1350 N o n re c i p roca l E x ch a n g e
Nonreciprocal Exchange
F W Stahl
Copyright ß 2001 Academic Press
doi: 10.1006/rwgn.2001.0905
When two chromosomes in meiosis interact to pro-
duce recombinants for markers that are more than a
kilobase (or so) apart, the recombinants usually arise in
complementary pairs. In a cross AB  ab, the recom-
binants aB and Ab arise in a single act of exchange.
That reaction, which involves breakage and rejoining
of the chromatids, is conservative (two chromatids in,
two chromatids out) and reciprocal (complementary
recombinants arise in the same, individual act).
With closer markers, the complementary recombin-
ant types often arise in separate acts, with each of the
following outcomes being comparably probable: in a
cross AB  ab, the products of recombination are
typically (AB ‡ aB) or (AB ‡ Ab) or (Ab ‡ ab) or
(aB ‡ ab). The rection is conservative but nonrecipro-
cal, involving the loss of a marker and replacement by
its alternative (gene conversion).
In prokaryotes, homology-dependent recombin-
ation is frequently nonreciprocal and sometimes
nonconservative (two DNA molecules in, one DNA
molecule out).
See also: Gene Conversion; Recombination,
Models of
Nonrepetitive DNA
Copyright ß 2001 Academic Press
doi: 10.1006/rwgn.2001.1929
Nonrepetitive DNA is DNA that demonstrates the
reassociation kinetics expected of unique sequences.
See also: DNA
Nonsense Codon
Copyright ß 2001 Academic Press
doi: 10.1006/rwgn.2001.1930
A nonsense codon is any one of three triplets ± UAA
(ochre), UAG (amber), and UGA ± that do not code
for an amino acid but act as signals for the termination
of protein synthesis.
See also: Amber Codon; Ochre Mutation
Nonsense Mutation
Copyright ß 2001 Academic Press
doi: 10.1006/rwgn.2001.1931
A nonsense mutation is any change in DNA that
causes a nonsense (termination) codon to replace a
codon representing an amino acid.
See also: Mutation; Nonsense Codon
Nonsense Suppressor
Copyright ß 2001 Academic Press
doi: 10.1006/rwgn.2001.1932
A nonsense suppressor is a gene coding for a mutant
tRNA with the ability to respond to one or more of
the nonsense codons.
See also: Nonsense Codon; Transfer RNA (tRNA)
Nontranscribed Spacer
Copyright ß 2001 Academic Press
doi: 10.1006/rwgn.2001.1933
A nontranscribed spacer is the region between trans-
cription units in a tandem gene cluster.
See also: Transcription; Transcribed Spacer
Northern Blotting
J Eberwine and Y Sugimoto
Copyright ß 2001 Academic Press
doi: 10.1006/rwgn.2001.0907
Northern blotting is a widely used procedure for ana-
lyzing the molecular size and abundance of mRNA.
This procedure requires the isolation of RNA from
tissue samples from cultured cells. There are a number
of RNA isolation procedures including those that use
chaotropic reagents (to inhibit endogenous RNAse)
and differential nucleic acid precipitation (to separate
RNA from DNA) that yield total RNA for character-
ization by Northern blot analysis. For Northern blot
analysis the RNA is denatured, loaded on a denaturing
agarose gel and the RNA species separated by electro-
phoresis. After electrophoresis the RNA is transferred

from the gel to a nylon membrane by either diffusion
blotting or by electroblotting. If diffusion blotting is
used to transfer the RNA from the gel to the mem-
brane then usually the transfer buffer is a high molar-
ity salt solution so that the charged nucleic acids will
move with the salt through the gel and onto the mem-
brane (Figure 1).
After transfer, the membrane is either placed in a
UV-crosslinker or vacuum oven at 808C to irreversibly
attach the RNA to the filter. The next step involves
prehybridizing the filter in a blocking solution which
provides reagents that bind to all of the reactive sites
on the membrane that are not already associated with
RNA. After prehybridization the filter is exposed to a
solution containing a suitable probe and hybridization
is begun. The types of probe can vary; either DNA or
antisense RNA can be used. The probe is usually radio-
actively labeled so that a hybridization signal can be
visualized on film or using a phosphoimager system.
Alternatively, probe can be made with a label that
permits antibody detection (e.g., digoxigenin). The
anti-digoxigenin antibody in turn is usually conjugated
Load RNA on denaturing gel
Place paper towels on top
of nylon membrane and allow
SSC to diffuse through the gel
to the paper towels carrying the
RNA to the nylon membrane where it
is deposited
Figure 1
N o r t h er n B l o t t i n g 1351
to an enzyme that will convert a substrate to product
at the site of antibody binding. In this way hybrid-
ization can be visualized by nonradioactive procedures
including chemiluminescence (Figure 2). An example
of a Northern blot is presented in Figure 3. In this
example, cDNA probes specific for prostaglandin
synthase-2 (COX-2, lanes 1 and 2) and for prostaglan-
din receptor EP4 (lanes 3 and 4) were used to screen
RNA from a macrophage-like cell line, RAW264.7, to
assess the abundance and size of the cognate mRNA.
Total RNA (20 mg) isolated from the nonstimulated
cells (lanes 1 and 3) or the LPS-stimulated cells (lanes 2
and 4) were electrophoretically separated on a 1.2%
agarose gel. Hybridization bands for COX-2 mRNA
are seen at a size of 4.1 kb and bands for EP4 mRNA are
at 3.7 kb. Expression of EP4 mRNA, but not COX-2
mRNA, is detected in nonstimulated cells. However,
COX-2 mRNA expression is highly induced in the
LPS-treated cells, while EP4 mRNA expression is con-
stant during stimulation.
As illustrated in Figure 3, total RNA can be used
in Northern blotting. It should be remembered that
Electrophorese the
RNA through the gel
Place nylon membrane on
gel for RNA transfer
SSC
1352 Nuclear Envelope , Transpor t

between 1 and 3% of total RNA is mRNA, which is
usually the class of RNAs being examined. Northern
blotting has a sensitivity of detection of approximately
1±5 ng of a particular species of mRNA. The amount
of mRNA that can be detected on Northern blots is
dictated by the specific activity of the probe and the
amount of RNA loaded on the denaturing gel. If an
mRNA cannot be detected in a total RNA sample the
Labeled probe is
hybridized to
Northern blot
poly (A) ‡ mRNA can be isolated from the total RNA
using oligo-dT as a `hook' to anneal to the poly (A) ‡
mRNA and remove it from the total RNA population.
The poly (A) ‡ mRNA can be concentrated and run
on a denaturing gel rather than total RNA. The
enrichment this offers is illustrated by the following
example: If 30 mg of total RNA is loaded on a denatur-
ing gel then approximately 1 mg of this is poly (A) ‡. If
the poly (A) ‡ is isolated from the total RNA sample,
concentrated, and 30 mg is loaded on the gel, a thirty-
fold increase in sample will be available for hybrid-
ization with the probe, thereby facilitating the
visualization of the previously undetectable mRNA
species.
The term `reverse Northern,' used recently in the
microarray literature, refers to a procedure in which
DNAs are attached to a nylon membrane followed
by hybridization with labeled RNA probes. This
procedure can provide quantitative data regarding
mRNA abundance but will not provide any infor-
mation concerning mRNA size.

See also: Messenger RNA (mRNA)

Probe hybridization
is detected by
appropriate procedure

Figure 2
Nuclear Envelope,
Transport
1 2 3 4 J D Aitchison and M P Rout
Copyright ß 2001 Academic Press
doi: 10.1006/rwgn.2001.0908

The nuclear envelope (NE) represents the boundary

of the interphase nucleus. It regulates the composition
and structure of the nucleus, by providing a selective
barrier to control the exchange of material between
28s the nucleoplasm and cytoplasm, and by being in-
volved in the maintenance of nuclear architecture
and chromatin organization.

18s Structure of the Nuclear Envelope

The NE consists of two continuous, distinct parallel
membranes, the inner and outer nuclear membranes,
enclosing a perinuclear space (Figure 1). The outer
nuclear membrane and perinuclear space are continu-
ous with the endoplasmic reticulum, and share its
functions. The inner nuclear membrane is composi-
tionally distinct from the outer nuclear membrane and
in many cells is lined with a fibrous nuclear lamina on
its nucleoplasmic face. The nuclear lamina is a lattice-
like sheet of variable width, consisting mainly of poly-
Figure 3 A Northern blot. mers of filamentous lamin proteins (which are related