced

to conclude

that the

extra

bases are inserted

during

or after transcrip-

tion.

A similar

discrepancy

between nRNA

and

genomic

sequences is found

in

genes

of the SV5

and measles paramyxoviruses,

in these

cases

involving

the

addition of G residues

in

the

mRNA.

Similar

editing of RNA

sequences

occurs

for

other

genes,

and includes

deletions as

well

as additions

of uridine. The

extraordinary

case

of the coxIII

gene

of Trypanosoma

brucei is sum-

marized

in

ai*:iRl

iI.I:.

More than

half of

the residues

in the wRNA

con-

sist of uridines

that

are not coded

in the

gene

Com-

parison

between

the

genomic

DNA

and the

mRNA

shows

that no stretch

longer

than seven

nucleotides

is represented

in

the mRNA

with-

out alteration,

and runs

of

uridine up

to seven

bases long

are inserted.

What provides

the information

for

the spe-

cific insertion

of

uridines? A

guide

RNA

con-

tains

a sequence

that is complementary

to the

correctly

edited

mRNA.

l'i*ijFlt

i,r.ii-i

shows a

model for its

action in the cytochrome

b

gene

of

Leishmania.

The sequence at

the top of the figure

shows

the original transcript,

or

preedited

RNA.

Gaps

show

where

bases will be inserted

in the

edit-

ing

process.

Eight uridines

must be inserted

into

this region to

create the valid mRNA

sequence.

The

guide

RNA is complementary

to the

mRNA for

a significant distance,

including

and

surrounding the edited region.

T\zpically

the

com-

plementarity

is more

extensive

on the 3'side

of

the

edited region and is rather

shon

on the 5'side.

Pairing

between

the

guide

RNA

and the

preed-

ited RNA leaves

gaps

where

unpaired

A residues

in the

guide

RNA do not find

complements

in

the

preedited

RNA. The

guide

RNA

provides

a tem-

plate

that

allows the missing

U residues

to

be

inserted

at these

positions.

When

the reaction

is

completed

the

guide

RNA

separates

from

the

mRNA,

which becomes available

for

translation.

Specification of

the

final

edited

sequence

can be

quite

complex.

In this example,

a lengthy

stretch of

the transcript is

edited by

the insertion

of a total

of

l9

U residues,

which

appears

to

require

two

guide

RNAs

that act

at adjacent

sites.

The first

guide

RNA

pairs

at the

3'-most

site, and

the edited sequence

then becomes

a

substrate for

further

editing by the next

guide

RNA.

The

guide

RNAs

are encoded

as indepen-

dent transcription

units.

fg*LlFtfl

;,-:.i::

shows

a

map of the relevant

region

of

the Leishmania

r:'i:r-iii{

il,!li: The

mRNA for

the

trypanosome

coxll

gene

has

a

frameshift

retative

to

the DNA;

the

correct reading

frame is

created

by the insertion

of four

uridines.

i;if;l.ifii;-:

rr

1'"1:1 Partof

themRNAsequence

of T.brucei

coxllf

showsmanyuridinesthatarenotcodedintheDNA

(shown

in

red)

or that are removed

from

the RNA

(shown

as

T).

ISSLGIKVE

AUA

UCA AGU

UUA GGU AUA

AAA GUA

GAG A

frameshift

AUA

UCA AGU

UUA GGU AUA

AAA GUA

GAU UGU AUA

CCU GGU AGG

UGU AAU

RNASEOUENCE

I

S S L

G I K V D

C I P

G R

C N Proternseouence

UAUAUGUUUUGUUGUUUAUUAUGUGAUUAUGGUUUUGUUUUUUAu,J,o,,,,,UAGAUUUAUUUAAUUUGUUGAU

A

AAUACAUUUUAUUUG

UAAUUUUUUUGUU

qEE

U

ru

UUGUGUUUUGGUUUA

UUUUUUGUUGUUGUUGUUUUGUAU

722

CHAPTER

27

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Inteins have characteristic leatures. They

are found as

in-frame insertions into coding

sequences.

They can be recognized as such

because

of the existence of

homologous

genes

that lack the

insertion. They have an N-termi-

nal serine or cysteine

(to provide

the

-XH

side

chain)

and a C-terminal asparagine.

A

typical

intein has a sequence

of

-I50

amino acids at

the N-terminal end and

-50

amino acids at the

C-terminal

end that are involved in catalyzing

the

protein

splicing

reaction. The

sequence

in

the center

of the intein can have other

functions.

An extraordinary

feature

of

many inteins

is that they

have homing endonuclease

activ-

ity. A homing

endonuclease cleaves a target

DNA to

create a site

into

which the

DNA

sequence

coding

for the intein can be inserted

(see

Figure

27.l2in

Section

27.5,

Some

Group

I Introns

Code for Endonucleases

That Spon-

sor Mobility).

The

protein

splicing and

homing

endonuclease

activities of an

intein are

independent.

We do not

really

understand the

connection

between

the

presence

of both these

activities

in an

intein, but two types of model

have been

suggested.

One

is to

suppose

that there was orig-

inally some

sort oI connection between

the

activities,

but that they

have

since become

inde-

pendent

and

some inteins have lost the

hom-

ing

endonuclease.

The

other

is to suppose that

inteins

may have originated as

protein

splicing

units, most of

which

(for

unknown

reasons)

were

subsequently

invaded by homing endonu-

cleases.

This is consistent with the

fact that hom-

ing endonucleases

appear to have invaded other

types of

units as well,

including, most notably,

group I introns.

l@

Summary

Self-splicing

is a

property

of two

groups

of

introns,

which are widely

dispersed in

lower

eukaryotes,

prokaryotic

systems, and

mitochon-

dria.

The

information necessary

for

the

reac-

tion

resides in the intron sequence

(although

the

reaction

is actually assisted by

proteins

in vivo)

.

For both

group

I and

group

II introns,

the reac-

tion

requires

formation of a specific secondary/

tertiary

structure

involving shott consensus

sequences.

Group

I intron RNA creates

a struc-

ture

in

which

the substrate

sequence

is held by

the

IGS region

of the intron, and

other con-

served sequences

generate

a

guanine

nucleotide

binding

site.

It occurs by a transesterification

involving

a

guanosine

residue as cofactor.

No

input of energy

is

required. The

guanosine

i:i{rilli:i

i:lr.r

l

Bonls

are

rearranged

through

a series

oftransesterifications

invotving the

-0H

g'oups

of

serine

or threonine

or

the

-SH

group

of cysteine

untjL

the exteins

ar€ connected

by

a

peptide

bond

and the

intein

is

reteased

with a circularized

(-terminus.

breaks the

bond

at the

5'exon-intron

junction

and

becomes

linked

to

the

intron;

the

hydroxyl

at the

free end

of the

exon

then

attacks

the

3'exon-intron

jtrnction.

The

intron

cyclizes

and

loses the

guanot;ine and

the

terminal

l5

bases'

A series

of relaled

reactions

can be

catalyzed

via

attacks

by the

terminal

G-OH

residue

of

the

intron

on intental

phosphodiester

bonds.

By

providing

appropriate

substrates,

it has

been

possible

to

engitreer

ribozymes

that

perform a

variety

of

catalytic

reactions,

including

nu-

cleotidyl

transferase

activities.

Some

group I and

group II

mitochondrial

introns

have opt:n

reading

frames.

The

proteins

coded by

group

J introns

ate endonucleases

that

make double-stranded

cleavages

in

target

sites

in DNA;

the cleavage

initiates

a

gene

converslon

process in whi<:h

the

sequence

of

the

intron

itself is copied

ilrto the

target

site.

The

proteins

X=SorO

o

tl

c-NH2

j.-

trx

H

CHz

I

\

/'

Exteinl

/

tl

27.13 Summarv

725

coded by

group

II introns

include

an endonu-

clease

activity

that initiates

the

transposition

process,

and

a

reverse

transcriptase

that

enables

an RNA

copy

of the intron

to

be copied into

the

target

site.

These

types of introns probably

orig-

inated

by insertion

events. The

proteins

coded

by both

groups

of

introns

may

include

maturase

activities

that assist

splicing

of the intron

by

sta-

bilizing

the formation

of the

secondary/tertiary

structure

of the active

site.

Catalytic

reactions

are

undertaken

by

fhe

RNA

component

of the RNAase

P ribonu-

cleoprotein.

Virusoid

RNAs

can undertake

self-cleavage

at a

"hammerhead"

structure.

Hammerhead

structures

can form

between

a

substrate

RNA

and a ribozyme

RNA, which

allows

cleavage

to be directed

at

highly

specific

sequences.

These

reactions

support

the view

that

RNA

can form

specific

active

sites that have

catalytic

activity.

RNA

editing

changes

the

sequence

of an

RNA

after

or during

its

transcription.

The

changes

are required

to create

a meaningful

coding

sequence.

Substitutions

of individual

bases

occur in

mammalian

systems;

they take

the form

of

deaminations

in

which

C is con-

verted

to

U or A is

converted

to L

A catalytic

subunit

cytidine

or adenosine

deam-

inase

functions

as

part

of

a larger

complex

that

has

specificity

for

a

particular

target

sequence.

Additions

and

deletions

(most

often

of uri-

dine)

occur

in trypanosome

mitochondria

and

in

paramyxoviruses.

Extensive

editing reactions

occur in

trypanosome

s in which

as many

as half

of the

bases in

an mRNA

are

derived

from

edit-

ing. The

editing

reaction

uses a

template

con-

sisting

of

a

guide

RNA

that

is complementary

to

the nRNA

sequence.

The reaction

is

catalyzed

by an

enzyme

complex

that includes

an endonu-

clease,

terminal

uridyltransferase,

and RNA li-

gase,

using free

nucleotides

as

the source

for

additions,

or releasing

cleaved

nucleotides

fol-

Iowing

deletion.

Protein

splicing

is an

autocatalytic

reaction

that

occurs

by bond

transfer

reactions

and input

of energy

is

not required.

The

intein

catalyzes

its

own

splicing

out

of the flanking

exteins.

Many

inteins

have

a homing

endonuclease

activity

that is

independent

of the

protein

splic-

ing

activity.

References

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Setf-Spticing

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Self-splicing

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(

1987)

. The chemistry

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-splicing

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236,

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Resea rch

Been, M. D.

and Cech, T.

R.

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One

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Te

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-rRNA

self

-

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/rans-

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CHAPTER

27

Catal.ytic

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rc h

Winkler, W. C., Nahvi,

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@

Some Group I Introns Code for

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Proteins

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A.

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tl.,

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RNA

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GluR-B:

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Navaratnam,

N e:.

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Powell. L.

M.. Wattis,

S. C.,

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R. J.,

Edwards,

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Aphasizhev

R.,

Slticego,

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M., Rivlin,

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Ttlpanosome

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h

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727

Benne,

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C.

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Blum,

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Direct

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Dansereau,

J. T., Dalgaard,

J.2., andBelfort,

M.

(1997).

Genetic definition

of a

protein-splicing

domain: functional

mini-inteins

support

structure

predictions

and

a model for

intein

evolution.

Proc. Natl. Acad.

Sci.

USA 94.

tt466-tt47t.

Perler,

F. B.

et al.

(L992).

Intervening

sequences in

an Archaea

DNA

polymerase

gene.

Proc. Natl.

Acad

Sct. USA 89, 5577-5581.

Xu, M.

Q.,

Southworth, M.

W., Mersha,

F. B.,

Hornstra,

L. J., and Perler,

F. B.

(19931

. in

vitro

prorcin

splicing

of

purified

precursor

and

the identification

of a branched

intermediate.

Cell

75,

liTl-1377.

728

CHAPTER

27

Catatytic

RNA

6ZL

aODd

lrau

uo

panuquoJ

'solnqnlorrrru

0l uorllauuol aql

apr^old

Aeu saxaqduor

oMl asaql

lrouuol 1eq1

suralold eq1

r

'uorllunJ

luoruorluor r0J

lprtuassa

s!

III-lCl

ol spurq

1eq1

xaldLuor

urelord

gg93

aq1

r

'II-l0l

]P

pau.loJ

sr alnllnrls urlPurolql

lPnsn

aql

ol anrlpurallp up sr

1eq1

xaldLuor uralord

pazrlerrads

y

o

xalduol uralord

p

spur.€ alauorluel eqf

'11-363

uorbar Llrp-I-V aql

)uplJ

tpq]

III-I0l

pue

I-l0l

soluonbos

pa^rasuol

iloqs

oql

lo

lslsuol

sluauala

Nll

o

'srsolrru

1e

[1e1elnrre ale6erEas o1

pruseld

e rvrolle

o1

fi1L1Lqe eq1

f,q

aostaatals u!

pagquepr

a.lp sluouala

[!]l

r

anLst^dil

's

ur saluanbas

vNo

iloqs

a^eH salauollual

'uMoul

lou

sr

yp6

enrlLladar oql

Jo

uor]lunJ aLlf

o

'yp6

enqLladar

lo

slunoue abrel

uleluol

seuosouro.lql rrlofile4na raq6rq uL se]euor]uol

o

vN0

a^qqaoau ureluol APhl salau.lorlual

'salu0n00s

vN0

alrtleles

ur

qlu

sr.

leql

urleur0rql0ralaq a^Pq uolJo s0.r0ruo.llu0l

o

'uorDal

lueuorlual slr ur suuoJ

lPql

eroqrolauq aql ol salnqnlo.llrur

Jo

luauqrPlle

oql

r\q

elpuLds

lrlolru aql uo

plaq

sr auosorxo.lql rrlofire1na

y

o

alnao uoqe6ar6as e sI aurosoruolql lqo^rPln3 aql

,,'sgnd,.

e,rLb o1

puedxe

sauos

-ourolql

euefiod uo

uoLssardxa auab

1o

salrs ale

]p{}

spuefl

r

uorssaldxl

auag

Jo

salrs

le

puedxl

sauosourolLll

eual^lod

'deu

1err6o1o1Ar

e se

pasn

oq upl

1eq1

spupq

lo

soues e eneq suereldLp

Jo

sauosoruo.lql ouolnlod

.

spuPg rulol saurosoruorql aual,{lod

'srxp

leu0s0ul0rql

aql ulorJ

popualxa

ale

leql

s00ol

MOqS seuosouroiqr

qsnlqduel

uo uoLsserdxa auab

Jo

solr$

r

papuaul

arv sauosouorql

LlsnlqouPl

'spueqlolut

qlP-l-9

aql ut

polellualuol

alP sOUO!

r

'spuPqlalut

aql ueql

}uolu0l

l-!

ut iaMol elP spuPq oql

r

'spueq-9

pallpr

ale

qlrqM

'suorlpuls

Jo

sauas

p

Jo

oluereadde aql

a^pq ol sauosoruo.lql

aq] asnPl sanbruqlal 6ururels ur.P|.lol

I

sulailed 6uLpueg a^eH sauosorxorql

'asPq0lelut

ln0

-qbnorql

palred Alasuap utPual

ugeuolqlolaloq;o

suor6eX

.

's0ruosou0rql

llloltul

upql

pelred A11q6q ssel

st

qrtqM

'utlsuolqlna

Jo

ulloJ

aql

ur sr. urleuorlll

Jo

sseu

lelauab

aql

'asPqdlalut

6uun6

r

'srsolrur

6uunp

r{1uo uaes

oq uPl

seuosouto.l!r

lPnpLnLpul

r

u tlPtuoJ

LllolalaH

pue

utlPtx0lqlnl

olut

papl^]o sI ullPtu0lql

'0luenDos

sns

-uesuol

llJDads

fiue aneq

lou

op

]nq

ql!-.l-V

ore

sl!fi otlf

r

'suvs

ro suvt/\l

pollPl

sesuanbes

llJt]eds

lP

xllleu .lPallnu

eql 01

poqrPne

s!

vNo

r

xulew aseqdralul

ue

o1

vNo

qlPnv

saluanbas

llJllads

'peqrellP

aie

vN0

paltoriadns

1o

sdool eq1

qrtq/v\

01

ploljels utalold P

aAeq sauosotllo.ltll

ose{oP}e}rl

o

'qI

98-

Jo

sutPulop

luepuadoput

olur

palrolradns [1art1e6au st

utlPtuo.lq]

asPqdra]ut

J0

vN0

.

plo#Prs

e

o]

paqlP]lv suteuo0

puP

sdool sPH

vN0

lqo,{rP)nl

'dq

gg1/rorredns

I-

sr. butttor.redns

;o

flrsuap

a6erane

aq1

r

'sureu0p

polrolladns [1anL1e6au

]uapuedaput

ool-

sPq

ptoellnu

0q1

.

paltolredns

{

auiouo!

leuape8

aqt

'polJquapt

uaoq

lou

e^eq

VN6

eql 6uLsuepuor

loJ alqtsuodsal

ale

lPql

suralotd

aql

r

'uralold

lo

VNU

uo

llP leql

sluabe

^q

paploJun

oq uel

pue

ssPu

nq

vNo

%08-

st

ptoallnu

lPua]leQ

aLll

o

ptoallnN

e sI auoua9

lPuellP8

otll

'llaqs

ut010lo

polquasseald P olut

vNo

oql

uesul

sasnlt^

vNo

lsluoqds

.

'1r

punole

llaqspeeq

aql alquassP

Aeql se

euouab

VNU

aqt

asuapuol

sasur^

VNU

sno]ueulelll

r

'pasuapuol

filaualya

st

lleqspPaq

aql uttlltM

plle

ltelrnI

o

'lloqspPaq

eq]

Jo

alnllnlls

oql

fq

paltultl

sr snlh

P olut

polPlod.lolu!

eq uPl

lPql

vNo;o

q16ua1 eql

r

sleol

ltaql olut

pabellPd arv

sauoua!

lPlL^

u0qlnpollul

3Nrtrn0

u3l-dvH3

rflfir

wm

Telomeres

Have

Simpte Repeating

Sequences

o

The

te[omere is required

for

the stabitity

of the

chromosome end.

r

A

tetomere consists

of a simpte repeat

where

a C + A-rich

strand

has

the seouence

cr(A/r)F4.

Te[omeres

Sea[ the

Chromosome Ends

.

The

protein

TRF2

catalyzes

a

reactjon

in

which

the 3'

repeating

unit of the G

+

T-

rich

strand

forms

a

loop by disptacing its

homotog

in an

upstream region

of the

telomere.

Telomeres Are

Synthesized

by a

Ribonucteoprotein Enzyme

o

Tetomerase

uses the 3'-0H

of the G + T

telomeric strand to

prime

synthesis of tan-

dem

TTGGGG reoeats.

.

The RNA component

of telomerase has

a

sequence that

pairs

with the

C

+

A-rich

repeats.

r

One of the

protein

subunits

is

a reverse

transcriptase

that uses the RNA

as temptate

to synthesize

the G

+ T-rich

sequence.

Te[omeres Are

EssentiaI for

SurvivaI

Summarv

@

Introduction

A

general

principle

is

evident in

the organiza-

tion of all

cellular

genetic

material. It

exists as

a compact

mass that is

confined

to a limited vol-

ume,

and its various

activities,

such as replica-

tion and

transcription,

must

be accomplished

within

this space. The

organization

of this mate-

rial

must accommodate

transitions

between

inactive

and

active states.

The

condensed

state of nucleic

acid results

from its

binding

to basic

proteins.

The

positive

charges

of these

proteins

neutralize

the nega-

tive

charges

of the nucleic

acid. The

structure

of

the nucleoprotein

complex

is determined

by

the interactions

of the

proteins

with the DNA

(or

RNA).

A common problem

is

presented

by the

packaging

of DNA

into

phages,

viruses,

bacre-

rial

cells, and

eukaryotic

nuclei. The

length

of

the DNA

as an extended

molecule

would vastly

exceed the

dimensions

of the

compartment

that

contains

it. the DNA

(or

in

the case

of some

viruses,

the RNA) must

be compressed

exceed-

ingly

tightly to fit into

the

space avallable.

Thus

in contrastwith

the customary

picture

of DNA

as an

extended doubk helix,

structural deformation

of DNA

to bend or

fold

it into

a mlre clmpact

form

is the rule

rather than

exceotion.

The magnitude

of the

discrepancy

between

the

length

of the nucleic

acid and

the size

of

its

compartment

is evident ftom

the examples

sum-

marized

in

FIGUHt

eS.1.

For bacteriophages

and

for eukaryotic viruses,

the nucleic

acid

genome,

whether

single-stranded or

double-stranded

DNA

or

RNA,

effectively fills

the

container

(which

can be rodlike

or spherical).

For

bacteria or for

eukaryotic

cell compart-

ments, the

discrepancy is hard

to calculate

exactly,

because the DNA is

contained in

a com-

pact

area

that occupies

only

part

of the com-

partment.

The

genetic

material

is

seen in

the

form

of the nucleoid

in bacteria

and

as the

mass

of chromatin in eukaryotic

nuclei

at inter-

phase (between

divisions).

The density

of DNA in these

compartments

is

high. In a

bacterium it is

-10

mg/ml,

in

a

eukaryotic

nucleus it is

-100

mg/ml,

and in

the

phage

T4 head

it is

>5O0mg/ml.

Such

a concen-

tration

in solution

would be equivalent

to a

gel

ftfi'-lHHf;S.t

TheLengthofnucleicacidismuchgreaterthanthedjmensionsofthesurroundingcom-

Dartment.

Compartment

TMV

Phage fd

Adenovirus

Phage T4

E.

coli

Mitochondrion

(human)

Nucleus

(human)

Shape

filament

icosahedron

cylinder

oblate

spheroid

0.008 x

0.3

pm

0.006 x 0.85

pLm

0.07

pm

diameter

0.065 x

0.10

pm

1.7 x

0.65

pm

3.0 x

0.5

pm

6

pm

diameter

Dimensions Type

of Nucleic

Acid

Length

One single-stranded RNA

One single-stranded

DNA

One

double-stranded DNA

One

double-stranded DNA

One double-stranded

DNA

-10

identical

double-stranded

DNAs

46 chromosomes

of

double-stranded

DNA

2pm=6.4kb

2pm=6.0kb

11

pm

=

35.0 kb

55

pm

=

170.0

kb

1.3mm=4.2x103kb

50

pm

=

16.9 16

1.8 m

=

6 x 1Oo kb

730

CHAPTER

28

Chromosomes

Lewin Benjamin (ed.) Genes IX

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