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

REACHING THE ENDS OF THE PHYSICAL MAP: CLONING TELOMERES

265

REACHING THE ENDS OF THE PHYSICAL MAP:

CLONING

TELOMERES

Ten years ago there was a tremendous flurry of activity in cloning mammalian telomere-

associated sequences. This was motivated by several factors. The simple sequence repeat

at the very end of mammalian chromosomes had been determined, but little was known

about what kinds of DNA sequences lay immediately proximal to this. Such sequences

should be of considerable biological

interest, since they ought to play a role in chromo-

some function,

and they might help determine the identity of different chromosomes.

Clones near telomeres will be extremely useful probes for partial digests, as we have al-

ready described. Finally telomeres are, by definition, the ends of all linear chromosome

maps, and until they are anchored to the remainder of a genetic or physical map, one can-

not say that that map is truly complete.

True telomeres would not be expected to be present in plasmid, bacteriophage, or cos-

mid libraries, and in fact it has

been all but impossible to find them there. There are at

least two explanations for this. First, the structure of the simple sequence telomere repeat

contains a hairpin rather than a normal duplex or single-stranded overhang (Chapter 2).

This hairpin is quite stable

under the conditions used for

ordinary

DNA cloning. It will

not ligate under these conditions, and thus one would expect that the telomeres would fail

to be cloned. Second, simple tandem repeats are not very stable in typical cloning vec-

tors; they are easily lost by recombination; the 10to 30-kb simple human telomeric re-

peat would almost

certainly

not be

stable in typical bacterial cloning strains. The se-

quences

distal to

the simple

repeat

are themselves rich in medium-size tandem repeats,

and whole blocks of such sequences are repeated. Thus these regions are also likely to be

quite unstable in

E. coli.

For all these reasons it is

not surprising that early attempts

to

find telomeres in conventional libraries failed.

To

circumvent

the problems discussed above and find mammalian

telomeric

DNA,

about six research groups simultaneously developed procedures for

selectively

cloning

these sequences in

the yeast,

S. cerevisiae.

The standard cloning vector used in yeast is a

yeast artificial chromosome (YAC; see Box 8.2). We and others have reasoned that be-

cause telomeres are such key elements in chromosome function, and because the proper-

ties of telomeres are so well conserved through most species, there was a chance that a

mammalian telomere can function in yeast even if its structure is not identical to the yeast

telomere and associated sequences. To test this idea, we used half YACs as cloning vec-

tors. To survive in yeast as a stable species, the half YAC would have to become ligated to

another telomere-containing DNA fragment.

We did not expect that the process of cloning telomeres would be very efficient. To en-

hance the chances for a human telomere to be successfully cloned, we developed the pro-

cedure shown in Figure 8.34. Total genomic human DNA was digested with the restric-

tion enzyme

Eco R I. The reaction mixture was diluted, and ligase was added. All normal

Eco R I DNA fragments have two sticky ends generated by the original restriction enzyme

digest. These are capable of ligation, and at the reduced concentrations the primary liga-

tion products become intramolecular circles (just as in the case of the jumping library

construction). The only fragments that do not lose their sticky ends by ligation are telo-

meres,

which have only one

sticky

end and cannot circularize, and very

short

Eco R I

BOTTOM-UP LIBRARY ORDERING

269

by

yeast

telomeric simple sequence repeats. This indicates

that the human simple se-

quence is unstable in yeast but that some feature of it is

eventually recognized

by the

yeast telomerase, which then converts the end of the clone into a yeastlike telomere.

The particular clone that we first isolated turned out to be useful in mapping a few Mb

of DNA at the long-arm telomeres of human chromosomes 4 and 21. However, it could

only be used in a hybrid cell line because no where on this clone could any single-copy

human DNA be found. Since this early work, others have systematically cloned and char-

acterized single-copy probes from a number of human telomeres. These findings promise

to be useful also for clinical diagnostics, since DNA rearrangements at telomeres are not

uncommon in diseases like cancer.

OPTICAL

MAPPING

Recently

an optical method for restriction mapping has been described that could speed

the process considerably (Kai et al., 1995; Samad et al., 1995). This method has success-

fully been applied to bacteriphage and YAC clones and to natural yeast chromosomes.

Recently it has been semi-automated and extended to even larger DNAs. In optical map-

ping DNA molecules are elongated by gentle flow as they are fixed by capture onto poly-

L -lysine

derivatized glass surfaces. The restriction enzyme

cleavage is used to fragment

the fixed molecules. A small portion of the stretched chain relaxes at each cleavage site.

This leaves a gap that is visible by fluorescence microscopy after staining the DNA sam-

ples. The contour length of each fragment seen indicates its size. However, a very signifi-

cant feature of this method is that the pattern of organization of the fragments is main-

tained by the initial fixation, and thus the order of the fragments is immediately known.

Because this is a single-molecule method, it can deal effectively with any sample hetero-

geneity. If sufficient numbers of molecules are examined, a complete map of each class of

species in the sample should be revealed.

BOTTOM-UP LIBRARY ORDERING

The conventional approach to constructing an ordered library of clones is to fingerprint a

dense set

of samples and look for clones that share overlapping

properties. A key variable

in designing such strategies is the minimum fraction of the clones that must be in common

in

order

for their overlap to be detectable. The smaller the overlap required,

the fewer

clones needed to produce an ordered set, and the faster the

process proceeds (Fig.

8.38).

Figure 8.38 The degree of overlap,

f,of two clones of length

L1 and L2 will determine the resolu-

270

PHYSICAL MAPPING

Typical methods usually require a fiveto tenfold redundant set of clones to ensure that

there is a good chance of sampling the entire target at sufficient density to allow overlap

detection. A simplistic estimate based on this redundancy would indicate that overlaps be-

tween clones should be perfectly scored at the 80 to 90% overlap level and perhaps half

detected at the 40 to 50% overlap level. The key thing is to avoid false positives: predict-

ing an overlap where none exists in fact. This is a serious error because it will result in the

assignment

of clones to incorrect regions of the

target. A number

of

different

methods

have been used to detect overlaps in past studies. In all of these

a

key

requirement is

to

have some sort of statistical way of determining the most likely set of overlaps in cases

where there are ambiguities or potential inconsistencies. One very effective approach for

evaluating overlap data is summarized in Box 8.3.

The earliest clone fingerprinting methods used restriction fragment sizes seen in single

and double digests with 6-base specific enzymes like

Eco

R I and

Hind

III. This approach

was first used to order bacteriophage lambda clones of

S. cerevisiae.

It

was actually

very

difficult because of inaccuracies in sizing DNA fragments in the 1 to 10 kb range, particu-

larly when results obtained on different gels electrophoresed on different days had to be

compared. More accurate sizing is possible with smaller DNA fragments, but the number

of such fragments that result from digestion of a bacteriophage or larger clone with a 4-

base specific restriction enzyme is too large to allow all of them to be separated cleanly.

One way around this problem is to use an end-labeling strategy as shown in Figure 8.39.

This was first developed to order a cosmid library from the nematode

C. elegans.

First the

clone is digested with a 6-base specific enzyme. The ends of the resulting fragments clone

are labeled. Then a second digest is done with a

4-base specific

enzyme. This

results in

DNA pieces

that can be analyzed with single-base

resolution of

DNA

sequencing

gels,

but their number is restricted to a manageable set.

An alternative approach for clone fingerprinting by restriction enzyme digestion is il-

lustrated

in Figure 8.40. This procedure was first used by

Kohara

in

constructing an

E. coli

library in bacteriophage lambda clones. Indirect end labeling from probes in the

vector sequence was used to determine the positions of restriction sites seen in separate

partial digests, each generated with one of eight different restriction enzymes. The key

advantage of this approach was that all eight digests were analyzed in a single gel elec-

trophoresis. Under these circumstances, even though the DNA size information

might be

272

PHYSICAL MAPPING

BOX

8.3

(Continued)

To use the

above equations

one must evaluate

P ( ,O ) and

P ( ,O ) before

P ( ,O ) can be calculated. When these results are considered for the entire set of

restriction digests or hybridization results seen, we can compute

the likelihood,

L,of

any particular overlap value,

f,for each pair of clones as

L( f)

P (X n ,O )

P (X n ,N )

n

where the outcome for the comparison of the two clones with the

n th test is

xn , and the

product is taken over the entire set of tests. The calculation is very time intensive be-

cause all pairs of probes must be considered separately for all probes and all overlap

values. The procedure can be simplified by using results with longer clones to arrange

the shorter clones into bins (see Fig. 9.4). Then overlap calculations need to be carried

out only within bins and between the edges of neighboring bins.

accuracy. This order information was

the major source of data used to fingerprint

the

clones.

Some of these approaches can be improved upon by using the power of current auto-

mated four-color DNA sequence readers (see Chapter 10). An example is shown in Figure

8.41. Here restriction fragments are

filled in by end labeling in order to reduce the

num-

and hybridized

with several different interspersed repeating sequence probes. These

probes provide a

signature that goes beyond pure size measurements and indicates those

Several

other

powerful variations of clone fingerprinting

have been

described, and

some of these are now being tested intensively. If repeats can be

nulled out, then the use

of clones or

sets of clones as hybridization probes against other clones or sets of clones

becomes an effective

fingerprinting method. This approach will be

described

in detail in