CHROMOSOME PURIFICATION |
49 |
BOX 2.2 (Continued)
|
|
|
|
|
|
|
|
Figure 2.18 |
Entrapment of a circular DNA molecule on agarose fibers. |
||
The |
linear |
DNA molecules that |
make up intact |
human chromosomes are so large |
|
that they |
do not appear able to enter agarose at |
all under any PFG conditions so far |
|||
tried. It |
will |
be shown in Chapter |
5 that these molecules do apparently enter the gel |
||
under SPFG conditions. No size fractionation is seen, but the molecules migrate well.
We |
|
reasoned that under these conditions a circular human chromosomal DNA would |
|
be |
unable to enter agarose; if this were the case |
we would have a simple bulk proce- |
|
dure |
for human chromosome purification. Thus far we |
have experimented to no avail |
|
with |
a cell line containing a chromosome 21 circle. This material seems to co-migrate |
||
in |
the gel with ordinary linear chromosome 21 DNA. The most likely explanation is |
||
that under the conditions we used, the molecule has been fragmented—by physical
forces during the electrophoresis itself, by |
nuclease contamination, or, much less |
|
likely, the molecule (unlike the morphological |
appearance of the chromosome) was |
|
never a |
circle to begin with. We will need to explore a wider range of conditions, and |
|
look at |
other circular human chromosomes. |
|
We can arrange this to solve for n and thus determine the |
number |
of clones |
needed to |
||
achieve a fractional coverage of f. |
|
|
|
|
|
n |
log |
(1 f) |
|
||
log (1 N |
/ C) |
||||
|
|||||
Typical useful libraries will have a redundancy of twoto tenfold.
In practice, however, most libraries are shown to be over-represented in some genome regions, under-represented in others, and totally missing certain DNA segments. Cloning biases can arise from many reasons. Some bacterial strains carry restriction nucleases that specifically degrade some of the methylated DNA sequences found in typical mammalian
cells. Some mammalian sequences if expressed produce proteins toxic to the host cell. Others may produce toxic RNAs. Strong promoters, inadvertently contained in a highcopy number clone, can sequester the host cell’s RNA polymerase, resulting in little or no growth. DNA sequences with various types of repeated sequences can recombine in the
host cell, and in many cases this will lead to loss of the DNA stretch between the repeats. The inevitable result is that almost all libraries are fairly biased.
BOX 2.3
PREPARATION OF SINGLE CHROMOSOME LIBRARIES IN
COSMIDS AND P1
Because flow-sorting produces only small amounts of purified single chromosomes, proce- |
|
|
|
||||||||||||
dures for cloning this material must be particularly efficient. The ideal clones will also have |
|
|
|
||||||||||||
relatively large insert capacities so that the complexity of the library, namely the number of |
|
|
|||||||||||||
clones needed to represent one chromosome equivalent of insert DNA, can be kept within |
|
|
|
||||||||||||
reasonable bounds. The earliest single-chromosome |
libraries were made in bacteriophage |
|
|
|
|||||||||||
or plasmid vectors (see Box 1.3), but these were rapidly supplanted by cosmid vectors. |
|
|
|||||||||||||
Libraries of each human chromosome in cosmids have been made and distributed by a col- |
|
|
|
||||||||||||
laboration between Lawrence Livermore National Laboratory and Los Alamos National |
|
|
|
||||||||||||
Laboratory. These libraries are available today at a nominal cost from the American Type |
|
|
|
||||||||||||
Culture Collection. Gridded filter arrays of |
clones from most of these libraries have also |
|
|
|
|||||||||||
been made by various genome centers. Interrogation of these filters by hybridization with a |
|
|
|
||||||||||||
DNA probe or interrogation of DNA pools with PCR primers will identify clones that con- |
|
|
|
||||||||||||
tain specific DNA sequences. The use of the same arrays by multiple investigators at differ- |
|
|
|
||||||||||||
ent |
sites |
facilitates |
|
coordination |
of |
a |
broad |
spectrum |
of |
genome |
research. |
|
|
||
|
Cosmids are chosen as vectors for single chromosome libraries because they have |
|
|
|
|||||||||||
relatively large inserts. A cosmid clone consists of two ends of bacteriophage lambda |
|
|
|
||||||||||||
DNA (totaling about 10 kb in length) with all of the middle of the natural vector re- |
|
|
|||||||||||||
moved. The ends contain all of the sequence information needed to package DNA into |
|
|
|
||||||||||||
viruses. Hence a cloned insert can replace the central 40 kb of lambda. Recombinant |
|
|
|||||||||||||
molecules are packaged in vitro using extracts from cells engineered to contain the |
|
|
|||||||||||||
proteins needed for this reaction. Lambda DNA packaged into a bacteriophage head is |
|
|
|
||||||||||||
a linear molecule, but the ends have 12 |
base |
complementary |
5 |
|
|
|
|
-extensions. Once the |
|||||||
virus infects an |
E. coli |
cell, the two ends circularize and are ligated together as the first |
|||||||||||||
step in the viral life cycle. The 5 |
|
|
-extensions are called |
COS sites, |
for cohesive ends, |
||||||||||
and the name of this site has been carried over to the vectors that contain them |
|
|
|||||||||||||
(COSmids). Cosmids propagate in |
|
|
|
E. coli |
cells as low-copy plasmids. |
|
|
||||||||
|
Bacteriophage P1 offers another convenient large insert cloning system. P1 pack- |
|
|
||||||||||||
ages its DNA by a headful mechanism that accommodates about 90 kb. Hence, if the |
|
|
|
||||||||||||
target DNA is much larger than 90 kb, it will be cut into adjacent fragments as it is |
|
|
|||||||||||||
packaged. A typical P1 cloning vector is shown below. The vector is equipped with |
|
|
|||||||||||||
bacteriophage SP6 and T7 promoters to allow strand-specific transcription of the in- |
|
|
|||||||||||||
sert. The resulting clones are called P1 artificial chromosomes (PACs). |
|
|
|
|
|||||||||||
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
PAC cloning vector. In the circle are shown cutting sites for some restriction nucleases and also locations of known sequences, PAC2, PAC4 suitable for PCR amplifications of the insert (see Chapter 4).
CHROMOSOME NUMBER |
51 |
CHROMOSOME NUMBER
There are two determinants of the total number of chromosomes in a cell. The first is the number of different chromosomes. The second is the number of copies of a particular chromosome. In general, in a species the number of different autosomes (excluding sex chromosomes) is preserved, and the number of copies of each chromosome is kept in a constant ratio, although the absolute number can vary in different cells. We use the term
ploidy |
to refer to the number of copies of the genome. Yeast |
cells are most typically hap- |
|||
loid with one genome copy, but after mating they can be diploid, with two copies. Human |
|
||||
gametes are haploid; somatic cells vary |
between diploid |
and |
tetraploid depending |
on |
|
what stage in the cell cycle they are in. |
|
|
|
|
|
When |
actively growing eukaryotic cells |
are stained for |
total DNA content and |
ana- |
|
lyzed by FACS, a complex result is seen, as shown in Figure 2.19. The three peaks repre-
sent diploid G1 cells, tetraploid G2 and M cells, and intermediate ploidy for |
cells in S |
phase. Plant cells frequently have much higher ploidy. In specialized tissues much |
higher |
ploidy is occasionally seen in animal cells as, for example, in the very highly polyploid
chromosomes of Drosophila salivary |
glands. When such events occur, it is a great boon |
for the cytogeneticist because it |
makes the chromosomes much easier to manipulate and |
to visualize in detail in the light microscope. |
|
Aneuploidy is an imbalance in the relative numbers of different chromosomes. It is of- |
|
ten deleterious and can lead to |
an altered appearance, or phenotype. Ordinarily gene |
dosage is carefully controlled by the constant ratios of different segments of DNA. An ex- |
|
tra chromosome will disturb this |
balance because its gene products will be elevated. In |
the human the most commonly seen |
aneuploidy is Down’s syndrome: trisomy chromo- |
some 21. The result is substantial physical and mental abnormalities, although the individuals survive. Trisomy 13 is also seen, but this trisomy leads to even more serious deformations and the individuals do not survive long beyond birth. Other trisomies are not seen in live births because fetuses carrying these defects are so seriously damaged that they do not survive to term.
In the human (and presumably in most other diploid species) monosomies, loss of an entire chromosome, are almost always fatal. This is due to the presence of recessive lethal alleles. For example, imagine a chromosome that carries a deletion for a gene that codes for an essential enzyme. As long as the corresponding gene on the homologous chromo-
some is intact, there may be little phenotypic effect of the haploid state of that gene.
Figure 2.19 FACS analysis of the amount of DNA in a population of rapidly growing and dividing cells. Peaks corresponding to particular cell cycle stages are indicated.
52 A GENOME OVERVIEW AT THE LEVEL OF CHROMOSOMES
However, monosomy will reveal the presence of any recessive lethal allele on the remaining chromosome. The result is not compatible with normal development.
Cell ploidy is maintained by mitosis, for somatic cells (see Box 2.4), and it is altered by meiosis (see Box 6.1) during gametogenesis. Errors during these processes or errors made
during DNA damage and repair |
can lead to cells that themselves |
are aneuploid or |
that |
|||||||
produce |
aneuploid |
offspring. |
Cells |
with |
defects |
in |
their |
division |
cycle |
frequently |
accumulate abnormal chromosome complements. This is particularly dramatic in many late- |
|
|||||||||
stage tumors which frequently have large numbers of different chromosome abnormalities. |
|
|||||||||
BOX 2.4
MITOSIS
During cell division, events must be carefully controlled to ensure that each daughter cell receives one copy of each of the two homologous chromosomes, namely receives one copy of the paternal genome and one copy of the maternal genome. An obvious potential source of confusion is the similarity of the two parental genomes. The way in which this confusion is effectively avoided is shown by the schematic
illustration of some of the steps in mitosis in Figure 2.20. Our example considers a cell with only two chromosome types. At the G1 phase the diploid cell contains one
copy of each parental chromosome. These are shown condensed in the figure, for clarity, but remember that they are not condensed except at metaphase. After DNA synthesis the cell is tetraploid; there are now two copies of each parental genome. However, the two copies are paired; they remain fused at the centromere. We call
these structures |
|
sister chromatids. |
Hence there is no chance for the two parental |
|||
genomes to mingle. During mitosis the paired sister chromatids all migrate to the |
||||||
metaphase plate |
of |
the cell. Microtubules |
form between each centromere and the |
|||
two centrioles that will segregate into the two daughter cells. As the microtubules |
||||||
shrink, each pair of sister chromatids is dragged apart so that one copy goes to each |
||||||
daughter cell. |
|
|
|
|
|
|
Errors can occur; one type is called nondisjunction. The sister chromatids fail to |
||||||
separate so that one daughter gets both sister chromatids; the other gets none. Usually |
||||||
such an event will be fatal because of recessive |
lethal alleles present on the sole copy |
|||||
of that chromosome present in the daughter. One additional complication bears men- |
||||||
tion. While sister |
chromatids are paired, a process called sister chromatid |
exchange |
||||
can occur. In this form of mitotic recombination, DNA strands from one sister invade |
||||||
the other; the eventual result is a set of DNA breaks and reunions that exchanges ma- |
||||||
terial between the two chromatids. Thus each final product is actually a mosaic of the |
||||||
two sisters. Since |
these |
should be identical |
anyway (except |
for any errors made in |
||
DNA synthesis) this process has no phenotypic consequences. We know of its exis- |
||||||
tence most compellingly through elegant fluorescence staining experiments conceived |
||||||
by Samuel Latt at Harvard Medical School. He used base analogues to distinguish the |
||||||
pre-existing |
and |
newly |
synthesized sister |
chromatids, and a |
fluorescent |
stain that |
showed different color intensities with the two base analogues. Thus each chromatid exchange point could be seen as a switch in the staining color, as shown schematically in Figure 2.21.
(continued)
BOX 2.4 (Continued)
Figure |
2.20 |
Steps in mi- |
||
tosis, the process of cell |
||||
division. For |
simplicity |
the |
||
chromosomes |
are |
shown |
|
|
condensed |
at |
all |
stages |
of |
the cell cycle. In actuality, |
||||
they are |
condensed only |
|||
during metaphase. |
|
|
||
(continued)
53
54 A GENOME OVERVIEW AT THE LEVEL OF CHROMOSOMES
BOX 2.4 (Continued)
Figure 2.21 Visualization of sister chromatid exchange, by fluorescence quenching. Newly synthesized DNA was labeled with 5-
bromoU which quenches the fluorescence of the acridine dye used to stain the chromosomes at metaphase.
Partial |
aneuploidy can arise in a number of different |
ways. The results are often se- |
vere. One common mechanism is illustrated in Figure 2.22. Reciprocal chromosome |
||
translocations |
are fairly common, and they are discovered by |
genetic screening because |
the offspring |
of such individuals frequently have genetic abnormalities. The example, |
|
shown in the |
figure, is a reciprocal translocation between |
chromosome 5 and chromo- |
some 20. Such translocations can occur by meiotic or mitotic recombination. Unless the |
||
break points interrupt vital genes, the translocation results in a normal phenotype because |
||
all of the genome is present in normal stoichiometry. This illustrates once again that the arrangement of genes on the chromosomes is not usually critical.
Now consider the result of a mating between the individual with a reciprocal transloca-
tion and a normal individual. Fifty percent |
of the |
children will have a normal dosage of |
all of their chromosomes. Half of these will have a |
totally normal genotype because they |
|
will have received both of the normal homologs originally present in the parent with the |
||
reciprocal translocation. Half will have received both abnormal chromosomes from that |
||
parent; hence their genome will still be |
balanced. The remaining 50% of the offspring |
|
will show partial aneuploidy. Half of these will be partially trisomic for chromosome 20, partially monosomic for chromosome 5. The other half will be partially monosomic for chromosome 20, partially trisomic for chromosome 5.
UNUSUAL CHARACTERISTICS OF SEX CHROMOSOMES
AND MITOCHONDRIA
In mammals a female carries two copies of the X chromosome; males have one X and one Y. However, this simple difference in karyotype (the set of chromosomes) has pro-
found effects that go beyond just the establishment of sex. The first thing to consider is why we need sex at all. In species with just one sex, each organism can reproduce clonally. The offspring of that organism may be identical. If the organism inhabits a wide ecological range, different selection processes will produce a geographical pattern of genetic differences, but there is no rapid way to combine these in response to a shifting environment. Sex, on the other hand, demands continual outbreeding, so it leads to much more efficient mixing of the gene pool of a species.
UNUSUAL CHARACTERISTICS OF SEX CHROMOSOMES AND MITOCHONDRIA |
55 |
Figure 2.22 Generation of partial aneuploidy by a reciprocal translocation, followed by segregation of the rearranged chromosomes to gametes and, after fertilization, generation of individuals
that are usually phenotypically abnormal.
We are so used to the notion of two sexes, that it often goes unquestioned why two and only two? Current speculation is that this is the result of the smallest component of mam-
malian genomes, the mitochondrial DNA. Mitochondria |
have |
a circular |
chromosome, |
much like the typical bacterial genome from which |
they |
presumably |
derived. This |
genome codes for key cellular metabolic functions. In the human it is 16,569 kb in size, and the complete DNA sequence is known. A map summarizing this sequence is shown in
Figure 2.23. Mitochondria have many copies of this DNA. What is striking is that all of an individual’s mitochondria are maternally inherited. The sperm does contain a few mitochondria, and these can enter the ovum upon fertilization, but they are somehow destroyed.
Bacterial DNAs carry restriction nucleases that can destroy foreign (or incompatible) DNA
(see Box 1.2). Perhaps, from their bacterial origin, mitochondria also have such properties.
56 A GENOME OVERVIEW AT THE LEVEL OF CHROMOSOMES
Figure 2.23 |
Map of human mitochondrial |
DNA. The tRNAs are indicated by their cognate amino |
|||
acid letter code. The genes encoded by the G-rich |
heavy (H) strand are on the |
outside of the circle, |
|||
while those for the C-rich light (L) strand are on the inside. The H- |
and |
L-strand origins (O |
|||
OL ) and promoters |
(P |
H and P L ) are shown. The common |
5-kb deletion associated with aging is |
||
shown outside the circle. (Adapted from Wallace, 1995.) |
|
|
|||
If they do, this would explain why one sex must contribute all of the mitochondria. It can be used as an argument that there should only be two sexes. In fact, however, cases are known
where organisms have more than two sexes. The slime mold, |
|
Physarum polycephalum, |
13 sexes. However, these turn out to be hierarchical. When two sexes mate, the higher one |
||
on the hierarchy donates its mitochondria to the offspring. |
This ensures that |
only one |
parental set of mitochondria survive. So one important thing about sex is who you get your |
||
mitochondria from. |
|
|
In the human and other mammals, the Y chromosome is |
largely devoid of |
genes. |
The long arm is a dark G band (Fig. 2.13), and the short arm is small. However, an excep- |
||
tion is the gene-rich tip of the short arm, which is called |
the |
pseudoautosomal region. |
H and
has
|
UNUSUAL CHARACTERISTICS OF SEX CHROMOSOMES AND MITOCHONDRIA |
57 |
||||
|
|
|
|
|
|
|
BOX |
2.5 |
|
|
|
|
|
MORE |
ON MITOCHONDRIAL |
DNA |
|
|
||
The pure maternal inheritance of mitochondria makes it very easy to trace lineages in |
|
|||||
human populations, since all of the complexities of diploid genetics are avoided. The |
|
|||||
only |
analogous situation is |
the Y chromosome which must be paternally inherited. |
|
|||
One region of the mitochondrial DNA, near the replication origin, codes for no known |
|
|||||
genes. This region shows a relatively fast rate of evolution. By monitoring the changes |
|
|||||
in the DNA of this region, Allen Wilson and his coworkers have attempted to trace the |
|
|||||
mitochondrion back through human prehistory to explore the origin of human ethnic |
|
|||||
groups and |
their |
geographic |
migrations. While considerable controversy still exists |
|
||
about some of the conclusions, most scientists feel that they can trace all existing hu- |
|
|||||
man groups |
to a single |
female |
progenitor who lived in Africa some 20,000 years ago. |
|
||
|
The mitochondrion has recently been implicated in studies by Norman Arnheim, |
|
||||
Douglas Wallace, and their coworkers as a major potential site of accumulated damage |
|
|||||
that results in human aging. In certain inherited diseases a large deletion occurs in mi- |
|
|||||
tochondrial DNA. This deletion drops out more than 5 kb of the genome between two |
|
|||||
repeated sequence elements (Fig. 2.23). It presumably arises by recombination. Small |
|
|||||
amounts of similar deletions have been detected in aging human tissue, particularly in |
|
|||||
cells like muscle, heart, and brain that undergo little or no cell division. While the full |
|
|||||
significance of these results remains to be evaluated, on the surface these deletions are |
|
|||||
striking phenomena, which provide potential diagnostic tools for what may be a major |
|
|||||
mechanism of aging and a way |
to begin to think rationally about how to combat it. |
|
||||
The |
mitochondrion |
is |
the site of a large amount of oxidative reactions; these |
are |
||
known to be able to damage DNA and stimulate repair and recombination. Hence it is |
|
|||||
not surprising that this organelle should be a major target for DNA aging. |
|
|||||
|
|
|
|
|
|
|
This region is homologous to the tip of the short arm of the X chromosome. A more detailed discussion of this region will be presented in Chapter 6. There are also a few other places on X and Y where homologous genes exist. Beyond this, most of the X contains genes that have no equivalent on the Y. This causes a problem of gene dosage. The sex chromosomes are unbalanced because a female will have two copies of all these X-linked
genes while the male will have only one. |
The |
gene dosage problem is solved by |
the |
process known as X-inactivation. |
|
|
|
Mature somatic cells of female origin |
have |
a densely staining condensed object |
called |
a Barr body. This object is absent in corresponding cells of male origin. Eligibility of female athletes competing in the Olympics used to be dependent on the presence of a Barr
body in their cells. Mary Lyon first demonstrated that the Barr body is a highly condensed X chromosome. Since we know that condensed chromatin is inactive in expression, this suggests that in the female one of the two X chromosomes is inactivated. This process, X- inactivation, occurs by methylation of C. It covers the entire X chromosome except for the pseudoautosomal region and other genes that are homologous on X and Y. The exact mechanism is still not understood in detail, but it seems to be a process that is nucleated
at some X-specific sequences and then diffuses (except where barriers limit its spread). Cells with translocations between the X chromosome and autosomes are known; in these
cases the inactivation can spread onto part of the adjacent autosome fragment.
58 A GENOME OVERVIEW AT THE LEVEL OF CHROMOSOMES
If X-inactivation occurred at the single-cell stage of an embryo; one of the two parental Xs would be lost, and males and females would have similar sex-linked genetic properties. However, X-inactivation occurs later in embryogenesis. When it occurs, the two parental X chromosomes have an equal probability of inactivation. The resulting female embryo then
becomes a mosaic with half the cells containing an active paternal X chromosome, half an active maternal X chromosome. When these two chromosomes carry distinguishable mark-
ers, this mosaicism is revealed in patterns of somatic differences in clones of cells that derive from specific embryonic progenitors. One spectacular example is the tortoise shell cat (Fig. 2.24). This X chromosome of this animal can carry two different color coat alleles. The male is always one color or the other because it has one allele or the other. The female can have both alleles and will inactivate each in a subset of embryonic ectodermal cells. As
Figure 2.24 A tortoise shell Himalayan female cat. A gene responsible for overall development of skin pigment is temperature sensitive. As a result, pigmentation occurs only in regions where the animal is normally cold such as the tips of the ears, nose, and paws. These colored regions are mottled because the cat is a mosaic of different color alleles because of random X-inactivation early in development. (Photograph courtesy of Chandran Sabanayagam.)