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

same DNA replication machinery as the

chromosome, and they are present in a copy

number that is equal to the chromosomal copy number. An example is the naturally oc-

curring F

DNA of

E. coli

which represents about 2% of the DNA present in the cell.

In some bacteria essential genes are located on two genetic elements. For instance, in

Rhodobacteria sphaeroides

the genes encoding the ribosomal RNAs (rRNAs) are located

on a 0.9

Mb chromosome,

whereas the

remainder of the genome is located on a 3 Mb

BACTERIAL CHROMOSOMES

31

is defined as all of the DNA

in a cell, plasmids and other extrachromosomal DNA ele-

ments must be counted as part of it.

Bacterial chromosomes contain bound protein molecules that are essential for normal

growth. Some of these promote an organization or packaging of DNA similar to that seen

in higher

organisms. However,

the proteins that appear to be responsible

for packaging

are present in such small amounts that can only interact with about 20% of the genomic

DNA. Furthermore the packaging does not seem to be as orderly or as stable as the pack-

aging of

DNA in eukaryotic

chromosomes. Bacterial chromosomal DNA is

organized

In condensed chromatin, loops of 300 Å fiber are attached to a scaffold. Although

the

DNA of

mammalian chromosomes is linear,

these

frequent attachment

points

make

each

constrained

loop topologically into a

circle. Thus topoisomerases are

needed for

the DNA in

chromatin to function.

Type

II topoisomerases are

a major

All higher organisms usually have linear chromosomal DNA molecules, although circles

can be produced under special circumstances. These molecules have a minimum of three

functional features, as shown in Figure 2.1. Telomeres are specialized

structures

at the

ends of the chromosome. These serve at

least two functions. They provide a mechanism

by which the ends of the linear chromosomes can be replicated. They stabilize the ends.

Normal double-strand DNA ends are very

unstable in eukaryotic cells. Such ends could

be the result of DNA damage, such as that caused by X rays, and could be lethal events.

Hence very efficient repair systems exist that rapidly ligate ends not containing telomeres

together. If several DNAs are broken simultaneously in a single cell, the correct fragment

pairs are unlikely to be reassembled,

and one or more translocations will result. If the

ends are not repaired fast enough by ligation, they may invade duplex DNA in order to be

repaired by recombination. The result, even for a single, original DNA break, is a re-

arranged genome.

Centromeres are DNA regions necessary for precise segregation of chromosomes to

daughter cells during cell division. They are the binding site for proteins that make up the

kinetochore, which in turn serves as the attachment site for microtubules, the cellular or-

ganelles that pull the chromosomes apart during cell division.

Another feature of eukaryotic chromosomes is replication origins. We know much less

about the detailed structural properties of eukaryotic origins than prokaryotic ones. What

is clear from inspecting the pattern of DNA synthesis along chromosomes is that most

chromosomes have many active replication origins. These do not necessarily all initiate at

the same time, but a typical replicating chromosome will have many active origins. The

presence of multiple replication origins allows for complete replication of entire human

genome in only 8 hours. It is not known how these replication processes terminate.

CENTROMERES

In

the yeast,

S. cerevisiae,

the centromere has been defined by genetic and molecular ex-

periments to reside in a small DNA region, about 100 bp in length. This region contains

several A–T rich sequences that may be

of key importance for function. The centromere

of

S. cerevisiae

is very different in size and characteristics than

the centromeres of more

advanced organisms, or even the yeast

S. pombe.

This is perhaps not too surprising in

view of the key role that the centromere plays in cell division. Unlike these other species,

S. cerevisiae

does not undergo symmetrical cell division. Instead, it buds and exports one

copy of each of its chromosomes to the daughter cell. In species that produce two nomi-

nally identical daughter cells, centromeres appear to be composed mostly of DNA with

tandemly repeating sequences. The most striking feature of these repeats is that the num-

ber of copies can vary widely. A small repeating sequence, (GGAAT)

n , has recently been

found to be conserved across a wide range of species. This conservation suggests that the

sequence may be a key functional element in centromeres. While not yet proved, physical

studies on this DNA sequence reveal it

to have rather unusual helical properties that at

least make it an attractive candidate for a functional element. As shown in Figure 2.2,

the

DYNAMIC BEHAVIOR OF TELOMERES

35

DYNAMIC BEHAVIOR

OF TELOMERES

The actual length of the simple telomeric repeating DNA sequence is highly variable both

within species and between species. This appears to be a consequence, at least in part, of

the way in which telomeres are synthesized and broken down. Telomeres are not static

structures. If the

growth

of cells is monitored through successive generations, telomeres

are observed to gradually shrink and then sometimes lengthen considerably. Nondividing

cells and some cancer cells appear to have abnormal telomere lengths. At least two mech-

anisms are known that can lead to telomere degradation. These are shown in Figure 2.5

a.

In one mechanism the single-strand extension is subject to some nuclease cleavage. This

shortens that strand. The alternate mechanism is based on the fact that the 5

-ended strand

must serve as a starting position for DNA replication. This presumably occurs by the gen-

eration of an RNA primer

that is then extended inward by DNA

replication. Because of

the tandem nature of the repeated sequence, the primer can easily be displaced inward be-

fore synthesis continues. This will shorten the 5

-strand. Both of these mechanisms seem

likely to occur in practice.

A totally different mechanism exists to synthesize telomeres and to lengthen existing

telomeres (Fig. 2.5

b ). The enzyme telomerase is present in all cells with telomeres. It is a

ribonucleoprotein. The RNA component is used as a template to

direct the synthesis

of

the 3 -overhang of the telomere. Thus the telomere is lengthened by integral numbers of

repeat units. It is not known how the complex and subtle variations seen in this simple se-

quence arise in practice. Perhaps there is a family of telomerases with different templates.

More likely, some of the sequences are modified after synthesis, or the telomerase

may

just be sloppy.

age: (a)Nuclease cleavage;

(b)downstream priming by telomerase;

(c)mechanism of telomere

growth.

36

A GENOME OVERVIEW AT THE LEVEL OF CHROMOSOMES

The total length of telomeric DNA in the human ranges from 10 to 30 kb. Because of

its heterogeneity, fragments of DNA cut from any particular telomere by restriction en-

zymes have a broad size range and appear in gel electrophoretic size

fractionations

as

broad, fuzzy bands. This is sufficiently characteristic of telomere behavior to constitute

reasonable evidence that the band in question is telomeric. In mice, the length of telom-

eres is much longer, typically 100 kb. We don’t know what this means. In general, the

message from both telomeres and centromeres is that eukaryotic cells apparently feel no

pressure to minimize the size of their genome or the sizes of these important functional

elements. Unlike viruses, which must fit into small packages for cell escape and reinfec-

tion, and bacterial cells which are under selection pressure

in

rich

media

to

replicate

as

fast as they can, eukaryotic cells are not mean and lean.

CHROMATIN

AND THE HIGHER-ORDER

STRUCTURE

OF CHROMOSOMES

A eukaryotic chromosome is only about half DNA by weight. The remainder is protein,

mostly histones. There are five related, very basic

histone

proteins

that

bind

tightly

to

DNA. The rest is a complex mixture called, loosely,

nonhistone

chromosomal proteins.

This mixture consists of proteins needed to mediate successive higher-order packaging of

the DNA, proteins needed for chromosome segregation, and proteins involved in gene ex-

pression

and regulation. An enormous effort has gone into characterizing

these

proteins

and the structures they form. Nevertheless, for the most part their role in higher-order

structure or function is unknown.

At the lowest level of chromatin folding, 8 histones (two each of four types) assem-

ble into a globular core structure that binds about 140 bp of DNA

forming

it

into

a

coiled structure called the nucleosome (Fig. 2.6). Not only

are

nucleosomes

very

similar

in

all

organisms

from

yeast

to

humans

but

in

addition

the four core his-

tones are among the most evolutionarily conserved proteins known. Nucleosomes pack

together to form a filament that is 100 Å in diameter and is known by this name. The

details

of the filament are different in different species

because

the

lengths

of

the

spacer DNA between the nucleosomes varies.

In

turn the

100

Å

filament

is

coiled

upon itself to make a thicker structure, called the 300 Å fiber. This appears to be

solenoidal in shape. Stretches of solenoid containing on average 50 to 100 kb of DNA

are attached to a protein core. In condensed metaphase chromosomes,

this

core

ap-

pears as a central scaffold of the chromosome; it can be seen when the chromosome is

largely

stripped

of other

proteins and

examined

by

electron

microscopy (Fig.

2.7).

A major component of this scaffold is the enzyme topoisomerase II (Box 2.1), which

probably serves a role analogous to DNA gyrase in

E.

coli

of acting as a swivel to

circumvent any topological problems caused by

the

interwound

nature

of

DNA

strands.

At other stages in the cell cycle, the scaffold proteins may actually attach to the nu-

clear envelope. The chromosome is then suspended from this envelope into the

interior

of the nucleus. During mitosis, if this picture is correct, the chromosomes are then

essentially

turned inside

out,

separated into

daughters,

and

reinverted.

The

topologi-

cal domains created by DNA attachment to the scaffold at 50 to 100 kb intervals are

similar

in size to those seen in bacteria, where they

appear

to

be

formed

by

much

simpler structures.

Figur

e 2.6 Hierarchy

of structural

elements

in

chromatin

and

chromosomes:

(a)The

nucleosome;

(b)a 100 Å fila-

ment;

(c)a 300 Å solenoid.

(d)chromosome

loop

anchored

to

a protein

scaf fold;

(e)successi

ve

loops

stack

ed

along

the

scaf fold; (f)the appearance

of a condensed

metaphase

chromosome.

The

path of

the scaf

fold

is

unkno

wn,

b ut

it is

not

straight.