Genomics- The Science and Technology Behind the Human Genome Project. Charles R. Cantor, Cassandra L / genomics1-10 / 2

CHROMOSOMES IN THE CELL CYCLE

39

CHROMOSOMES IN THE CELL CYCLE

Bacteria are organisms without nuclei. They are continuously synthesizing DNA and di-

viding if food is plentiful. In contrast, nucleated cells are often quiescent. Sometimes they

are even frozen forever in a nondividing state; examples are cells in the brain or heart

muscle. Most eukaryotic cells proceed through a similar cycle of division and DNA syn-

thesis, illustrated in Figure 2.8. Cell division is called

mitosis.

It occurs at the stage la-

beled M in the figure. After cell division, there is a stage, G1, during which no DNA syn-

thesis occurs. Initiation of DNA synthesis, triggered by some stimulus, transforms the cell

to the S phase. Not all of the DNA is necessarily synthesized in synchrony. Once synthe-

sis is completed, another resting phase ensues, G2. Finally, in response to a mitogenic

stimulus, the cell enters metaphase, and mitosis occurs in the M stage. In different

cell

types the timing of the cycle, and the factors that induce its progression, can vary widely.

Only in the M stage are the chromosomes compact and readily separable or visualiz-

able under the light microscope. In other cell cycle stages, most portions of chromosomes

are highly extended. The extended regions are called

euchromatin.

Their

extension ap-

pears to be a prerequisite for active gene expression. This is reasonable considering the

enormous steric and

topological barriers that would

have

to be

overcome

to express

a

DNA

sequence

embedded

in the highly condensed chromatin structure

hierarchy. There

are

regions,

called

heterochromatin,

unusually

rich in

simple repeated sequences that do

not decondense after metaphase but instead remain condensed throughout the cell cycle.

Heterochromatin is characteristic of centromeres but

can

occur

to

different

extents in

other regions

of the

genome. It is particularly prevalent on the

human

Y chromosome

which contains the male sex determining factor but relatively

few

other

genes.

Heterochromatic regions are frequently heterogeneous in size within a species. They are

generally sites where little or no gene expression occurs.

In most cases the level of expression of a gene does not depend much on its position in

the genome, so long as the cis-acting DNA regions needed for regulation are kept reason-

ably near the gene. In fact typical eukaryotic genes are bracketed by sequences, such as

enhancers or nuclear scaffold sites that eliminate transcriptional cross talk between adja-

cent genes. However, there are some striking exceptions to this rule,

called

positional

variation.

Using genetic or molecular methods, genes can be moved from euchromatic re-

Figure 2.11 Chromosome banding.

(a)A typical preparation of banded chromosomes. Cells are

arrested in metaphase and stained with Geimsa. Individual chromosomes are identified by the pat-

tern of dark and light bands, and rearranged manually for visual convenience. The particular indi-

vidual in this case is a male because there is one X and one Y chromosome, and he has Down’s syn-

drome because there are three copies of chromosome 21.

(b)Typical properties of bands.

42

A GENOME OVERVIEW AT THE LEVEL OF CHROMOSOMES

The molecular origin of the stained bands is not known with certainty. Dark bands seen

with a particular stain, Geimsa, appear to be slightly richer in A

T, while light bands are

slightly

richer in G

C. It is not clear how

these base composition differences can yield

such dramatic staining differences directly. At one time

the light and dark bands

were

thought to have different densities of DNA packing. As progress is made in mapping exten-

sive regions of the human genome, we can compare the DNA content in different regions.

Thus far, although there is still some controversy, not much strong evidence for significant

differences in the DNA packing density of light and dark bands can be found. The most ten-

able hypothesis that remains is that the bands reflect different DNA accessibility to reagents,

perhaps as a result of different populations of bound nonhistone proteins.

While the physical origin of chromosome bands is

obscure, the

biological

differ-

ences that have been observed between bands are dramatic. There are two general phe-

nomena (Fig. 2.11b). Light Geimsa bands are rich in genes, and they replicate early in

the S phase of the cell cycle. Dark Geimsa bands are relatively poor in genes, and they

are late replicating. Finer distinctions can be made. Certain light bands, located adja-

cent to telomeres, are extremely rich in genes and have an unusually high G

C con-

tent. An

example

is

the

Huntington’s

disease

region

at the

tip of

the

short

arm

of hu-

man chromosome 4.

The

appearance

of chromosome bands is not fixed. It depends

very

much

on

the

method that was used to prepare the chromosome. Different procedures focused on ana-

lyzing chromosomes from earlier and earlier stages in cell division yield more elongated

chromosomes that reveal increasing numbers of bands. In general, it is customary to work

with chromosomes that show a total of only about 350 bands spanning the entire human

genome

because

the

more extended forms are more difficult to

prepare

reproducibly.

Some examples are shown in Figure 2.12. One particular annoyance in studying chromo-

some banding patterns is that it complicates the naming of bands. Unfortunately, the

nomenclature

in

common

use

is based on

history.

Early

workers saw few bands and

named them outward from the centromere as p1, p2, etc., for the short (p

petit) arm and

q1, q2, etc., for the long arm (q comes after p in the

alphabet). When a particular band

could be resolved into multiplets, its components were named q21, q22,

etc. If in later

work, with more expanded chromosomes, additional sub-bands could be seen, these were

renamed as q21.1, q21.2, etc. More expansion led to more names as in q21.11, q21.12.

This nomenclature is not very systematic; it is certainly not a unique naming system, and

it risks obfuscating the true physical origins of the bands. However, we appear to be stuck

with it.

Like

the

Japanese system for assigning street addresses in the order

in

which

houses were constructed, it is wonderful for those with a proper historical perspective, but

treacherous for the newcomer.

Humans

have

22

pairs of autosomes and two sex chromosomes (XX or XY). Their

DNAs range in size from chromosome 1, the largest with 250 Mb to chromosome 21, the

smallest, with 50 Mb. One of each pair of autosomes and one sex chromosome is inher-

ited from each parent. The overall haploid DNA content of a human cell is 3

10 9 bp. At

660 Da per base pair, this leads to a haploid genome molecular weight of about 2

10 12 .

The chromosomes are distinguishable by their size and unique pattern of stained bands. A

schematic representation of each, in relatively compact

form, is given in Figure

2.13.

There are a few interesting generalizations from this genome overview. All human telom-

eres,

except Yq,

19q, and 3p, are Geimsa light bands. The ratio of

light to

dark banding

on different chromosomes can vary quite a bit from 19 which is mostly light, and appears

to have a very large

number of genes, to chromosomes 3 and 13 which are mostly dark,

and are presumably relatively sparse in genes.

CHROMOSOME PURIFICATION

43

rescence activated

cell sorter). However, it was

soon

found

to be applicable for chromo-

somes, especially

if more powerful lasers were used,

and

these

were focused more tightly.

CHROMOSOME PURIFICATION

45

FACS instruments can be used to determine a profile of chromosome sizes or other charac-

teristics, by pulse height analysis of the emission from large numbers of chromosomes, or

they can be used for actual fractionation, one chromosome at

a

time, as

shown

schemati-

cally in Figure 2.14.

In FACS, fluorescently stained metaphase chromosomes

are

passed

in a

collimated

flowing liquid stream, one at a time past a powerful focused laser beam. After passing the

laser, the stream is broken into uniform droplets by ultrasonic modulation. Each emission

pattern is captured and integrated, and the resulting pulse

height is stored

as an event. If

chromosomes

are more

fragile and

more easily

broken

than

smaller

chromosomes.

Thus they appear in substoichiometric amounts, and debris from their breakage can cont-

aminate

fractions designed to contain only a particular small chromosome. The other lim-

itation

of

chromosome

purification

by FACS is

that it

is

a single

molecule method.

Typical sorting rates are

a few thousand chromosomes per second. Even if the yield of a

particular chromosome were

perfect, this would imply the capture of only a few hundred

per second. In practice,

observed yields are often much worse than

this. Several high-

speed sorters have been

constructed that increase the throughput by

a factor of 3 to 5.

CHROMOSOME

PURIFICATION

47

However, these are not yet generally available instruments. Thus FACS affords a way of

obtaining fairly pure single chromosome material, but usually not in as large quantities as

one would really like to have. Alternatives to FACS purification of chromosomes are still

needed. One possibility is discussed in Box 2.2.

The problem of contamination of small chromosomes with large, and the problem of

resolution of certain chromosomes, can be circumvented by the use of rodent-human hy-

brid cells. These will be described in more detail later. In ideal cases they consist of a sin-

gle human chromosome in a mouse or hamster background. However, even if more than

one human chromosome is present, they are usually an improved source of starting mater-

ial. FACS is performed on hybrids just as on pure human cells. Windows (bounds on par-

ticular fluorescence signals) are used to select the desired human chromosome. Although

this will be contaminated by broken chromosome fragments, these latter will be of rodent

origin. The degree of contamination can be easily assessed by looking for rodent-specific

DNA sequences.

FACS-sorted chromosomes can be used directly by spotting them onto filters, prepar-

ing DNA in situ, and using the resulting filters as hybridization targets for particular DNA

sequences of interest (Chapter 3). In this way the pattern of hybridization of a particular

DNA sequence allows its chromosome assignment. This procedure is particularly useful

when the probe of interest shows undesirable

cross-hybridization with other human or

with rodent chromosomes. However, for most applications, it is necessary to amplify the

flow-sorted human chromosome material. This

is done either by variants of the poly-

merase chain reaction, as described in Chapter 4, or by cloning the DNA from the sorted

chromosome into various vectors. Plasmids, bacteriophages like lambda (Box 1.2), P1, or

cosmids (Box 2.3), and bacterial or yeast artificial chromosomes (BACs or YACs, Box

8.1) have all been used for this purpose. Collections of such clones are called single chro-

mosome libraries. While early libraries were often heavily contaminated and showed rela-

tively uneven representation of the DNA along the chromosome, more recently-made li-

braries appear to be much purer and more representative.

Single-chromosome libraries represent one of the most important resources for cur-

rent genome studies. They are readily

available in the United States from

the

American Type Culture Collection (ATCC). The first chromosome-specific libraries

consisting of small clones were constructed in plasmid vectors. A second set of chro-

mosome-specific libraries consists of larger

40 kb cosmid clones. One way in which

such libraries are characterized is by their coverage, the probability that a given region

is included on at least one clone. If the

average insert size cloned in

the

library

is

N

base pairs, the number of clones is

n,

and

the size of

the

chromosome

is

C base

pairs,

the redundancy of the library is just

Nn

/C. Assuming that the

library

is

a

random se-

lection of DNA fragments of the chromosome, one can compute from the coverage the

probability that any sequence is represented in the library. Consider a region on the

chromosome. The probability

that it will be contained on the first clone

examined

is

N /C. The probability that it will not

be contained on this clone is 1

N /C. After

n

clones have been picked at random the probability that none of them will contain the

region selected is (1

N /C)n . Thus we

can

write that the fraction,

f,

of

the

chromo-

some covered by the library is