Genomics- The Science and Technology Behind the Human Genome Project. Charles R. Cantor, Cassandra L / genomics1-10 / 6

Figure 6.29

Genetic maps

of

human

chromo-

somes.

(a,b)

Status of sex-averaged maps

of

chro-

mosomes

20

and

21

several

years ago.

(c)

Schematic

summary

of

current

genetic

map

(from

The problem

this raises is that the X and Y are very different in size,

and much of these

two chromosomes appear to share little homology. How then does recombination occur

between them? It turns out that there are at least two regions of the Y chromosome that

share close

enough of the X to allow recombination. These are called

pseudoautosomal

regions,

for reasons that will become apparent momentarily.

The major psuedoautosomal region of the Y chromosome is located at the tip of the

short arm. It is about 2.5 Mb in length and corresponds closely in DNA sequence with the

2.5 Mb short arm terminus of the X chromosome. These two regions are observed to pair

up during meiosis, and recombination presumably must occur in this region (Fig. 6.30). If

we imagine an average of 0.5 crossovers per cell division, this is a very high recombina-

tion rate indeed compared to a typical autosomal region.

Since the Y chromosome confers a male phenotype, somewhere on this chromosome

must lie a gene or genes responsible for male sex determination. We know that this region

lies below the pseudoautomal boundary, a place about 2.5 Mb from the short term telo-

mere. Below

this boundary, genes appear to be sex linked because, by definition, these

genes must not be able to separate away from the sex determination region in meiosis. A

genetic map of the pseudoautomal region of the Y chromosome is shown in Figure 6.31.

There is a

gradient of recombination probability across the region. Near the p

telomere,

GENETICS IN THE PSEUDOAUTOSOMAL REGION

199

have a 50% probability of being inherited with each sex. As one nears the pseudoautoso-

mal boundary, the recombination frequency of genes with the sex determination region

approaches

a more normal value, and these genes appear

to be almost completely sex

linked.

Recently data have been obtained that indicate that a second significant pseudoautoso-

mal region

may

lie at

the extreme ends of the

long arms

of the X and Y chromosomes.

About 400

kb

of DNA

in these regions appears

to consist

of homologous sequences, and

BOX

6.4

MAPPING FUNCTIONS: ACCOUNTING

FOR

MULTIPLE RECOMBINATIONS

If

two markers are not close, there is

a

significant

chance

that multiple

DNA

crossovers may occur between them in a particular meiosis. What one observes experi-

mentally is the net probability of recombination. A more accurate measure of genetic

distance will be the average number of crossovers that has occurred between the two

markers. We need to correct for the occurrence of multiple crossovers in order to com-

pute the expected number of crossovers from the observed recombination frequency.

This is done by using a mapping function.

The various possible recombination events for zero, one, and two crossovers are il-

lustrated schematically in Figure 6.32. In each case we are interested in correlating the

observed number of recombinations between two distant markers and the actual aver-

age

number of crossovers among the DNA strands. In all the examples discussed be-

low it is important to realize that any crossovers that occur between sister chromatids

(identical copies of the parental homologs) have no effect on the final numerical re-

sults. The simplest case occurs where there are no crossovers between the markers;

clearly in this case there is no recombination between the markers. Next, consider the

case

where there is a single crossover between

two

different

homologs

(Fig. 6.32

b ).

The

net result is a 0.5 probability of recombination because half

of the sister chro-

matids will have been involved in the crossover and half will not have been.

When two crossovers occur between the markers, the results are much more com-

plex. Three different cases are illustrated in Figure 6.32

c . Two single crossovers can oc-

BOX 6.4

(Continued)

Figure 6.32

Possible crossovers between pairs

of genetic markers and the resulting meiotic re-

combination frequencies that would be observed between two

markers (vertical arrows) flanking

the region.

(a)

No crossovers.

(b) One crossover.

(c) Various ways in which two crossovers can

occur.

DETECTION OF HOMOZYGOUS REGIONS

203

number of alleles in the actual human population, and thus they are very powerful genetic

markers. See, for example, Box 6.5. Most individuals are heterozygous for these alleles,

and most parents have different alleles. Thus the particular homologous chromosomes in-

herited by an offspring can usually be determined unambiguously.

BOX 6.5

A HIGHLY INFORMATIVE

POLYMORPHIC MARKER

A particularly elegant example of the power of VNTRs is a probe described by Alec

Jeffries. This single-copy probe which detects a nearby minisatellite was originally

called MS32 by Jeffries (MS means minisatellite). When its location was mapped on

the genome, it was found to lie on chromosome 1 and was assigned by official desig-

nation D1S8. Here D

refers to the fact that

the marker is a DNA sequence, 1

means

that it is on chromosome 1, S means that it

is a single copy DNA sequence,

and 8

means that this was the eighth such probe assigned to chromosome 1. The power of

D1S8 is illustrated in Figure 6.35. The minisatellite contains a

Hin f I

cleavage site

within each 29 base

repeat. In addition some repeats contain an internal

Hae

II cleav-

determinations.

(a) Repeating DNA structure of the marker.

(b) PCR production of probes to an-

alyze the marker.

(c) Typical results observed when the marker is analyzed after separate, partial

Hae

II and Hin

f I digestions.

204

GENETIC ANALYSIS

BOX

6.5

(Continued)

age site. When appropriately chosen PCR primers are used, one can amplify the region

containing the repeat and radiolabel just one side of

it. Then

a partial digestion

with

the

restriction

enzyme

Hae

II or Hin

f I generates a

series of DNA bands whose sizes

reflect all of the enzyme-cutting sites within the repeat. This reveals not only the num-

ber of repeats but also the locations of the specific

Hae

II sites. When this information

is combined, it turns out that there are more than 10

70

possible alleles of this sequence.

Almost every member of the human population (exempting identical twins) would be

expected to have a different genotype here; thus this probe is an ideal one not only for

genetic analysis but also for personal identification.

By comparing the alleles of D1S8 in males and in sperm samples, the mutation rage

of this VNTR has been estimated. It is extremely high, about 10

3 per meiosis or 10

5

higher than the average rate expected within the human genome. The mechanism be-

lieved to be responsible for this very high mutation rate can be inferred from a detailed

analysis of the mutant alleles. It turns out that almost all of the mutations arise from

interallelic events, as shown in

Figure

6.36. These

include

possible

slippage

of

the

DNA during DNA synthesis, and unequal sister chromatid exchange. Only 6% of the

observed mutations arise from ordinary meiotic recombination events between homol-

ogous chromosomes.

Figure 6.36

Recombination events that generate diversity in the marker D1S8.

(a) Intra-allelic

recombination or polymerase slippage, a very common event.

(b) Inter-allelic recombination, a

relatively rare event.