Genomics- The Science and Technology Behind the Human Genome Project. Charles R. Cantor, Cassandra L / genomics1-10 / 8

TABLE

8.1 Restriction Enzymes Useful for Genomic Mapping

Enzyme a

Recognition Site (5

–3 )b

Source a,c

Enzymes with Extended Recognition Sites

I-Sce

I

TAGGGATAA/CAGGGTAAT

B

VDE

TATSYATGYYGGTGY/

O

GGRGAARKMGKKAAWGAAWG

I-Ceu

I

TAACTATAACGGTCCTA/AGGTAGCGA

N

I-Tli

I

GGTTCTTTATGCGGACAC/TGACGGCTTTATG

N

I-Ppo

I

CTCTCTTAA/GGTAGC

P

Enzymes with

6-bp Recognition Site

Pac

I

TTAAT/TAA

N

Pme

I

GTTT/AAAC

N

Swa

I

ATTT/AAAT

B

Sse

83888t1

CCTGCA/GC

T

Enzymes with

6-bp Recognition that Cut in CpG Islands

Rsr

II (Csp

*)

CG/GWCCG

N, P*

SgrA

I

CR/CCGGYG

B

Not

I

GC/GGCCGC

B, N, S, P

SrfI

GCCC/GGGC

P, S

Fse

GGCCGGCC

B

Sfi

Id

GGCCNNNN/NGGCC

B, N, P

Asc

I

GG/CGCGCC

N

Enzymes that cut in CpG Islands: Fragments Average

200 kb

Mlu

I

A/CGCGT

B, N, P, S

Sal

I

G/TCGAC

N

Nru

I

TCG/CGA

N

Bss

HII

G/CGCGC

Sac

II

CCGC/GG

N

Eag

I (EcI

XI*,Xma

III)

C/GGCCG

B*, N

Enzymes that cut in CpG Islands: Fragments Average

200 kb

Nar

I

GG/CGCC

N, P

Sma

I

CCC/GGG

N

Xho

I

C/TCGAG

N

Pvu

I

CGAT/CG

N

Apa

I

GGGCC/C

N

Enzymes with TAG in their Recognition Sequence

Avr

II (Bln

I*)

C/CTAGG

N, T*

Nhe

I

G/CTAGC

N, P, S

Xba

I

T/CTAGA

B, N, P, S

Spe

I

A/CTAGT

B, N, P, S, T

Nhe

I

G/CTAGC

P

Dra

I

TTT/AAA

B, P, S

Ssp

I

AAT/ATT

P

HTF ISLANDS

245

Note that the recognition site for

Sfi

I contains no CpG’s, while the site recognized by

Not I contains 2 CpG’s. In Chapter 1 we showed that the frequency of occurrence of this

dinucleotide sequence is reduced in mammalian DNA to about 1/4 of the level expected

statistically. On this basis

Not

I can be expected to behave like a ten-base specific enzyme

rather than an eight-base specific enzyme; as a result the expected fragment sizes are pre-

dicted to lie around 1 Mb, in agreement with experimental results. However, this is an

oversimplified argument for it ignores the effect of DNA methylation. Overall, in the hu-

man genome about 80% of the CpG sequences are methylated to 5-meC, and

Not

I (and

most other restriction nucleases) is unable to cleave at sites that are methylated. If we fac-

tor this effect into the calculation, we can now predict that for random methylation only

about 1/25 of the

Not

I

sites

have no

m C and thus are cleavable. This yields

an average

DNA fragment size of 25 Mb, making

Not I all but useless for conventional macrorestric-

tion mapping. Fortunately the distribution of CpG methylation is not random. In practice,

it appears that about 90% of the

Not I sites in the human genome are not methylated, and

so they are accessible to cleavage by the enzyme. This explains why

Not

I can

generate

fragments that average about 1 Mb in size.

HTF

ISLANDS

The peculiar distribution of methylated CpG, which has such a dramatic effect on the

frequency of

Not I cutting sites, is a reflection of a more general statistical unevenness

in mammalian genomes. This was first discovered when genomic digestions were car-

ried out with a much less specific restriction enzyme,

Hpa

II. A typical genomic digest

generated by this

enzyme that

recognizes

the sequence CCGG is shown in

Figure

8.9.

A statistically random genome would be expected to give a roughly Gaussian distribu-

tion of fragment sizes. Instead, the two-phase distribution observed in practice is strik-

ing.

Hpa

II

is inhibited by DNA methylation. It

cannot cut the sequence C

m CGG. To

as good approximation, the fragment sizes shown in Figure 8.9 can be fit by assuming

that

the

genome

is divided

into

regions

where no methylation occurs and

regions

where most of the CpG’s are methylated. The large fragments in the latter regions are

what were expected from an

Hpa

II digest. The small

fragments

were

unexpected.

They

were

named

Hpa

II tiny fragments (HTFs), and the

regions that

contain

them

have

been

called

HTF islands.

As we have learned more about

the properties

of

these

regions, many researchers have preferred to call them

CpG islands,

but for others,

the

original term has stuck.

Figure 8.9

Distribution of the sizes of the DNA fragments generated by a complete

Hpa

II digest

of a typical mammalian genome.

246

PHYSICAL

MAPPING

HTF islands have a number of very interesting properties. They tend to be located near

genes, most often at the 5

-edge of genes. They are very rich in G

C. The frequency

of

CpG is as expected from binomial statistics—that is, there is no suppression of CpG in

these regions and no elevation of TpG (which results from

m

CpG

mutagenesis as de-

scribed in Chapter 1.) In HTF islands the CpG sequences are unmethylated. These results

are self-consistent: If there is no methylation in these regions, there should be no progres-

sive loss of CpG by mutation, and thus the frequency of this sequence should just reflect

the

local

C

G content. More than 90% of the known

Not

I sites appear to be located in

HTF islands; this is understandable. To produce a cleavable

Not

I site requires two nearby

unmethylated CpG’s. This is an event most unlikely to occur in the bulk of the genome

where CpG’s are both very rare and methylated.

Many HTF islands have been studied by sequencing the DNA flanking

Not

I sites.

This is relatively easy to do because, as we will discuss later in the chapter, there are

straightforward ways to clone DNA that contains a cutting site for this enzyme (or al-

most any other enzyme for that matter). Two representative human DNA sequences

flanking

Not

I

sites on chromosome 21 are described in

Figure 8

.10, which shows

the

local base composition as a function of distance from the

Not I site. The first of these

examples is extraordinarily G

C rich throughout a 600–800 base region. The second

example

shows a

transition from an HTF island to more ordinary genomic DNA; the

Not

I site is near the edge of the island. The distribution of CpG, GpC, and TpG se-

quences in the

first

of these examples

is plotted as a function of position

in Figure

8.11. It is evident that GpC’s and GpC’s are extraordinarily prevalent; more signifi-

cantly, their prevalence is roughly equivalent, showing the lack of any significant CpG

suppression.

ORDERING

RESTRICTION

FRAGMENTS

The simplest way to view top-down

mapping is by projecting a low-resolution

map

with ill-defi

ned

distances

onto

a higher-resolution map with more carefully

defined

distances. Suppose that a genetic

or cytogenetic map already exists for a chromosome

of interest, and this is actually the case today for almost all regions

of the

human

genome, at least at 2 Mb resolution (Chapter 6). Each genetically mapped or cytoge-

netically

mapped

DNA marker can be radiolabeled and used as a hybridization probe

to

identify the

corresponding large DNA fragments that it resides on, as

shown

in

Figure 8.12. If DNA fragments can be generated that are comparable in size to

the

density of available DNA markers, then most of the construction of a restriction map

would be accomplished with a relatively

small number of direct DNA hybridizations.

In reality, for almost all regions

of the genome, the probes available

today

are not

dense enough to order a complete set of restriction fragments. Instead one must utilize

procedures that allow the restriction map to be extended outward from the position of

known fragments

into

neighboring regions. A second problem is that the DNA frag-

ment sizes generated by total digestion with available rare-cutting enzymes are quite

diverse. Thus, although the average fragment size seen with

Not I is about 1 Mb, many

fragments

are 3

Mb

in size,

and many are 0.2 Mb or smaller. Available

DNA probes

are

very

likely

to

be found that

recognize the 3-Mb fragments; it is much less

likely

5. Any tiny fragments generated

by chance, by enzymes that on average yield very

large fragments, must not be ignored.

Methods now exist that deal with all

of these problems reasonably efficiently. Most will

be described in this chapter; a few

will be deferred to Chapter 14 where we deal with

DNA FRAGMENTS GENERATED BY A RARE-CUTTING RESTRICTION ENZYME

249

60-second pulses for fragments 200

to 1000 kb, 1000-second pulses at lower field

strengths for 1 to 3 Mb pieces, and 3600-second pulses or secondary PFG for fragments

larger than this.

The entire distribution of DNA fragments can be visualized by staining the PFG frac-

tionated material in the gel with ethidium bromide. This dye binds in between every other

base pair (total stoichiometry 0.5 dye per base pair), and shows a more than 25-fold fluo-

rescence enhancement when bound. Ethidium bromide appears to have no significant

specificity for any particular base compositions or base sequences. It is sensitive enough

for all routine use. Recently a number of

other dyes have been reported that may offer

the promise of higher-sensitivity DNA detection than ethidium bromide. However, the

interactions of these dyes with very large DNA molecules have not yet been fully

characterized.

If the genome under analysis is a relatively simple one, ethidium staining allows all the

pieces to be visualized, as we showed earlier in Figure 8.5. Since the amount of ethidium

bound is proportional to the size of the DNA

fragment, the resulting distribution of fluo-

rescence

intensity

in a

result like Figure 8.5 gives

the weight

average distribution

of

DNA. Small fragments are very hard to see. In general, a monotonic increase in fragment

intensity is expected as fragment size increases. Deviations from this pattern indicate

multiplets: size fractions that contain two or more unresolved DNA pieces, or heterogene-

ity; DNA fragments present in substoichiometric amounts because they arise from a con-

taminant

in the

sample,

from a restriction site that has

been only partially cleaved, or

from DNA partially degraded (by nuclease or even by electrophoresis itself). These com-

plications aside, a quantitative analysis of the pattern of ethidium staining will usually al-

low an accurate analysis of the number of DNA fragments in the genome. When these

sizes are summed, an estimate for the total genome size is produced. Indeed, when ethid-

ium staining is carried out very carefully, the stained intensity of DNA bands is not just a

monotonic function of their size; it is very close to a linear function of their size

up

to a

few Mb. Thus from the relative staining intensity alone it is sometimes possible to make

reliable

estimates

of DNA

sizes,

even when

proper size

standards, for

some

reason,

are

not present or useable.

With complex

genomes

only a smear of ethidium staining

intensity

is generally

seen.

At

the highest

obtainable

PFG resolution, the entire human

and mouse

genome actually

show a very discrete and reproducible banding pattern in ethidium-stained gels of DNA

digested

with

Not

I or other rare-cutting restriction enzymes (Fig. 8.13). The significance

of

this

pattern

has never

been

explained—but

it

does

allow

DNA from

different

species

to be identified. In those unusual cases

where

a

major

repeating

sequence

has

a

rare-

cutting site in it, a bright DNA band will sometimes be seen

above

the

background ethid-

ium smear.

To

develop

a

restriction

map

of

a

chromosome

from

a

complex

genome,

like

the

human genome, it is best to

start

with

that chromosome in a hybrid cell. Even

better,

for

the

larger

mammalian

chromosomes,

are

hybrid

cells

that

contain

only

a

10

to

DNA FRAGMENTS GENERATED BY A RARE-CUTTING RESTRICTION ENZYME

251

Figure 8.14

PFG fractionation of

Not

I-digested DNA from a mouse cell line that contains chro-

mosome 21 as the only human component. After electrophoresis

the gel was blotted, and the blot

was hybridized with the highly repeated human-specific

Alu sequence. Shown in some of the panels

are size standards used to estimate the length of particular human DNA fragments. (Taken from

Sainz et al., 1992.)

The smallest fragments of much interest in human macrorestriction mapping are around

50 kb; the least

Alu

-dense

regions of

the

genome

have a 10-kb spacing

between

Alu ’s;

thus in these regions the chance that a 50-kb fragment has no

Alu

is exp(

5). The chance,

at random, that a 1 Mb fragment should lack an

Alu

is infinitesimal. We have actually

characterized the

distribution of

Alu

’s

on

more

than

50

Not

I fragments of

human chro-

mosome 21. In practice, all but one contain

Alu

as detected by hybridization experiments.

The sole exception, however, is a 2.3 Mb fragment.

The probability

of seeing

such

an

event at

random is

e 230 assuming it

derives from

an

Alu

-poor region of the chromosome.

Clearly we will have to refine our statistical picture of human DNA sequences quite con-

siderably as more cases like this surface.

When human DNA fragments are detected by hybridization with human-specific re-

peated sequences the resulting distribution of intensity should still be generally propor-

tional to fragment size. Note, however, that considerable variations around this mean will

occur because repeated sequences tend to cluster and because small fragments with small

numbers of repeats on average will show typical small number fluctuations. One way to

generate

labeling

intensity

that is

independent

of

fragment

size

is

to

label

the

ends.

252

PHYSICAL MAPPING

This can

be done

with enzymes like polynucleotide kinase that place a

phosphate on the

5 -end

of a DNA

chain, or it can be done by filling in any inset 3

-ends of duplex DNA

digests, double

digests become hopelessly complicated to analyze.

With

mammalian

DNA, in contrast to DNA from simple genomes, one cannot

easily access

each

purified

fragment because it

is contaminated by other human or rodent

fragments. PCR can help

GENERATION

OF LARGER

DNA

FRAGMENT

SIZES

253

GENERATION OF LARGER DNA FRAGMENT SIZES

Some of the problems illustrated by the example in Figure 8.15

would

be

alleviated

if

there were a systematic way to generate large DNA fragments in a region of interest. One

general approach for doing this will be discussed here; others, like the RARE method, are

deferred to Chapter 14.

It

has

already

been

mentioned

that

most

restriction

enzymes

are

inhibited

by

DNA methylation. One can take advantage of this to increase the specificity of certain re-

striction enzymes by methylating a subset of their cutting sites. Methylation can also be

used as a general way to promote partial digests by using a methylase that recognizes all

the cutting sites but does not allow the reaction to go to completion. Here, however, we

deal with methylation reactions that are carried to completion.

Consider the DNA se-

quence shown below. It contains a

Not

I

site flanked

by

additional G–C

pairs

shown

in

boldface:

CG

CGGCCG

CG

: m CG

CGGC

m

CGCG

GC

GCCGGC

GC

GC

G

m

CCGGC

G

m C

Roughly one-quarter of all

Not

I sites

will

have

an extra

C

at

their

5

-end; an additional

quarter will have an extra G at their 3

-end

as shown

in

the

above example. These

extra

residues

generate a

recognition site

for

the

Fnu

D

II

methylase that converts CGCG to

m CGCG. Since some of the methylation is within the

Not I cutting site, this inhibits any

subsequent attempts to cleave the site with

Not I. Thus by methylation one can inactivate

about half of all the

Not

I cleavage sites and double the average

fragment size generated

by this enzyme. Many variations on this theme exist.

Methylation also plays a key role in a whole set of potential schemes for site-selective

cleavage of DNA employing the unusual restriction endonuclease

Dpn

I. This enzyme

recognizes the sequence GATC, but it requires that the A

be

methylated

in

order

for

cleavage to occur. The preferred substrate is

G m

ATC

CT

m

AG

A major complication is that the monomethyl

derivative

is

also

cut,

but

much more

slowly. Hence, in the schemes described below, it is essential to expose the DNA sub-

strate to

Dpn

I

for

the minimum

time

needed

to

cleave

at

the desired

sites before

the

background of undesired additional cleavages becomes overwhelming.

Dpn

I is converted to an infrequently cutting enzyme by starting with unmethylated

target DNA,

which

Dpn

I cannot

cleave

at

all, and

selectively

introducing

methyl

groups

by treatment with methylases with recognition sequences that overlap

part

of

the

Dpn

I

site. The simplest example, shown below, employs the

Taq

I methylase. This enzyme rec-

ognizes the sequence TCGA and converts it to TCG

m

A. If

two

such

sequences

lie adja-

cent to each other in a genome, the result, once both are methylated is

TCG

m ATCG

m

A

m AGCT

m

AGCT