GENETICS AND THE HUMAN GENOME ~
OCR for page 8
example is Down syndrome, in which an extra copy of one of the smallest
human chromosomes causes mental retardation, congenital heart cle-
fects, and increased susceptibility to infection. Other diseases of this
type arise from a(lditional copies of other chromosomes, missing chro-
mosomes, or chromosomes in which the genetic message has become
garbled through insertions, (leletions, or other obvious rearrangements.
Another category of hereditary diseases is macle up of those diseases
associated with a defect in a single gene. These diseases are generally
livide(1 into two groups, dominant and recessive, depending on their
mode of inheritance. The DNA in human cells is clivided into 46
chromosomes organized into 23 pairs (Figure A-. Each pair consists
of a copy of one of the 23 chromosomes in the father's sperm cell and
a copy of one of the 23 chromosomes in the mothf~rtc ~ ~11 Ul;~h
~ 1 ~. · ~1 =r ~ ~
~ ~ ~ ~ ..~ · ~ ~ 1 ~ 1 1
one exception ot the ~ and Y chromosomes in males, the two members
of each pair are very similar. They contain virtually the same genes in
virtually the same orcler. But the pairs of genes are not identical. Over
30 percent of the corresponding genes in a chromosome pair (1iffer in
some way, reflecting the overall genetic variability of the human pop-
ulation.
A dominant genetic (lisease occurs when an individual ren~eivec ~
defective gene from either parent. in other words, the presence of a
functioning gene on one member of a chromosome pair is not enough
to overcome the effects of the defective gene on the other member. An
example is familial hypercholesterolemia, in which an inability to clear
cholesterol from the blood can cause children to suffer fatal heart attacks
as early as 18 months. A person with a dominant genetic disease has
a 50-50 chance of passing it on to a chilcl.
Recessive genetic defects require that a person inherit defective
copies of a gene from both parents. A person who has one defective
and one functioning version of such a gene, known as a carrier, suffers
little or no ill effects. But each child of two carriers has a one in four
chance of inheriting two (defective genes and suffering from the (lisease.
Examples of recessive diseases are sickle cell anemia, cystic fibrosis,
Duchenne muscular dystrophy, and Tay-Sachs clisease.
Over 3,000 single-gene, or monogenic, (diseases have been identified.
Although individually rare, together they account for a great clear of
human suffering. They affect more than ~ percent of liveborn infants
anti cause almost TO percent of deaths among children.
The role of genetics is not as clear-cut in m~1ti~enir. I thick
~ _ 1 _ . 1 . . · ~
~ I, ~= ~
1~lvolvlng Ine interactions ot a number of genes with the environment.
Examples of multigenic diseases include "hypertension, schizophrenia,
manic (repression, juvenile diabetes' heart (liseases, rheumatoid ar
8 SHAPING THE FUTURE
OCR for page 9
thritis, ant] a host of others "
according to Berg. Biologists know that
these diseases can be inherited, because they can cluster in families.
But they clo not yet know the number or identity of most of the genes
involves} in these diseases.
Cancer should also be cllasse(1 as a genetic disease, Berg argues,
even though it is not a hereditary disease. Cancer is causer] by defects
in the genetic signals that regulate cell growth ant! reproduction, and
human families clearly have genetic predispositions to certain kinds of
A
C
D
_
-
_.
2
it::
~ a
6 7 8
: ~
~&-&
~_ IF
13 14 15
~ ~5
I.
B - _
4
. -
~-
s
9 10 11 12
~-
hi:
16 17 18
F '-' l-'- G i-`-i-l Of
At, .
19 20 21 22 X / Y
FIGURE 1-1 The DNA in human cells consists of 23 pairs of chromosomes.
Twenty two of these pairs, which are numbered roughly in order of descending length,
have very similar members, reflecting the equal genetic contributions of mother and
father. In addition, each cell carries either two X chromosomes (in the case of a
female) or an X and a Y chromosome I in the case of a male). Chromosome photographs
such as the one shown here, known as karyotypes, are produced by arresting a cell
during division (the chromosomes have doubled but not split apart), staining the
chromosomes, and arranging photographs of the chromosomes by size. Karyotype
courtesy of Dr. Patricia N. Howard-Peebles, Genetics & IVF Institute, Fairfax,
Virginia.
GENETICS AND THE HUMAN GENOME 9
OCR for page 10
tumors. Also, genetic defects in the human immune system, in DNA
repair systems, and in the ability to metabolize carcinogens are asso-
ciatec] with a higher frequency of certain tumors.
Even infectious diseases, according to Berg' can be seen as genetic
diseases. But in this case' the genes of the infecting organism and their
relationship with the host ant] with the environment determine the course
of the disease.
Tracking Disease to Its Source
Molecular biology has already clemonstrated the amazing precision
it can offer in analyzing genetic diseases. Research has shown, for
instance, that sickle cell disease, which is characterized by anemia,
impaired growth, and increaser] susceptibility to infection, is caused
by a change in a single genetic base. This change lea(ls to the substi-
tution of one amino acid for another in hemoglobin, the protein that
carries oxygen in the blood, causing the molecule to misfunction when
oxygen is scarce. Another example is phenylketonuria (PKU), which
can cause severe mental retardation if uncliagnosed, it is caused by a
efect in the gene co(ling for an enzyme that converts one amino acid
into another. ``Every few months another gene is isolated and the struc-
tural defect is identified," says Berg. "It's not too optimistic, ~ think,
to predict that the genes and their (lefects for many of the monogenic
diseases will be known within the next five years."
The impressive accomplishments of the past emphasize how much
remains to be learned, however. Of the over 3,000 monogenic (diseases
now recognized, the responsible gene has been identified in only about
100 cases. The genes involved in multigenic diseases and disease
susceptibilities remain largely unknown.
The ideal situation would be to know the gene or genes involved in
every human disease, their location on the chromosomes, the nature of
the defect associated with the disease, and the way in which the defect
contributes to the disease. This information is known for very few
diseases, and attaining it for the majority of diseases will take many
decades. But biologists are starting to systematically pursue some of
the early stages of such a program. They are constructing maps of the
human chromosomes that give the locations of known genes. By mapping
these locations, researchers can clevelop genetic probes to (letermine
whether a person has a normal or a defective gene, leading to great
advances in the diagnosis and treatment of disease. Genetic maps can
also reveal patterns in the way genes are organized and regulate(l,
10 SHAPING THE FUTURE
OCR for page 11
leading to a greater understanding of how genes function in health and
disease.
The scientific community has also been considering a much more
ambitious proposal: a plan to determine the exact sequence of the
billions of genetic bases making up the human genome. This information
would be an invaluable resource for biologists. It would allow them to
identify all of the genes within a given region of a chromosome, including
many currently unknown genes. Genetic differences among inclivicluals
could be compared, revealing a great deal about the functioning of
normal and defective genes. The mechanisms that control the expression
of genes and the processes involved in development could be deci-
pherecI. The structure and organization of genes in different species
could be contrasted, leacling to insights into the evolutionary processes
that resulted] in those species.
The complete sequence would not answer every question in biology.
it would not establish exactly how genes are controller! or how gene
products function within a cell or organism. It would not completely
~rnl~in how n'?onle are different or how humans have evolved. But it
is unlikely that these questions can be answered without a creep un-
clerstanding of the human genome.
AN ~ ~141 1~J ~ r~r ~ At, ~ __
Putting Genes on a Map
Geneticists were mapping genes to chromosomes well before they
knew how chromosomes are constructed. The first genetic map was
made in 1913, for five traits carried on the X chromosome of the fruit
fly Drosophila melar~ogaster. Today, maps of human chromosomes are
being made using the same principles.
The method used to make such maps involves analyzing how traits
are passed down from generation to generation. in a parent's repro-
ductive system, each chromosome pair separates during the formation
of egg and sperm cells, reducing the original 46 chromosomes to 23.
If this were all that occurred during reproduction, inheritance would
be a straightforward matter, chromosomes would remain intact and be
passed unchanged! between generations. But chromosomes do not re-
main intact. Rather, before the formation of egg and sperm cells, each
chromosome pair can exchange parts through a process known as cross
~ng-over.
Imagine three different genes located on a pair of chromosomes
(Figure T-2~. Each chromosome can have different versions of these
genes, which interact according to the usual rules for dominant ant!
.
GENETICS AND THE HUMAN GENOME 11
OCR for page 12
Without Crossing-Over
~ -~/~J'-~:
Chromosome Each Chromosome \
Pairs Come Duplicates, Remaining \
Together in Linked at Centromere Cell \~_ a ~
Reproductive Divides l
Cells Once _ b I
Cells Divide
Again to Yield
Four Sperm Cells
or Egg Cells
With Crossing-Over
~ o ~ B\e bib
~/
~J `1
Chromosome
Pairs Come
Together in
Reproductive
Cells
Duplicated
Chromosomes
Exchange Pieces
/~
Cell
Divides
Once
1~]
1 'A
\
Cells Divide
Again to Yield
Four Sperm Cells
or Egg Cells
FIGURE 1-2 During the formation of egg and sperm cells, a process known as
crossing-over can unlink versions of genes located on the same chromosome. In the
above diagram, A and a are versions of a specific gene located on a chromosome
pair, as are B and b and C and c. During crossing-over, A becomes unlinked from
B and C in two of the four sperm or egg cells produced. If the resulting cells produce
offspring, the genetic linkage will differ between parent and child. This information
can be used to deduce the relative distance between the different genes.
12 SHAPING THE FUTURE
OCR for page 13
recessive genes. For instance' gene B in Figure T-2 may be for brown
eyes a dominant trait while gene b is for blue eyes.
Tf crossing-over ant] other forms of genetic recombination never oc-
curred, the versions of genes located on a given chromosome wouIc!
always be inherited! together. In genetic terms, they would be perma-
nently linked (top half of Figure Tub. But crossing-over can reshuffle
the genes on a chromosome pair, resulting in new genetic combinations.
The trick in genetic mapping is to observe how often this process
separates the versions of genes on a chromosome. As shown in the
~ ~ ~ r r ~ ~ ~
bottom half of Figure T-2, genes that are close together tend to stay
together, whereas distant genes are easier to separate. By noting the
frequency of separation, researchers can calculate the distance between
genes and assign them relative locations on a chromosome.
Another way to map genes draws on the appearance of chromosomes
under a microscope. If the chromosomes are stainer} during cell division
with certain chemicals, alternating bands of light and `dark regions
appear, with up to several (dozen bands on a single chromosome (Figure
T-3~. These cytogenetic bancis distinguish the chromosomes and provide
broad lanclmarks along their length. But they are still quite large' with
each band containing an average of 100 genes.
-
.~
.~ -
: -
Neuroblastoma
| ~ Gaucher Disease, Type 1
FIGURE 1-3 Several dozen disease-causing genes have been mapped to human
chromosome 1. The gene responsible for Gaucher disease, type 1, a recessive disease
characterized by an enlarged spleen, skin pigmentation, and bone lesions, has been
mapped to a section of the q arm of the chromosome. One or more genes that control
the spread of tumors in neuroblastoma, a cancer of nerve cells, have been localized
to part of the chromosome's p arm. Karyotype of chromosome 1 courtesy of Dr. Patricia
N. Howard-Peebles, Genetics & IVF Institute, Fairfax, Virginia.
GENETICS AND THE HUMAN GENOME 13
OCR for page 14
Some people with genetic disorders have recognizable abnormalities
in their cytogenetic banding patterns. For instance, the gene for Duch-
enne muscular (lystrophy was mapped to a specific region of the X
chromosome by noting that some sufferers of the disease were missing
that portion of the chromosome. TransIocations, in which part of one
chromosome has broken away and become attacher] to another chro-
mosome, and fragile sites that are susceptible to breakage have also
pointer] to the locations of specific genes.
If the base sequence of the gene being mapped is known, it can be
located on a chromosome by making radioactive copies of part or all
of the gene (by synthesizing or cloning the gene using radioactive
constituents). These DNA probes can then be mixed with human chro-
mosomes under chemical conditions that cause the DNA strands to
temporarily separate. When a single-stran~le<1 probe encounters a matching
single strand of DNA on a chromosome, the two combine or hybridize
to produce double-stranclecl DNA. The radioactivity given off by the
probe then functions as a marker to find the chromosome and the
approximate location of the gene.
Another method of locating genes on chromosomes involves an in-
triguing technique known as somatic cell hybridization. When human
cells and mouse tumor cells are grown together under the proper con-
clitions, they tend to fuse and form hybrid cells. As these hybrid ceils
grow and divide, they lose most of their human chromosomes. But often
one or a few human chromosomes will become stably established in a
particular cell. The result is a mouse tumor cell line containing specific
human chromosomes. By looking for human proteins pro(luced by a
given gene, that gene can be assigned to a chromosome carried in a
cell line. Genes with known sequences can also be located using nNA
hybridization.
All of these techniques can establish the relative or approximate
location of a gene on a chromosome. But to map a gene to an exact
chromosomal location requires the techniques of recombinant DNA.
~_ .
A Genetic Scalpel
The ability of biologists to recombine DNA at will originated in the
discovery of enzymes in bacteria that can cut DNA at specific sequences
of four to ten genetic bases. These so-called restriction enzymes, which
bacteria evolved to fight invasions of foreign DNA, were a marvelous
gift to biologists. They allow researchers to slice DNA at specific lo-
cations, so that [Large DNA molecules can be cut into smaller, more
14 SHAPING THE FUTURE
OCR for page 15
manageable pieces. Then, using a class of enzymes known as ligases,
biologists could splice together any two pieces of DNA formed with the
same restriction enzymes. In this way, they couIcI create mosaics of
DNA, known as chimeras, that have never before existed in nature. (In
Greek mythology, the chimera is an animal with the head of a lion, the
body of a goat, and a serpent for a tail.)
Recombinant DNA has transformed the mapping of the human gen-
ome. It has enabled researchers to cut complex genomes into pieces'
each of which can then be cloned many times over. These pieces can
be organized into a library, creating a complete collection of all the
DNA in an organism. Indiviclual volumes in the library can be further
analyzecl to map specific genes or pieces of DNA. The logical conclusion
of this process is the next step beyond mapping: the sequencing of
genetic bases in part or all of an organism's genome.
One of the products of recombinant DNA technology has been a
family of genetic markers much more precise and powerful than the
banding patterns on chromosomes. Human beings are much more alike
genetically than they are unalike. Only about one in every hundred
base pairs of DNA are different between any two people. Many of these
differences have no effect on the functioning of the genes, but others
contribute to the crucial differences that make us unique: differences
in physical appearance, aspects of personality susceptibility to disease.
Without these genetic differences, people would all be as alike as
identical twins.
Using recombinant DNA, genetic differences among individuals can
also serve as markers along human chromosomes. First DNA from a
person's cells is cut into pieces using a specific restriction enzyme,
and the resulting pieces are placed along the edge of a gel. Under
~ ~ , ~ ~ ~
~ · · LIT · 1 ~ · 1 ~_ · ~. · ~
normal ColldltlOnS' L'1NIA has a sllgntly negative electric charge, so when
an electrical potential is applied across the gel the DNAis attracted
to the positive charge on the other side of the gel. The smaller pieces
of DNA move faster through the gel than do the larger ones. When the
DNA pieces are spread across the gel, this process, known as electro-
phoresis, is stopped. The result is a virtually continuous series of bands,
with each bane] corresponding to a DNA fragment of a different length.
~1 .] ~ · ~ 1 ~ .
~cat - - -
.r. ~
C~7
Researchers can then highlight specific bancts using radioactive DNA
probes that combine with given DNA sequences.
If two individuals have a difference, known as a polymorphism, in
the region of DNA being tested, that difference can cause the restriction
enzyme to produce DNA fragments of different lengths; For instance
~ -
. .
say a person has a difference in a sequence recognized by a restriction
enzyme, causing the sequence to remain uncut (Figure T-4~. If so, the
GENETICS AND THE HUMAN GENOME 15
OCR for page 17
RFLPs and Disease Diagnosis
The implications for genetics of RF~Ps are "extraordinary," accord-
ing to Berg. Geneticists have aIreacly located hundreds of RF[Ps scat-
tered throughout the human genome. At first, a RAP may be used as
a flag to indicate the presence of a clisease-causing gene closely linked
to the REAP. Later, RF~Ps may be used to track clown the actual
location of that gene, so that the nature of its defect can be cletermined.
The use of RF~Ps ant! other genetic probes to diagnose genetic
iseases "will change medicine in a very profound way," according to
Hood. For instance, it will eventually be possible to detect the genes
responsible for virtually every monogenic ~lisor(ler. These genes could
be detected after birth or prenatally, giving parents an option to ter-
minate a pregnancy. Carriers of defective genes conic! also be identifiecI,
allowing them to decide whether to have children and risk passing on
the disease.
The identification of carriers anc! prenatal testing conic] greatly reduce
the toll that many monogenic diseases take on the human population,
Berg points out. An example is Tay-Sachs disease, a recessive disease
occurring most frequently among Jews of European crescent that causes
retardation, paralysis, and early death. A test for Tay-Sachs disease
that can identify carriers and affecter! fetuses has been available for a
number of years. This test "has virtually eliminated Tay-Sachs disease
from the Jewish population," Berg says.
As scientists learn more about the role of genes in multigenic clis-
eases, it will increasingly be possible to (letermine an inclividual's
susceptibility to such diseases. In time, diagnostic tests shouIc} be
available to determine a person's susceptibility to such common diseases
as cancer, heart disease' ant! diabetes.
Such tests coul(1 provide "a new opportunity for preventive medicine,"
says Hood. Now, genetic testing is confiner! largely to fetuses and
expecting parents. But in the future it will be possible to diagnose the
disease susceptibilities of anyone ant! enter that information into a
medical record. That information could help a person to avoid certain
habits, cliets, or environmental conditions that might leac! to the disease.
New therapeutics might also be clevelopec! that could tower the chance
of contracting a disease when a susceptibility exists. "We can start to
think about changing some fundamental aspects of human health care,"
says Hood.
As an interesting siclelight, RF~Ps and other DNA probes have also
assumed a growing role in forensic medicine. The patterns in a person's
DNA are just as distinctive as a fingerprint, and in some crimes DNA
GENETICS AND THE HUMAN GENOME 17
OCR for page 18
samples are easier to find than fingerprints. Already, genetic tests have
been used to identify rape suspects, murcler suspects, and the parents
of children applying for immigration. ``Very soon we will be in a position
to translate genetic patterns into electronic databases and compare
particular DNA fingerprints with any others that come up'" says Hood.
``This raises challenging social and ethical questions."
The Price of Knowledge
The social and ethical questions posed by genetic analysis extend
far beyond DNA fingerprinting. As testing for diseases and disease
susceptibilities increases, it will be possible for people to learn a great
deal about themselves and their futures. if it were possible for a person
to know what he or she was most likely to die of, would that person
want to know? Today, genetic testing is often done as the prelude to
some therapeutic intervention, from the abortion of a severely affected
fetus to dietary or pharmacological interventions. But treatment may
not be possible for some of the diseases that will be detectable in the
future, at least until researchers apply new understandings of genetic
information to the development of new therapies.
A stark example of the dilemma posed by the gap between diagnosis
and treatment is Huntington's disease, which threatens about 125,000
people in the United States. The disease is caused by a dominant gene
inherited from either the mother or the father, therefore, a person with
an affected parent has a SO-SO chance of also having the disease. The
symptoms of the disease, beginning with loss of control over movement
and proceeding to dementia and eventual death, generally do not appear
until after the childbearing years. As with many monogenic diseases,
there is no treatment for Huntington's disease.
Through extensive analyses of RFI~Ps from families afflicted by Hun-
tington's disease, geneticists have narrowed the location of the Hun-
tington's gene to a million-base-pair region at the end of chromosome
4. By studying the RF~Ps of individual families, researchers can usually
find markers that signal the presence of the defective gene with a high
degree of accuracy. The question then becomes, Will a person at risk
of Huntington's disease want to know whether he or she carries the
gene for the disease? if a person is trying to decide whether to have a
child and risk passing the gene on to the next generation, the information
Is clearly useful. But if a person is past the childbearing years or does
~~~ ~ undergo a genetic test that has the
not want to have children, why ~ v
potential to predict future illness and early death?
18 SHAPING THE FUTURE
OCR for page 19
.
AS genetic testing becomes more powerful, similar dilemmas will
emerge. The ability to test for the presence of a gene is an inevitable
step in the process of unclerstanding a disease and developing ways to
treat it. But some diseases will always remain untreatable, and for
r 1 . ~
others the ability to diagnose a disease is imminent whereas the treat-
ment for it is much less certain.
Information from genetic testing will also raise a number of conten-
tious social questions. Should employers or insurance companies have
access to such information, even though in some cases it could lead to
loss of a job or loss of insurance coverage? What kinds of traits or
conditions should prospective parents be able to test for in deciding
whether to have chilciren or abort a pregnancy? Guidelines will have
to be established for the use of such information to preserve individual
confidentiality and autonomy.
Increaser] genetic testing will also require that people understand
the information they are being given. An increased susceptibility to a
disease does not mean that a person will inevitably get that disease.
In fact, the chance of getting the (disease may be quite small. Yet such
a diagnosis couIcl greatly increase a person's anxiety and affect important
~ · .
declslons.
Even in cases in which a diagnosis is more certain, the potential for
misinterpretation will be large. People who have a disease caused by
a single gene can show enormous variations in the manifestations an
severity of the (lisease. This variation is so great, says Berg, '`that some
physicians who see these patients cannot accept that they're all causer]
by the same genetic defect." In part, says Berg, these differences are
caused by a person's broac! genetic background, which probably will
not enter into the cliagnosis. They are also caused by the environmental
influences a person experiences, which can be endlessly variable. Peo-
ple will therefore have to understand the full range of outcomes that
could accompany a specific (liagnosis.
These are not new issues in human genetics. Similar situations have
already been addressed by researchers, clinicians, patients, and pol-
icymakers. But the issues will become more numerous and wiclespread
as biologists learn more about the connection between a person's genetic
heritage and disease.
From Mapping to Sequencing
From a purely scientific standpoint, biologists generally agree that
a program to map the human genome through the construction of a large
GENETICS AND THE HUMAN GENOME 19
OCR for page 20
library of RF[Ps and other genetic markers is a worthwhile goal. But
the new techniques of molecular biology allow more to be done. Within
a few years it should be possible to begin systematically sequencing
the 3 billion base pairs in the human genome. Tt would be the largest
project ever undertaken in biology' with characteristics much different
than those of normal biological research. There is much less consensus
among biologists about how to undertake such a program.
One thing is clear' however: it will be a formiciable task. Apart from
the technical difficulties involved in sequencing' the project will pro-
cluce a tremendous amount of information to store, analyze, and dis-
seminate. There are about 3 billion bases that would neec! to be sequenced
in the human genome (technically, only one member of each chromo-
some pair needs to be sequenced, since the members of a pair are so
similar) if every letter in this book represented one of these bases, it
would take roughly 10,000 of these books to represent the entire se-
quence. Furthermore, sequencing programs will leave to acquire se-
quences from different individuals and from other species to make the
best use of the human sequence. So far, only about 2 million base pairs
of the human genome have been sequenced ant] storer! in a central data
Building a Better Sequencer
The standard laboratory method used to
sequence DNA involves labeling fragments
of the unknown DNA with a radioactive
marker and passing the fragments through
four different electrophoretic gels, one for
each kind of base. Once the fragments have
been separated on the gels, it is possible to
read the base sequence from the order in
which the fragments appear.
The problem with this method is that it
is tedious and expensive, requiring skilled
scientists and the use of hazardous chem-
icals and unstable radiosotopes. If se-
quencing the entire human genome is to
be practical, sequencing methods must be-
come more efficient.
One way to increase efficiency has been
developed by Leroy Hood and his col-
leagues at the California Institute of Tech-
nology and Applied Systems, Inc. Instead
20 SHAPING THE FUTURE
of labeling each DNA fragment with the
same radioactive marker, the fragments are
labeled with four distinct flourescent mark-
ers, one for each kind of base. This makes
it possible to run all of the fragments through
a single electrophoretic gel. As fragments
of different size move down the gel, a laser
causes the passing bands of DNA to flu-
oresce, and the emitted light is recorded
by a computer (see figure). Since each flou-
rescent marker produces a different color,
the computer can read the DNA sequence
from the passing DNA bands.
This technique offers a number of ad-
vantages over the conventional sequencing
method. For one thing, it reduces the er-
rors inherent in using four different gels.
Also, because the sequencing is auto-
mated, the process proceeds much more
quickly. Commercial versions of the device
can sequence about 6,000 bases per day, or
approximately 1 million bases per year.
OCR for page 21
base' less than 0.T percent of the total. Without a special effort to
sequence the human genome' it will not be (lone for many years, if
ever.
There are several broad objections to a sequencing program that must
be answered for the project to proceed' according to Hood. The first
asks whether obtaining the full sequence would in fact be scientifically
uninteresting ant] therefore a misallocation of resources. Hood agrees
that much of the mapping and sequencing would be routine, repetitive
work. Icleally' he says' much of it can be automated (see box' pages
20-211. It is the sequence itself that will generate lithe staggering anc]
exciting kinds of scientific endeavors." Also, Hood questions whether
the United States can afford] not to undertake a sequencing program.
"I would argue that the impetus proviclec! by this program is going to
have a major impact on whether the United States stays competitive in
the inclustrial biotechnology area."
A second objection is that such a project will draw funds from other
areas of biology, and HoocI finds this to be "a much more serious
objection.'' But the technologies and information developed through a
sequencing program will find applications throughout biology, Hood
Gel
DNA Fragments
Electrophorese
Down the Gel
Laser
Beam ~/
Scanning
Laser Excites
Fluorescent Dye
(
Filters
Wheel
Computer By using four different fluorescent markers
Final Output to label the DNA fragments, the fragments
can be run on a single gel and detected by a
laser beam linked to a computer.
GENETICS AND THE HUMAN GENOME 21
OCR for page 22
points out. He also believes that the project will be "sufficiently com-
pelling" to generate new sources of funding for itself. Several committees
have studied sequencing proposals and have conclucled that funding in
the neighborhood of $200 million per year would be appropriate for
such a program. While this is a substantial amount of money' it is only
about 3 percent of the total amount of funds spent by the federal
government on biological research each year.
A third objection is that the project will be "big science" anc] therefore
at ocIds with the traditional approach to biological research, in which
most work is done by small, indepenclent groups of scientists. But Hood
contends that "it is not big science in the same sense that projects such
as the superconducting supercollider and the space shuttle are big
science." The costs of the instruments required are modest, and the
instruments can be widely disseminated. Research proposals for aspects
of mapping and sequencing will be peer reviewed, and this research
will also be widely distributed. Furthermore, once the information is
available, it will greatly increase the power and range of what small
groups can do, and they will no longer need to spend great amounts of
time on routine mapping and sequencing.
The final objection is that the technology is not yet adequate to Lo
the job, and this is the one objection that Hood finds convincing. As
currently performed, sequencing is tedious, time-consuming, labor-
intensive, and expensive, according to Hood. '`Technolo~v development
; ~Al ~11_ ~1~. 1 ~1
~ all ~ ~-~4 ~
1;~ Wlla'L WE Wholly ~llQUlU co~luenlrale on, ne says. "This is an area of
major deficiency at this time."
Sequencing the human genome will involve building up a library of
overlapping pieces of the genome, which can be stored in a central
location, easily reproduced, and sent to investigators to be sequenced
and studied. This is now relatively easy to do with small pieces of
DNA. They can be cut with restriction enzymes, inserted into pieces
of bacterial or viral DNA known as vectors, and maintained in culture
as clones. The problem is one of scale. Conventional vectors can hold
a maximum of about 40,000 bases, meaning that with an average overlap
of 10,000 bases, it would take over 100,000 different clones to store
the entire human genome. Maintaining a culture library of this size
would require an administrative structure much larger than any in
existence today. Furthermore, some parts of the human genome are
difficult to clone, and there are concerns about the stability of DNA in
a culture library.
Progress is being macie on many of these problems. Researchers are
developing new vectors in yeast that may be able to carry 500,000 to
~ million DNA bases, over ten times the size of the fragments that can
22 SHAPING THE FUTURE
OCR for page 23
be maintained with current systems. Instead of needing over 100,000
clones to encompass the entire human genome, it could be done with
several thousand. Restriction enzymes have recently been discovered
that cut DNA into very large pieces, and a new method of electrophoresis
· ~ 1 1 . · r. ~ 1
using Pusey electric Feces can separate DNA fragments 200 times
larger than the maximum possible with conventional electrophoresis.
These are the kinds of technological developments that will be necessary
to make sequencing the human genome economically and scientifically
feasible. (The box on pages 24-25 discusses several other technologies
that will be essential to sequencing efforts.)
Hood advocates that en effort to sequence the human genome proceed
In stages. During the first stage, new technologies would be developed
to increase the efficiency of DNA sequencing five- to ten-fold. At the
same ume, detailed mapping of the human genome could be under
way, which would provide a framework for the sequencing effort. A
phased approach would also allow systems to be developed to collect
and disseminate DNA clones and to store and analyze the huge amounts
of data that will be generated. Once this technological infrastructure
is in place, Hood says, the complete sequencing of the genome could
begin.
.
.
Prospects for Human Gene Therapy
The knowledge provided by mapping and sequencing the human
genome may make it possible to achieve one of the most provocative
of the new biotechnologies: human gene therapy. Interest in human
gene therapy arises from a depressing fact: for the majority of monogenic
diseases, no effective therapies are available. For some single-gene
defects, dietary restrictions, the use of drugs or biologic agents, or
transplantation of tissues or organs may alleviate part or all of the
symptoms. But for many such genetic defects there is no alternative to
"debilitating and progressive disease leading to suffering and early
death," according to Berg.
Any disease to be treated with human gene therapy must meet a
number of "very formidable" criteria, Berg says. The gene responsible
for the disease and the molecular nature of its defect must be known.
The disease must involve cell types that are accessible and well-char-
acterized. The disease cannot begin to exert its harmful effects until
after birth, since a variety of technical and ethical constraints prohibit
gene therapy before birth. Also, the defect must be limited to a single
gene. `'We certainly enter the realm of wishful thinking when the therapy
GENETICS AND THE HUMAN GENOME 23
OCR for page 24
aims to moclify more than one gene or to rectify the defects resulting
from chromosomal abnormalities," notes Berg.
The gene therapy being considered for humans involves placing nor-
mal genes into somatic, or bocly, cells, not into germ line, or sex, cells.
In this way, `'the therapy stays with the treater} patient and is not
transmitted to offspring," Berg says, Also any undesirable effects of the
integration are not going to be propagated to f~,t~re ~en~r~tion~ "
~ · . ~ ·. · 1 . 1 · 1 1
Liven tne criteria tnat canct~ciate creases must meet, only a handful
have received serious consideration. The most attention has focused on
a rare disease caused by defects in the enzymes necessary for normal
immune system development. ChilcIren with the disease, known as
severe combiner! immunodeficiency disease, must live in totally sterile
Protein Sequencers and Synthesizers
Recombinant DNA technology is not the
only driving force behind the rapid ad-
vances now occurring in genetics. Another
powerful influence has been the develop-
ment of microchemical instruments that can
analyze and synthesize genes and proteins
with remarkable proficiency.
One such instrument is known as a pro-
tein sequenator. Essentially, this device is
a chemical scissors that can clip off one
amino acid at a time from the end of a
protein and determine its identity. Since
the development of the first sequenator in
1967, the amount of a protein needed for
sequencing has steadily declined. Today,
biologists are on the verge of being able to
sequence the proteins purified by the most
sensitive technique now available two-
dimensional electrophoresis. This tech-
nique separates complex protein mixtures
in one dimension by size and in the other
dimension by charge (see figure). Within a
few years, it should be possible to sequence
virtually every protein that appears in a
two-dimensional gel. The result will be a
dramatic increase in the number of pro-
teins that can be analyzed.
Once the sequence of amino acids in a
protein is known, a synthetic gene can be
made for the protein by converting the pro-
tein's amino acid sequence into the corre-
sponding base sequence. DNA synthesizers
can now construct DNA fragments up to
200 bases long, and these fragments can be
joined together to make longer sequences.
A synthetic gene or part of a gene can be
hybridized with chromosomal DNA to lo-
cate the original gene for the protein. A
synthetic gene or the cloned original gene
can also be introduced into bacteria or other
hosts to manufacture large quantities of the
protein for research or therapeutic uses.
Biologists are also using microchemical
instrumentation to explore one of the most
fundamental issues in biology: how the se-
quence of amino acids in a protein deter-
mines its structure and hence its function.
Using DNA synthesizers, researchers are
making synthetic genes that are slightly
different from the original genes, resulting
in proteins with different amino acids in
particular locations. By observing how these
modified proteins fold and operate, re-
searchers hope to uncover the general prin-
ciples that govern the structure and function
of proteins. Researchers can also directly
synthesize protein units over 100 amino
acids long to examine which amino acids
24 SHAPING THE FUTURE
OCR for page 25
``bubbles ''7 since their immune systems cannot protect them from com-
mon viruses' bacteria' and fungi.
Researchers have developed an experimental procedure in mice that
could be applier! to humans if it proves effective and safe (Figure T-5~.
First' bone marrow ceils are removed from the animals and mixed with
an infectious agent known as a retrovirus. When retroviruses infect a cell,
they insert a copy of their genetic information into the genome of the host.
By genetically engineering the retroviruses to contain a normal version of
the defective gene' scientists can insert the normal gene into the bone
marrow cells. The ceils are then reimplanted into the mice where' pre-
sumably, they will produce the missing gene product.
Unfortunately' says Berg7 the results to date Shave been rather dis
are important for particular functions. In
time, such information may make it pos-
sible to construct proteins with new and
useful properties, inaugurating an era of
protein engineering that is likely to have
an even greater impact on science and
medicine than genetic engineering has had.
MOLECULAR CHARGE
Two-dimensional electrophoresis can sepa-
rate thousands of proteins extracted from a
few hundred cells. Each dot in this separation
indicates the presence of a single kind of pro-
tein. Photograph courtesy of Leroy Hood.
GENETICS AND THE HUMAN GENOME 25
OCR for page 26
Retrovirus
^;W
Hu man Cel I with Functional Gene
(: DNA
bye Nuclei
·DNA Equivalent of Retroviral ~
RNA with Ma for Genes Deleted DNA Segment Containi ng
functional Gene
Recombinant Retrovirus
-
Bone Marrow Cell with Defective Gene
Bone Marrow Cell Containing Functional Gene
FIGURE 1-5 Much of the research on human gene therapy has focused on re-
troviruses, infectious agents that can insert their own genetic material into the DNA
of cells they infect. The genome of a retrovirus consists of RNA, which is enzymatically
copied into DNA when the virus invades the cell. In the scenario shown above,
involving bone marrow cells with a defective gene, the major genes in a DNA copy
of the retroviral RNA can be deleted and replaced with a functional version of the
defective gene, along with the appropriate regulatory signals to ensure the expression
of the gene. Once the recombinant molecule has been reconverted to RNA, the
bioengineered retroviruses can be used to infect bone marrow cells withdrawn from
an organism with the defective gene. The retroviruses insert the functional gene into
a random location in the cells' DNA, and the transformed cells are reimplanted into
the organism.
26 SHAPING THE FUTURE
OCR for page 27
couraging and may prompt others to begin looking at other potential
vectors." The genes can be inserted into mouse bone marrow cells and
macle to produce the gene product, but only for a limited time. `'For
reasons that are totally mystifying at the moment, the gene that has
been introducec] by the virus is turned off over some varying perioc! of
time. Although the animal has acquired the foreign DNA, it no longer
expresses the gene of interest." Much effort has gone into trying to
maintain the expression of the introduced gene, but without success.
"My feeling is that this probably reflects our lack of knowledge about
the development of the cell," says Berg.
Human gene therapy has other potential problems that are only be-
ginning to be addressed, notes Berg. One is that the retrovirus inserts
its genetic message into the cell's DNA at random and uncontrollable
locations. It is possible that the foreign DNA would integrate into the
micIdIe of a gene essential for the survival of the cell, which wouIc]
destroy it. Or the gene could] insert itself in such a way that it increases
the activity of a gene regulating cellular growth, leading to tumors.
`'The inability to control the site at which the vector DNA integrates
is one of the major handicaps at the moment in this approach,'' Berg
points out. As a result, he and his coworkers are examining ways to
target the introduced DNA to specific locations. Perhaps vectors can
be developed that would recognize sequences within the cell's genome
and integrate its DNA in a predictable way around that sequence. Even
more desirable would be some sort of agent that homes in on the
defective gene ant] somehow repairs it in place. But knowing whether
any of these approaches are feasible will require a much more sophis-
ticatec! understanding of the workings of the human genome.
GENETICS AND THE HUMAN GENOME 27
Representative terms from entire chapter:
amino acids
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