Lewin Benjamin (ed.) Genes IX

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Phylum Species

Genome

(bp)

Algae Pyrenomas

salina

6.6 x 10s

Mycoplasma M.

pneumoniae

1 .0 x 100

Bacterium E. coli

4.2 x 106

Yeast

S. cerevisiae

1 .3 x 107

Slime mold D. discoideum

5.4

x 102

Nematode

C. elegans

8.0 x 107

lnsect D. melanogaster

1 .4 x 108

Bird

G. domesticus

1 .2 x 10e

Amphibian X. laevis

3.1

x 10e

Mammal H. sapiens

3.3

x 10s

FlSiJgf

r+"7

The

genome

sizes of some

common experi-

mentaI

orqanisms.

does not imply a

vast

increase in

genome

size;

yeast

may have a

genome

size of

-1.3

107

bp, which is only about twice the size of an aver-

age bacterial

genome.

A further twofold increase in

genome

size

is adequate

support the slime mold Dic-

tyostelium discnideum, which is able to Iive in either

unicellular

or multicellular modes. Another

increase in complexity is necessary to

produce

the

first fully multicellular organisms; the nem-

atode worm

Caenorhabditis elegans has

DNA

content of 8

107

bp.

We also can see the steady increase in

genome

size

with

complexity

the

listing in

rIG*Rg +.f

of some of the most commonly ana-

Iyzed organisms.

It is

necessary to increase the

genome

size

in order to make insects, birds or

amphibians, and

mammals.

After this

point,

though, there is

good

relationship between

genome

size

and morphological complexity of

the organism.

know that

genes

are much larger than

the sequences

needed to code for

proteins,

because

exons

(coding

regions) may comprise

only a small

part

of the total length of a

gene.

This explains why there

is much more DNA

than

is needed to

provide

reading frames for all

the

proteins

of the organism. Large

parts

of an

interrupted

gene

may not be concerned with

coding

for

protein.

In addition, there also may

be significant

lengths

DNA

between

genes.

it is not

possible

to deduce

from

the overall size

of the

genome

anything about the number of

genes.

The C-value

paradox

refers

to the

lack of

correlation

between

genome

size and

genetic

complexity.

There are some extremely curious

variations

in relative

genome

size. The toad

Xenopus and

man have

genomes

of essentially

the same size.

We assume,

though,

that man

more complex

terms

genetic

developmentl

some

phyla

there are extremely

large varia-

tions in DNA content

between

organisms

that

do not vary

much in complexity

(see

Fig-

ure 4.5).

(This

is especially

marked

in insects,

amphibians, and

plants,

but does

not occur

birds,

reptiles, and

mammals,

which all show

lit-

tle variation within

the

group,

with

-2x

range

genome

sizes.)

A cricket

has a

genome

I lx

the size of a

fruit fly. In

amphibians,

the small-

est

genomes

are

<l0e

bp,

whereas

the largest

are

l01r

bp.

There

is unlikely

to be a

large dif-

ference in the

number of

genes

needed to spec-

ify

these amphibians.

It is not

understood

why

natural selection

allows

this

variation

and

whether

it has evolutionarv

consequences.

Eukaryotic

Genomes

Contain

Both

Nonrepetitive

and

Repetitive

DNA

Sequences

The kinetics of

DNA

reassociation

after a

genome

has been denatured

distinguish

sequences

by their

frequency

repetition

in the

genome.

Genes

are

generatty

coded by

sequences

nonrepetitive DNA.

Larger

genomes

within a

phylum

do not contain

genes.

but

have [arge

amounts

repetitive

DNA.

A large

part

repetitive

DNA

may be

made up of

transposons.

The

general

nature

of the

eukaryotic

genome

can be assessed

by the

kinetics

reassociation

of denatured

DNA.

This technique

was used

extensively

before

large

scale

DNA sequencing

became

possible.

Reassociation

kinetics

identifies

two

gen-

eral types of

genomic sequences:

Nonrepetitive

DNA

consists

of se-

quences

that

are unique:

there

is only

one copy

in a

haploid

genome.

Repetitive

DNA

describes

sequences

that are

present in more

than one

copy

in each

genome.

Repetitive

DNA often

is divided

into

two

general

types:

Moderately

repetitive

DNA

consists

relatively

short

sequences

that

are

repeated

typically

l0-1000x

the

4.6

Eukaryotic Genomes

Contain

Both

Nonrepetitive

and

Repetitive

DNA Sequences

1010

10e

in8

10',

106

s37

The

proportions

of different

sequence components

vary in

eukaryotic

genomes.

The absotute

content of non-

repetitive

DNA

increases

with

genome

size but reaches

ptateau

-2

10e bp.

genome.

The

sequences

are dispersed

throughout

the

genome

and are respon-

sible for

the high degree

of secondary

structure formation

pre-nRNA,

when

(inverted)

repeats

in the introns

pair

form

duplex regions.

Highly repetitive

DNA

consists

of very

short

sequences

(typically

<100

bp) rhar

are

present

many

thousands

of times in

the

genome,

often organized

as long

tandem repeats

(see

Section

l, Satel-

lite

DNAS Often

Lie in

Heterochro-

matin).

Neither

class represents protein.

The proportion

the

genome

occupied

nonrepetitive

DNA varies

widely.

:,r,,i:.r

,,,:-

summarizes

the

genome

organization

of some

::ilT:"-'::-:;:T,Tr?iii,i#I:Til.";.l.l

most

the DNA is nonrepetitive;

<204

falls

into

one

or more moderately

repetitive

com-

ponents.

animal

cells, up

to half of

the DNA

often is

occupied

by moderately

and highly

repetitive

components.

plants

and

amphib-

ians,

the moderately

and highly

repetitive

com-

ponents

may

account for

to 80%

of the

genome,

so that

the nonrepetitive

DNA is

reduced

a minority

component.

CHAPTER

The

Content

the Genome

A significant

part

the moderately repet-

itive

DNA consists

of transposons,

short

sequences

DNA

(-l

kb) that have

the ability

to move

to new locations in

the

genome

and/or

to make additional

copies of themselves

(see

Chapter

21,

Transposons,

and Chapter 22,

Retroviruses

and

Retroposons).

some higher

eukaryotic

genomes

they may

even

occupy

than half of the

genome (see

Section 5.5,

The Human

Genome Has Fewer

Genes Than

Expected).

Transposons are

sometimes viewed

as fit-

ting the

concept of selfish DNA,

which

defined

as sequences that

propagate

themselves

within a

genome

without

contributing

to the

development

of the organism.

Transposons

may

sponsor

genome

rearrangements,

and these

could

confer selective advantages.

It is fair

say, though,

that we do not really

understand

why selective forces

do not

act against

trans-

posons

becoming such a large

proportion

of the

genome.

Another

term that is

used to describe

the apparent

excess of DNA is

junk

DNA,

mean-

ing

genomic

sequences

without any

apparent

function.

Of course, it

is likely

that there is

balance in the

genome

between

the

generation

of new

sequences

and the

elimination

unwanted

sequences, and

some

proportion

DNA

that apparently lacks

function

may

be in

the

process

being eliminated.

The length

of the nonrepetitive

DNA

com-

ponent

tends

to increase

with overall genome

size as we

proceed

up to a total

genome

size

-3

10e

(characteristic

of mammals).

Further

increases in

genome

size,

however,

generally

reflect

increase

the amount

and

propor-

tion of the repetitive

components,

that it is

rare

for an organism

to have

a nonrepetitive

DNA

component

>2 x

l0e.

The nonrepetitive

DNA

content

genomes

therefore

accords

bet-

ter

with our sense

of the relative

complexity

the

organism. E. colihas

4.2

I06

bp,

C. elegans

increases

an order

of magnitude

6.6

107

bp, D. melanogasler

increases

further

-108

bp,

and mammals increase

another

order

of magnitude

-2

lOe

bp.

What

type of DNA

corresponds

protein-

coding

genes?

Reassociation

kinetics

typically

shows that mRNA

is derived

from

nonrepetitive

DNA. The

amount

of nonrepetitive

DNA is

therefore

a better

indication

than

the total

DNA

of the coding

potential.

(However,

detailed

analysis

based

genomic

sequences

shows

that

many

exons have related

sequences

other

exons

[see

Section

].5, Exon

Sequences

Are

Conserved

but Introns

Varyl.

Such

exons evolve

by a duplication

give

copies

that initially are

identical, but which

then diverge in

sequence

during evolution.)

Genes Can Be Isolated

the Conservation

Exons

Conservation of exons can

be used as the basis for

identifuing

coding regions

identifuing

fragments whose sequences

are

present

muttipte

organisms.

Some

major

approaches to identifying

genes

are based on the contrast

between

the conser-

vation

of exons and the

variation of introns. In

a region containing

gene

whose function has

been

conserved among a range

of species, the

sequence

representing

the

protein

should have

two distinctive

properties

It must have

an open reading frame,

and

it is likely

to have

sequence

in other

species,

These features

can be used

isolate

genes.

Suppose we know

genetic

data that a

particular genetic

trait

located in a

given

chro-

mosomal region. If we lack

knowledge about

the nature

of the

gene product,

how are

we to

identify

the

gene

in a region

that may be, for

example,

Mb?

A heroic approach

that has

proved

success-

ful with

some

genes

of medical importance is to

screen relatively short fragments

from the region

for the two

properties

expected

of a conserved

gene.

First we seek

identify

fragments that

cross-hybridize

with the

genomes

of other

species. then we examine these fragments for

open reading frames.

The first

criterion is applied

performing

a zoo blot. We use

short fragments from the

region

(radioactive) probes

to test for related

DNA from a variety of species

by Southern blot-

ting. If we find hybridizing fragments in

several

species related to that

of the

probe-the probe

usually

human-the

probe

becomes a candi-

date for an exon of the

gene.

The

candidates are sequenced, and if they

contain open

reading

frames they are used to

isolate surrounding

genomic

regions. If these

appear

part

of an exon, we may then use

them to identify the entire

gene,

to isolate the

corresponding cDNA or mRNA, and ultimately

identify

the

protein.

This

approach

especially important when

the target

gene

spread out because it has

many

l:"iiil&i:

The

gene

involved in

Duchenne muscular

dystrophy

was tracked down

by chromosome

mapping

and

"watking"

to a region

in which deletions

can be

identi-

fied with the occurrence

ofthe disease.

large introns. This

proved

be the case

with

Duchenne muscular

dystrophy

(DMD),

degen-

erative disorder

muscle that

is X-linked

and

affects t in 3500

human

male births.

The steps

in identifying

the

gene

are summarized

:l:::iil

Linkage analysis

localized

the DMD

locus to

chromosomal

band

Xp2l. Patients

with the

dis-

ease often

have chromosomal

rearrangements

involving this band.

By comparing

the ability of

X-linked DNA

probes

hybridize

with DNA

from

patients

with

normal

DNA, cloned

frag-

ments were obtained

that

correspond

to the

region that was rearranged

deleted

patients'

DNA.

Once some

DNA

in the

general vicinity of

the target

gene

has

been obtained,

it is

possible

"walk"

along the

chromosome

until

the

gene

is reached.

A chromosomal

walk

was used

construct a

restriction

map of

the region

either side of

the

probe, which

covered

a region

4.7

Genes

Can

Be Isotated by

the Conservation

Exons

i:.

i:,

The Duchenne

muscutar

dystrophy

gene

was

characterized

zoo

btotting,

cDNA

hybridization.

genomic

hybridization,

and identification

ofthe

protein.

>100

kb. Analysis

of the DNA from

a series

patients

identified large

deletions in

this

region

that extended in

either

direction. The

most

telling deletion

is one that

is contained

entirely

within

the

region,

because this delin-

eates a segment

that must

be important in

gene

function

and indicates

that the

gene-or

at least

part

it-lies

this

region.

Having now

come into

the region

of the

gene,

we need

identify

its

exons and introns.

A zoo

blot identified

fragments

rhat

cross-

hybridize

with

the mouse X

chromosome

and

with other

mammalian

DNAs. As

summarized

;rir,3l,ii:i

,i,.t,r,

these were scrutinized

for

open

reading frames

and the sequences typical of

exon-intron

junctions.

Fragments

that met

these criteria were used as

probes

to identify

homologous

sequences in a cDNA library pre-

pared

from muscle mRNA.

The

cDNA corresponding to the

gene

iden-

tifies an unusually large nRNA

of approximately

l4 kb. Hybridization

back to the

genome

shows

that the mRNA is represented in >60

exons.

which are

spread over

-2000

kb of DNA. This

makes

DMD the longest

gene

identified.

The

gene

codes for

protein

-500

called dystrophin, which is a component

of mus-

cle and is

present

in rather low

amounts. All

patients

with

the disease

have

deletions at

this

locus and lack

(or

have

defective) dystrophin.

Muscle

also has the distinction

of having

the largest known

protein,

titin, with

almost

27,000

amino acids. Its

gene

has the largest

number

of exons

(178)

and the longest

single

exon in

the human

genome (17,000

bp).

Another

technique that

allows

genomic

fragments to be

scanned rapidly for

the

presence

of exons is called

exon trapping.

:ili;ri!

.:,,:;

shows that it

starts with

a vector

that

contains a strong

promoter

and has a sin-

gle

intron

between two exons.

When this

vec-

tor is transfected into

cells, its transcription

generates

large

amounts of an RNA

containing

the

sequences of the two exons.

A restriction-

cloning site lies

within the

intron

and is

used to

insert

genomic

fragments from

region

of inter-

est. If

fragment

does not

contain an exon,

there is no

change in the

splicing

pattern,

and

the RNA contains

only the same

sequences as

the

parental

vector. If the

genomic

fragment

contains

an exon flanked

by two

partial

intron

sequences,

though, the splicing

sites on either

side of this

exon are recognized

and

the

sequence

of the exon is inserted into

the RNA

between

the two exons of

the vector. This

can

be detected readily

by reverse transcribing

the

cytoplasmic

RNA into

cDNA and

using PCR to

amplify the

sequences between

the two exons

of the

vector. So the appearance

in the

ampli-

fied

population

of sequences from

the

genomic

fragment

indicates

that an exon

has been

trapped. Because

introns are

usually large

and

exons

are small in animal

cells, there

is a high

probability

that a random

piece

genomic

DNA

will

contain the required

structure

of an exon

surrounded

partial

introns.

In fact,

exon trap-

ping

may mimic

the events that

have

occurred

naturally

during evolution

genes (see

Sec-

tion

3.8, How Did Interrupted

Genes Evolve?).

CHAPTER

The

Content

of the

Genome

The

vector

contains

two exons

that are

spliced together in the transcript

promoter

5' splice

junction

3' splice

junction

InIron

Transcription

and

splicing

to remove intron

Genomic fragment

intron

exon

intron

Insert

genomic

fragment

into

intron

exonl

exon

rexon

intron

Transcription

and

splicing to remove

intron

il*{Jli[ *.31

speciaI spticing

vector is

used

for

exon trapping.

exon

present

in the

genomic

fragment,

its sequence

wit[ be

recovered in

the cyto-

plasmic

RNA. If

the

genomic

fragment

consists

sotety of sequences

from within

an intron,

though,

spticing

does not

occur, and the mRNA is not exported

the

cytoplasm.

The

Conservation

Genome

0rganization

Helps

Identify

Genes

Atgorithms

for identifying

genes

are not

perfect

and

many

corrections must

made

the

initiat

data set.

Pseudogenes

must

be distinguished

from

active

genes.

Syntenic

relationships

are extensive

between

mouse

and human

genomes,

and most

active

genes

are

a syntenic region.

Once we have

assembled

the sequence

of a

genome.

we still have

to identify

the

genes

within it. Coding

sequences

represent a

very

small fraction. Exons

can

be identified as

unin-

terrupted open reading

frames flankedby

appro-

priate

sequences. What

criteria

need to be

satisfied to identify an

active

gene

from

a series

of exons?

i":g';t.t5{!.

+..:: shows that

an active

gene

should

consist

of a series

of exons

for which the first

exon immediately follows a

promoter,

the inter-

nal

exons are

flanked by appropriate

splicing

junctions,

the last exon

is followed by 3'process-

ing

signals, and a single

open

reading frame

starting with an initiation

codon and ending

with

a termination

codon can

be deduced by

joining

the exons together.

Internal exons

can

be identified as open

reading frames

flanked by

splicing

junctions.

In the simplest

cases, the first

and Iast exons contain the

start and end

of the

coding region, respectively

(as

well as the

5'and

untranslated

regions).

In more complex cases,

the first

last exons may

have only untrans-

lated regions

and

may therefore

be more diffi-

cult to identify.

The algorithms that

are used to

connect

exons are not completely

effective

when

the

genome

very

large and the

exons maybe sep-

arated

by very

large distances.

For example,

the

initial

analysis of

the human

genome

mapped

4.8 The Conservation

of Genome

0rganization

Hetps to

Identify Genes 65

i'ii.l-li.:i

r;.

i.l

Exons of

protein-coding genes

are

identified as coding sequences

ftanked

by appro-

priate

signals

(with

untranstated regions at both ends).

The series of exons

must

generate

open

reading frame with

appropriate

initjatjon and termination codons.

170,000

exons into 12,000

genes.

This is

unlikely

to be correct because it

gives

an aver-

age of 5.1 exons

per gene,

whereas the average

of individual

genes

that have

been

fully char-

acterized is 10.2. Either

have missed many

exons, or they should be connected differently

into a

smaller

number

qenes

the whole

genome

sequence.

Even

when the organization of a

gene

correctly identified, there is the

problem

of dis-

tinguishing active

genes

from

pseudogenes.

Many

pseudogenes

can

recognized

by obvi-

ous defects in

the

form

of multiple mutations

that create an inactive coding

sequence.

Pseudo-

genes

that have arisen more recently have not

accumulated

so many mutations and thus may

be more

difficult to

recognize.

In an extreme

example,

the mouse has only one active Gapdh

gene (coding

for

glyceraldehyde phosphate

dehydrogenase),

but has

-400

pseudogenes.

Approximately

100 of these

pseudogenes

ini-

tially appeared

to be active in the mouse

genome

sequence,

and

individual

examination

was

nec-

essary to exclude them from

the list of active

genes.

Confidence that

gene

active can be

increased

comparing

regions

of the

genomes

of different

species.

There

has been extensive

overall reorganization

of sequences between

the mouse

and

human genomes,

seen

in the

simple fact

that there are 23 chromosomes in

the

human haploid

genome

and 20

chromosomes

the mouse haploid

genome.

However, at the

local level

the order of

genes

generally

the

CHAPTER

The

Content

of the

Genome

same:

When

pairs

of human and mouse homo-

logues are compared, the

genes

located

on either

side also

tend to be homologues. This relation-

ship

called synteny.

i].i:i-i3":

ji.

+.;i3

shows

the relationship

between

mouse

chromosome

I and the human chromo-

somal set.

can recognize

segments in this

mouse chromosome that

have

syntenic coun-

terparts

in human chromosomes. The extent of

reshuffling

that

has occurred between the

genomes

shown by

the fact that

the segments

are spread among six different

human

chromo-

somes.

The

same

tlpes of relationships are found

in all mouse chromosomes except for the X

chromosome, which

syntenic only with the

human X chromosome.

This is

explained by the

fact that the X

a special

case,

subject to dosage

compensation to adjust

for

the difference

between

males

(one

copy)

and

females

(two

copies)

(see

Section 3L5, X Chromosomes

Undergo Global Changes). This may apply

selec-

tive

pressure

against the translocation of

genes

to and from the X chromosome.

Comparison of the

mouse

and human

genome

sequences

shows rhal

>90oh

of each

genome

lies in syntenic blocks that range

widely

in size

(from

300 kb to Ol Mb). There is

a total

of 342 syntenic segments,

with an average

Iength of 7 Mb

(O.3%

the

genome).

Ninety-

nine

percent

mouse

genes

have a homologue

the human

genome;

for 96o/"

that

homo-

logue is

in a syntenic region.

Comparing

the

genomes provides

interest-

ing

information about the evolution

of species.

10 20

90 t00 Mb

Mouse

chromosome 1

llffi

...'.

:tEH

1 2145

Corresponding human

chromosome

-IG*8f

i.13 Mouse

chromosome

t has

21 segments

to 25 Mb that

are syntenic with

regions

corresponding

parts

of six human

chromosomes.

The

number

gene

families

in the

mouse and

human

genomes

the same,

and a major

dif-

ference

between

the species

is the

differential

expansion

particular

families

in one

of the

genomes.

This is

especially noticeable

genes

that affect

phenotypic

features

that

are unique

to the species.

families

for

which the size

has been

expanded in

mouse, l4

contain

genes

specifically involved

in rodent

reproduction,

and

5 contain

genes

specific

to the immune

system.

validation

of the importance

of syntenic

blocks comes from pairwise

comparisons

of the

genes

within them. Looking

for

likely

pseudo-

genes

on the

basis of sequence

comparisons,

gene

that is not in

a syntenic location

(that

is,

its

context is different

in the

two species) is

twice

as likely

to be a

pseudogene.

Put

another way,

translocation away from

the

original locus

tends

to be associated

with

the creation

pseudo-

genes.

The lack

of a related

gene

in a

syntenic

position

therefore

grounds

for

suspecting that

apparent

gene

may

really be

pseudogene.

Overall,

>l0o/o

of the

genes

that are initially

identified by analysis

ol the

genome

are

likely

turn out to be

pseudogenes.

As a

general

rule,

comparisons

between

genomes

add significantly

to the

effectiveness

gene prediction.

When

sequence features

indicating

active

genes

are conserved-for

example,

between Man

and mouse-there

increased

probability

that they identify

active

homologues.

Identifying

genes

coding for

RNA is more

difficult because we

cannot

use the criterion of

the open reading frame.

It also is

true that com-

parative genome

analysis increased

the rigor

the analysis. For example,

analysis

of either the

human

or the

mouse

genome

alone identifies

-500

genes

coding for

IRNA,

but comparison

of features

suggests that

of these

genes

are

in fact active in each

qenome.

0rganelles

Have DNA

Mitochondria

and chtoroptasts

have

genomes

that

show non-Mendetian inheritance.

Typicatly

they

are maternatty inherited.

Organelle

genomes

may undergo somatic

segregation in

plants.

Comparisons

mitochondrial DNA suggest that

humans

are descended

from a singte female who

tived 200,000

years

ago in Africa.

The

first evidence for the

presence

genes

out-

side the nucleus was

provided

by non-

Mendelian inheritance in

plants (observed

the early

years

of the twentieth

century,

just

after

the

rediscovery

Mendelian inheritance).

Non-Mendelian inheritance sometimes

is asso-

ciated with

the

phenomenon

of somatic segre-

gation.

Both have a similar

cause:

Non-Mendelian

inheritance is defined

by the failure of the

progeny

of a mat-

ing to display Mendelian segregation

for

parental

characters.

It reflects

lack

association between the segregating

character and

the meiotic spindle.

Somatic segregation

describes a

phe-

nomenon in which

parental

characters

segregate

in somatic cells

and therefore

display heterogeneity

in the organism.

This is a notable

feature of

plant

devel-

opment, as

it reflects

lack

association

between the segregating

character

and

the mitotic spindle.

Non-Mendelian

inheritance and somatic

segre-

gation

are therefore taken to

indicate the

presence

genes

that reside

outside

the nucleus

and do not uti-

lize

segregation on the

meiotic and mitotic spindles

distribute replicas to

gametes

or to daughter

cells,

respectively. f,3.{;#Ri

.,!.3:+

shows

that this

happens

when

the mitochondria

inherited

from the male

and female

parents

have different

alleles,

and

by chance

a daughter

cell

receives an unbal-

anced distribution of

mitochondria

that repre-

sents only one

parent

(see

Section 17

.12, How

Do Mitochondria Replicate and

Segregate?).

The

extreme

form of non-Mendelian

inher-

itance is uniparental

inheritance, which occurs

when the

genotype

of only one

parent

inher-

ited and

that of the

other

parent

permanently

lost. In Iess

extreme

examples,

the

progeny

one

parental genotlpe

exceed those

of the other

genotype.

Usually it is the

mother whose

geno-

type

preferentially (or

solely)

inherited.

This

effect is

sometimes

described

as maternal

inheritance. The important

point

that

the

genotype

contributed

by the

parent

of one

4.9 0rqanettes

Have

DNA

Cell has mitochondria

from

both

parents

:,:

r.-

When DaternaL and maternat

mitochondrial

a[[e[es differ, a cetl has two sets of mitochondriat DNAs.

Mitosis usua[y

generates

daughter celts with both sets.

Somatic variatjon may resutt if unequaI

segregation

gen-

erates daughter cetts with onty one set.

particular

sex

predominates,

seen

in abnor-

mal

segregation

ratios

when a cross is made

*::ilff

:ffii[lx?i^:i["IJ"",]::ff Tn:';

occurs

when reciprocal crosses show the

contributions

of both

parents

to be equally

inherited.

The

bias in

parental genotypes

established

or soon after the formation of a zygote. There

are

various

possible

causes. The

contribution of

maternal

paternal

information

to the

organelles of the zygote may

be unequal;

the

most extreme case,

only one

parent

contributes.

ln other

cases the contributions are equal, but

the information

provided

by one

parent

does

not survive.

Combinations of both effects are

possible.

Whatever

the cause, the unequal rep-

resentation

of the

information

from the two

parents

contrasts with nuclear

genetic

informa-

tion, which derives

equally from each

parent.

Non-Mendelian

inheritance

results from the

presence

in mitochondria

and chloroplasts of

CHAPTER 4 The

Content of

the Genome

DNA

genomes

that are inherited independently

nuclear

genes.

In effect,

the

organelle

genome

comprises

a length of

DNA

that

has

been

physi-

cally sequestered

in a defined

part

of the cell and

is subject

to its own form of expression and reg-

ulation. An organelle

genome

can code for some

or all of

the RNAs, but codes

for

only some of the

proteins

needed to

perpetuate

the organelle. The

other

proteins

are coded

the

nucleus,

expressed

via the cytoplasmic

protein

synthetic apparatus,

and imported

into the organelle.

Genes not

residing within

the

nucleus are

generally

described as extranuclear

genes;

they are transcribed and

translatedintLre

same

organelle

compartment

(mitochondrion

chloroplast)

which

they reside. By contrast,

nuclear

genes

are expressed by

means

cyto-

plasmic protein

synthesis.

(The

term cytoplas-

mic inheritance sometimes

used to describre

the behavior

genes

in organelies. We shall

not use this description,

though, because it is

important to be able to distinguish between

events

in the

general

cytosol and those

spe-

cific organelles.

)

Higher animals show

maternal

inheritance,

which can be explained

if the mitochondria are

contributed entirely by the ovum and not at all

the sperm.

tiii:r,Jiii:

,1r.

i.-.

shows that the sperm

contributes only

a copy of the nuclear DNA.

Thus the

mitochondrial

genes

are

derived exclu-

sively

from the mother, and in males they are

discarded each

generation.

Conditions

the organelle are different

from those in the

nucleus,

and organelle DNA

therefore evolves at its own distinct rate. If inher-

itance is

uniparental,

there can

recombi-

nation between

parental genomes.

In fact,

recombination

usually does

not

occur in those

cases for which organelle

genomes

are inher-

ited

from both

parents.

Organelle DNA has a

different replication system from that of the

nucleus; as a result, the error rate

during repli-

cation may be different. Mitochondrial DNA

accumulates mutations more rapidly

than

nuclear DNA in mammals,

but

plants

the accu-

mulation in

the

mitochondrion

is slower than

in the nucleus

(the

chloroplast is intermediate).

One

consequence

of maternal

inheritance

is that the sequence of mitochondrial DNA

more sensitive than nuclear DNA

to reductions

the size of the breeding

population.

Compar-

isons of mitochondrial DNA

sequences in a

range

human

populations

allow an evolu-

tionary tree to be constructed. The

divergence

among human mitochondrial

DNAs spans

0.57%. A tree can be constructed in

which the

0rgane[Le

Genomes

Are

Circular DNAs

That

Code

for

Organe[[e Proteins

Organelle

genomes

are usualty

(but

not atways)

circutar motecutes

of DNA.

Organe[[e

genomes

code

for

some, but not

at[. of

the

proteins

found

the organetle.

Most organelle

genomes

take

the form of a sin-

gle

circular molecule of DNA

of unique

sequence

(denoted

mtDNA

in the mitochondrion

and

ctDNA in

the chloroplast).

There

are

a few

exceptions for which mitochondrial DNA

Iinear

molecule; these

generally

occur

in lower

eukaryotes.

Usually

there are

several copies of the

genome

the individual

organelle. There are

multiple

organelles

per

cell, therefore

there are

many

organelle

genomes per

cell. Although the

organelle

genome

itself is unique,

constitutes

a repetitive

sequence

relative to any

nonrepet-

itive nuclear

sequence.

Chloroplast

genomes

are

relatively large,

usually

-140

kb in higher

plants

and

<200 kb

in lower

eukaryotes.

This is comparable to the

size of a large bacteriophage,

for

example,

-165

kb. There are

multiple copies of the

genome

per

organelle, typically

20 to 40 in a

higher

plant,

and

multiple copies of the organelle

per

cell, typically 20 to

40.

Mitochondrial

genomes vary in

total size

by more than an order of

magnitude. Animal

cells have small mitochondrial

genomes-

approximately 16.5 kb

in mammals. There are

several hundred mitochondria

per

cell. Each

mitochondrion

has

multiple copies of the

DNA.

The total

amount of

mitochondrial

DNA rela-

tive to nuclear DNA

small;

it is estimated to

<l%.

yeast,

the

mitochondrial

genome

is much

larger. In Saccharomyces cerevisiae,

the exact size

varies

among different

strains but

averages

-80

kb. There are

-22

mitochondria

per

cell,

which

corresponds

-4

genomes per

organelle.

growing

cells,

the

proportion of mitochon-

drial DNA

can be as

high as

l8o/".

Plants show an extremely

wide

range

variation in mitochondrial

DNA size, with a

minimum of

-100

kb. The size of the

genome

makes it

difficult

to isolate

intact, but restric-

tion mapping

several

plants

suggests

that the

mitochondrial

genome is usually a single

sequence that

organized

as a circle.

Within

this circle there are

short

homologous

sequences. Recombination between

these ele-

ments generates

smaller,

subgenomic

circular

molecules

that coexist

with the complete,

"mas-

ter"

genome-a good

example of

the apparent

complexity of

plant

mitochondrial

DNAs.

With

mitochondrial

genomes

sequenced

from many organisms,

we can

now

see

some

general pattems

in the representation

functions

in mitochondrial DNA.

il:iilt.iiilil +.i

summarizes

the distribution of

genes in mitochondrial

genomes.

The

total

number of

protein-coding

genes

is rather small

and does

not correlate

with the size of

the

senome.

Mammalian

4.10 0rganetle Genomes

Are

Circular

DNAs That Code

for

Organelte

Proteins

irii:l-:*i:

.,.::i

DNA from

the sperm

enters

the oocyte to

form

the mate

pronucteus

the

fertil.ized

egg, but al.l.

the mitochondria

are

provided

by the

oocyte.

mitochondrial

variants

diverged

from

a com-

mon

(African)

ancestor. The

rate

at which mam-

malian mitochondrial

DNA

accumulates

mutations

is2"h Io 4o/o

per

million

years,

which

is >l0x

faster than

the rate for

globin.

Such a rate

would

generate

the

observed divergence

over

an evolutionary

period

of 140,000 to 280,000

years.

This implies

that the

human race is

descended from

a single female

who lived

Africa

-200,000

years

ago.

Species Size

Protein- BNA-

(kb)

coding

genes

Fungi 19-100

8-14

10-28

Protists

6-100 3-62

2-29

Plants 186-366 27-34 21-30

Animals 16-17 13 4-24

.:.:.:-:i:i

:, M'itochondriaL

genomes

have

genes

cod-

ing for

(mostly

complex

I-IV)

proteins.

rRNAs. and tRNAs.

mitochondria

use their

l6 kb

genomes

to code

for l3

proteins,

whereas

yeast

mitochondria use

their

60 to 80

genomes

to code

for

few as

eight

proteins.

Plants, which have much larger

mitochondrial

genomes,

code for more

proteins.

Introns are found in most mitochondrial

genomes,

although

not in

the

very

small

mam-

malian

genomes.

The two major rRNAs

are always coded by

the mitochondrial

genome.

The number of

tRNAs coded

the mitochondrial

genome

varies from none

to the

full

complement

(25

26 in

mitochondria). This accounts for the vari-

ation in Figure 4.16.

The

major

part

of the

protein-coding

activ-

ity is devoted to the components

the multi-

subunit assemblies of respiration complexes

I-IV.

Many ribosomal

proteins

are coded in

pro-

tist and

plant

mitochondrial

genomes,

but there

are few

or none in {ungi and animal

genomes.

There are

genes

coding for

proteins

involved in

import in many

protist

mitochondrial

genomes.

MitochondriaL

DNA

0rganization Is

VariabLe

Animal

cet[

mitochondrial

DNA is extremety

compact and typical.ty codes for 13

proteins.

rRNAs,

and 22 tRNAs.

Yeast mitochondrial DNA is

5x longer than animal

ceLt mtDNA

because of the

presence

of long

introns.

Animal

mitochondrial DNA is

extremely com-

pact.

There are extensive

differences in the

detailed

gene

organization found in different ani-

mal

phyla,

but the

general principle

is maintained

of a small

genome

coding for a restricted num-

ber of functions.

In mammalian mitochondrial

genomes,

the

organization is extremely compact.

Ihere

are no introns, some

genes

actually over-

lap,

and almost every

single base

pair

can be

CHAPTER 4 The

Content

of the Genome

ND3

c03

ATPase

co2

uvl

IRNAgenes

ffi

Cooing

regions

+ Indicates direction of

gene,

CO: cytochrome

oxidase

ND: NADH dehydrogenase

IltSFi{

;;.-1

:r Human

mitochondriaI

DNA has 22 tRNA

genes,

2 rRNA

genes,

and 13

protein-coding

regions.

Four-

teen

ofthe 15

protein-coding

rRNA-coding

regions are

transcribed

in the same direction.

Fourteen

of the IRNA

genes

are expressed

the clockwise direction and 8 are

read counter-ctockwise.

assigned to

gene. With

the exception

of the

loop,

region

concerned

with the initiation

DNA replication, no more than 87 of the 16,569

bp of the

human mitochondrial

genome

can be

regarded as lying in

intercistronic

regions.

The complete nucleotide sequences

mito-

chondrial

genomes

in animal cells

show exten-

sive homology

organization.

The

map of the

human mitochondrial

genome

summarized

fjii;l"rFii,

+.i.r.

There are l3

protein-coding

regions. All of the

proteins

are components of

the apparatus concemed

with

respiration. These

include cytochrome b, three subunits

cytochrome oxidase, one of the

subunits of

ATPase, and

seven subunits

(or

associated

pro-

teins) of NADH dehydrogenase.

The fivefold

discrepancy

size between

the S. cerevisiae

(84kb)

and mammalian

(16

kb)

mitochondrial

genomes

alone alerts

us to the

fact that there must be a

great

difference

in their

genetic

organization

spite of their common

function. The number of endogenously

synthe-

sized

products

concerned with mitochondrial

enzymatic functions appears to be

similar.

Does

the additional

genetic

material

yeast

mito-

chondria represent

other

proteins, perhaps

con-

cerned

with

regulation.

it unexpressed?