RESEARCHHIGHLIGHTSHIGHLIGHT ADVISORSSEAN B. CARROLL
UNIVERSITY OF WISCONSIN,USA
NANCY J. COX
UNIVERSITY OF CHICAGO, USA
RALPH J. GREENSPAN
THE NEUROSCIENCESINSTITUTE, CALIFORNIA, USA
YOSHIHIDE HAYASHIZAKI
RIKEN GENOMIC SCIENCES
CENTER, JAPANPETER KOOPMAN
UNIVERSITY OF QUEENSLAND,AUSTRALIALEONID KRUGLYAKFRED HUTCHINSON CANCERRESEARCH CENTER, USA
BARBARA MEYER
UNIVERSITY OF CALIFORNIA,
BERKELEY, USA
JOHN QUAKENBUSH
THE INSTITUTE FOR GENOMICRESEARCH, USA
NADIA ROSENTHAL
EMBL MONTEROTONDO, ITALYNORIYUKI SATOH
KYOTO UNIVERSITY, JAPAN
MARC VIDALDANA-FARBER CANCERINSTITUTE, BOSTON, USA
VIRGINIA WALBOTSTANFORD UNIVERSITY, USA

GENE EXPRESSION

A new take on expression
Variation in gene expression can bestudied just like any other quantitative genetic trait. This is the conclusion published in Nature by Morley,
Molony et al., who used a combination of microarray analysis andgenome-wide linkage analysis.
Natural variation in gene expression is widespread. Realizing thatherein lies the key to the inter-individual phenotypic variation, including
susceptibility to complex diseases, theauthors set out to see how amenable
this variation would be to genetic
study. First, they used microarrays toidentify the variable loci. To do this,they measured differences in theexpression of ~8,500 human genes
from immortalized B cells, andselected the 3,554 most variable genes
for subsequent mapping. Second,
they used a computer program called
SAGE to carry out genome-wide
linkage analysis in 14 families fromthe CEPH (Centre d’Étude duPolymorphisme Humain) panel,
which was also the source of theexpression data. Using two stringency
criteria (genome-wide p = 0.001 and0.05), they detected linkage for 142
and 984 expression phenotypes,
respectively.
The regions that are linked to theexpression phenotypes — the regulators — can be in cis (according to theauthors’ criteria, these have to lie
within 5 Mb of the target gene) or intrans. Although most target genes areassociated with regulators in trans,
few have multiple regulators. The
authors point out that rather thanreflecting a biological phenomenon,
this result might simply be due to thedifficulty in detecting multiple regulators, as each of them would have
only a weak effect on its target.
The authors found what they term
‘master regulators of transcription’ —transcriptional regulators that influence many expression phenotypes.

DETLEF WEIGELMAX PLANCK INSTITUTE FOR
DEVELOPMENTAL BIOLOGY,GERMANY
PHIL ZAMORE
UNIVERSITY OF
MASSACHUSETTS, USA
LEONARD I. ZON
CHILDREN’S HOSPITAL,BOSTON, USA

Two hot spots of such regulators were
found: one on chromosome 14 andone on chromosome 20. Furthermore, some targets of the master regulators showed coordinated levels of
expression.
For the handful of genes, theexpression levels of which are regulated by determinants in cis, theauthors used the results of linear
regression to estimate the proportionof the variation that can be attributed
to these cis factors. It turns out that forsome genes, they account for morethan half the variation in expression.
The authors point out that the remainder of the variation is probably caused
by a variety of factors, including theenvironment.
So, the combination of expression profiling and genome-wide
mapping has allowed the authors to
identify regulatory elements without
any previous knowledge of the regulatory mechanism. It also uncovered
a complex network of multiple regulators, including master elements.
Morley and Molony et al. haveshown us that it is possible to movebeyond the experimental manipulation of gene expression, towards
revealing the genetic interactions
that underlie natural variation in
expression.
Magdalena Skipper
References and links
ORIGINAL RESEARCH PAPER Morley, M. &Molony, C. M. et al. Genetic analysis of genomewide variation in human gene expression. Nature
21 July 2004 (doi:10.1038/nature02797)
WEB SITE

Vivian Cheung’s laboratory:
http://genomics.med.upenn.edu/vcheung

NATURE REVIEWS | GENETICSVOLUME 5 | SEPTEMBER 2004 | 6 3 7©2004 Nature Publishing GroupRESEARCH HIGHLIGHTSWEB WATCH
Integr8 genome andproteome browser• http://www.ebi.ac.uk/integr8
With the explosion in
genomic and proteomic
data stored in a variety of
databases, there is anincreasing need for thisinformation to be brought
together in a way that iseasy to access and intuitive
to use. In response to thisneed, the European
Bioinformatics Institutehas recently launched theIntegr8 web site, providing
a one-stop shop for
researchers exploring geneand protein function.
Integr8 currently houses
data on more than 180
species of archaeans,bacteria and eukaryotes,
providing information on
taxonomy, genome structure
and proteomics. For eachspecies, proteins areclassified according tosequence similarity, structureand function, giving the useran overview of proteome
content. There is also theoption of comparing thisinformation across several
species. Alternatively, if you
have a particular gene orprotein in mind, a simple
search pulls out a wealth of
information, including links toentries in gene and protein
databases, structural data,
key references from thepublished literature and asummary of expression
patterns and function.
The site also provides
several other useful
resources. For example, theBioMart tool allows complex
searches of several
databases using a range of
criteria, such as species
name, chromosomal region,and protein structure andfunction. There is also accessto FASTA similarity andhomology searching,enabling comparisons withsequences from proteome
and genome databases.
So, Integr8 truly lives up to
its name, and should allow
researchers from diverse
fields to make the most of theincreasing availability ofgenomic and proteomic
information.

Louisa Flintoft

G E NO M E EVOLUT ION

Give and takeWith some parasites it’s just “take…
take…take” and this can include thehost’s genes! However, other unwelcome guests actually make generous
DNA donations to their hosts.
Independent studies of Trypanosoma cruzi — the protozoan thatcauses Chagas disease — and theRafflesiaceae — a family of plants that
rely entirely on their hosts for nutrition — show that there have beenhorizontal gene transfers between
these parasites and their respective
vertebrate and plant hosts.
Following up on the hypothesisthat frequent integration of T. cruzi
DNA into the host genome mightunderlie Chagas disease, Nadjar Nitz
and colleagues extracted genomic
DNA from 13 patients with Chagas
disease. A probe derived from components of the T. cruzi mitochondrial
DNA (kinetoplast minicircles; kDNA)
was then Southern hybridized to these
extracts. This hybridization showedthat there were smaller fragments of
kDNA than would be expected if itwere present in its native form, whichindicated that kDNA had integrated
into the host genome.
Using 5′-RACE (rapid amplification of cDNA ends), the authors isolated the genomic integration sites ineach patient that they studied, identifying integration sites in a total of 5 loci.
But could such integration events beobserved in an experimental system?
To address this question, theauthors examined rabbits that had
been experimentally infected with
T. cruzi for up to 3 years. These rabbits had de novo kDNA integrations, which indicated that horizontaltransfer of parasite DNA to the host
could be a normal part of the infection process. The rabbit and humandata also indicated that β-globin lociand long interspersed nuclear elements (LINE-1) are frequent targets
for kDNA integrations.

R NA WORLDRNA stories on a mythical scale
RNA interference (RNAi) hardly
disappears off the science headlines.
Most recently, the spotlight has beenon Argonaute proteins — some ofwhich are involved in the smallinterfering RNA (siRNA) andmicroRNA (miRNA) pathways.
Okamura and colleagues reveal aninteresting division of labour between
the Argonautes in Drosophilamelanogaster: Argonaute1 (AGO1) isrequired for miRNA maturation andmiRNA-directed RNA cleavage,
whereas Argonaute2 (AGO2) acts inthe siRNA pathway.
siRNA… miRNA… the difference
is more than semantic. siRNAs are
the key agents for RNAi and mediate
RNA destruction in a sequencespecific manner. Although miRNAs

can direct RNA cleavage, they canalso block translation of theirtargets. Both species of small RNAs
carry out their functions as part of
the RNA-silencing complex(RISC), a multi-protein complex
that mediates RNA cleavage. Given
that both types of small RNAs
associate with the same RISC, do
the two pathways converge at thislevel, or do they differ?
Prompted by previous studies in
the worm, Okamura and colleagues
decided to resolve the issue bylooking at Ago1 and Ago2 mutantsin Drosophila. They mobilized
P-elements to delete Ago2 andfound that it is required for RNAiin vivo and for RISC assembly; inparticular, AGO2 is required for the

6 3 8 | SEPTEMBER 2004 | VOLUME 5Interestingly, the authors went on
to show that kDNA was also integrated in the genomes of the offspring of chronically infected rabbits
and in chickens hatched from T. cruziinnoculated eggs. Importantly, theyalso found kDNA integrations in
the germline of F2 chickens without
persistent infections, conclusively
showing vertical transfer to infectionfree progeny and so quashing any
chance that their results could be
artefactual.
unwinding of the siRNA, which is aprerequisite for siRNA-mediatedcleavage. Although the RISC is
associated with both siRNAs andmiRNAs, Ago2 is required
exclusively for the siRNA pathway.
AGO1, on the other hand, seems
to be involved only in the miRNA
pathway. Ago1 mutant flies die as
larvae with many developmental
defects, and miRNA cleavage does
not occur in lysates from Ago1
mutant embryos. In vivo analysis
indicates that AGO1 directlyinteracts with Dicer-1, which is
required for miRNA production
from larger precursors, and that therole of Ago1 is to stabilize mature
miRNAs.
In mammals, siRNA andmiRNA pathways seem toconverge downstream of Dicer.
This is not the case in otherorganisms, such as worms andflies. Okamura and colleagues
have elegantly shown that it is
Ago1 and 2 (as part of RISC) at

www.nature.com/reviews/genetics©2004 Nature Publishing GroupRESEARCH HIGHLIGHTS

that a section of the host mitochondrial genome was transferredto the parasite.
The huge significance of horizontal gene transfer for the evolution ofprokaryotes has been known for along time, as has the large contribution that intracellular endosymbiont ancestors of mitochondria andchloroplasts have made to eukaryotic
genomes (see further reading). Studies
such as these show that ongoing horizontal gene transfer from a range ofparasites and endosymbionts might
be more important for eukaryotic
evolution than we previously realized — just how important remains
to be seen.
Nick Campbell
References and links

Charles Davis and Kenneth
Wurdack took an entirely different,
but no less convincing, approach totheir analysis of horizontal genetransfer between plants. Their phylogenetic analyses showed that,
although Rafflesiaceae are close relatives of members of the order
Malpighiales, the rafflesian mitochondrial nad1B-C gene is moreclosely related to the homologue in
its obligate host, Tetrastigma (Vitaceae). Clearly, this is strong evidence

ORIGINAL RESEARCH PAPERS Nitz, N. et al.

Heritable integration of kDNA minicircle sequencesfrom Trypanosoma cruzi into the avian genome:
insights into human Chagas disease. Cell 118,
175–186 (2004) | Davis, C. C. & Wurdack, K. J.
Host-to-parasite gene transfer in flowering plants:
phylogenetic evidence from Malpigiales. Science
15 July 2004 (doi:10.1126/science.1100671)
FURTHER READING Brown, J. R. Ancienthorizontal gene transfer. Nature Rev. Genet. 4,
121–132 (2003) | Timmis, J. N. et al.
Endosymbiotic gene transfer: organelle genomes
forge eukaryotic chromosomes. Nature Rev.
Genet. 5, 123–135 (2004)
WEB SITE

Multidisciplinar de Pesquisa em Doença de
Chagas: http://www.unb.br/ib/cel/pg/ptbr/laboratorios/chagas.php

IN BRIEF
PLANT GENETICS

Plant Reproduction
Plant Cell 16 Supplement (2004)

This supplement to the June issue of Plant Cell is entirely devoted toplant reproduction, and comprises 19 review articles on flowering, fertilization and on the development of the gametophyte, seed and fruit.
The previous supplement was published in 1993, so this is a valuable
update on our mechanistic understanding of plant reproduction andits application to agriculture. It will be a great resource for those whoteach developmental biology or genetics, for researchers in the plant
field and those generally interested in sexual reproduction.
GENE EXPRESSION

Role of transposable elements in heterochromatin andepigenetic control.
Lippman, Z. & Gendrel A.-V. et al. Nature 430, 471–476 (2004)

Making use of microarrays, these authors show that, as predicted fromresearch on other organisms, Arabidopsis heterochromatin is composed of transposable elements (TEs) and other related tandem
repeats. Their expression is regulated by a chromatin-remodelling protein DDM1 that might be guided by small interfering (si) RNAs thatare complementary to the heterochromatic sequences. In addition,
TEs can epigenetically regulate individual genes if they lie close to thelocus in question, indicating that euchromatic loci such as FWA might
be imprinted by a siRNA-guided DDM1-dependent mechanism.
PHARMACOGE N ETICSthe heart of the difference
between siRNA- and a miRNAmediated RNA cleavage.
Undoubtedly, the small RNA
world has many more equally
elegant stories to tell us.
Magdalena Skipper

References and links
ORIGINAL RESEARCH PAPER Okamura, K.

et al. Distinct roles for Argonaute proteins insmall RNA-directed RNA cleavage pathways.
Genes Dev. 18, 1655–1666 (2004)
FURTHER READING Liu, J. & Carmell, M. A. et al.
Argonaute2 is the catalytic engine of
mammalian RNAi. Science 29 July 2004
(doi:10.1126/science.1102514)

Breed distribution and history of canine mdr1-1∆, apharmacogenetic mutation that marks the emergenceof breeds from the collie lineage.
Neff, M. W. et al. Proc. Natl Acad. Sci. USA 101, 11725–11730 (2004)

When treated with the anti-parasitic drug ivermectin some dogs,
including collies, can die from neurotoxicosis. To investigate the originof the 4-bp-deletion in the drug transporter gene mdr1 that underlies
this adverse response, Mark Neff and colleagues surveyed dog populations. The causal allele was isolated in seven new dog breeds and foundto have originated prior to dog breeds being isolated.
GENETIC DISEASE

Pms2 is a genetic enhancer of trinucleotide CAG·CTG
repeat somatic mosaicism: implications for themechanism of triplet repeat expansion.
Gomes-Pereira, M. et al. Hum. Mol. Genet. 13, 1815–1825 (2004)

The expansion of trinucleotide repeats causes several progressive
genetic disorders, but the genetic mechanism underlying repeat
instability is unclear. These authors have addressed the issue by
studying a transgenic CAG·CTG repeat in somatic tissue in themouse. The rate of expansion is reduced by 50% in animals that are
mutant for the DNA mismatch repair gene Pms2, implicating inappropriate mismatch repair in repeat instability.

NATURE REVIEWS | GENETICSVOLUME 5 | SEPTEMBER 2004 | 6 3 9©2004 Nature Publishing GroupRESEARCH HIGHLIGHTST E C H N O LO G Y

No-bake recipe for DNA
Modern genetics relies so heavily on PCR
that it is hard to imagine laboratory life
without it. However, although PCR was
indisputably one of the biggest
breakthroughs in genetics — transforming
fields from forensic science to diseasediagnosis — the thermal cycling at the heartof this technique makes it hungry for energy,
time and special equipment. By using abacterial helicase instead of heat to denature
the template, Huimin Kong and colleagues
have now overcome the limitations of PCR bydevising a means to amplify DNA at a single
temperature. This opens the door to thedevelopment of quick, hand-held diagnostic
devices that could move DNA analysis fromthe bench into the field.
The new amplification technique, called
helicase-dependent amplification (HDA),
mimics a process that occurs in nature. A
helicase — in this case the Escherichia coli
UvrD helicase — is first used to separate thedsDNA template molecules. The separated
strands are then coated with ssDNA binding
proteins to keep them apart, allowing primers

to anneal and be extended by a polymerase.
The reaction occurs at 37°C, from start tofinish, and can achieve over a million-foldamplification of genomic DNA. It is also
sensitive enough to pick up DNA from intact
cells and to detect pathogen DNA in blood.
HDA is not the first isothermal reaction to
be proposed, but the alternatives involve
more complex reactions and rely on an
initial denaturing step that requires heat.
Essential tweaks are now being carried out
to improve the efficiency of HDA. By
experimenting with the reaction
components — nature has hundreds ofhelicases to choose from — and theirconcentrations, Kong and colleagues hope tobring reaction times down to 15 minutes.
Machine-free DNA amplification ‘whileyou-wait’ might be just around the corner.
Tanita Casci
References and links
ORIGINAL RESEARCH PAPER Vincent, M., Xu, Y. & Kong,

H. Helicase-dependent isothermal DNA amplification. EMBO
Reports 9 July 2004 (doi:10.1038/sj.embor.7400200)
WEB SITE

New England Biolabs: http://www.neb.com

G E NO M E EVOLUT ION

Mutations: more common than you thought

Mutation rate, whether per locus or genomewide, features as a variable in many calculations
that underlie evolutionary genetic hypotheses.
But a true experimental estimate of mutation
rate is difficult to obtain. To this end, several
groups have used ‘mutation-accumulation’lines in both Drosophila melangaster andCaenorhabditis elegans: in an attempt to minimize selection pressure, genetically identical flies

or worms are maintained for many generations
in a benign environment. Such assays are longand tedious: for one collection, scientists evenseparated each worm in each generation fromeveryone else to avoid any selection pressure
induced by the stressful dating scene, andinstead, self-fertilize.
Previous studies have used fitness-based
assays to assess the number of mutations thatdeveloped in each line, yielding an indirect estimate of mutation rate. For a more direct estimate¸
Denver and colleagues have now revisited a collection of these special worm lines and sequenced more than 4 Mb of loci scatteredaround the genome. In a recent paper in Nature,they report the total haploid genomic mutation
rate to be approximately 2.1 mutations per
genome per generation — an estimate that is anorder of magnitude higher than previous best
guesses, and 2 orders higher than the indirect
estimate from the same collection (although
the previous estimates referred to deleterious
mutation rate, whereas these authors estimate

6 4 0 | SEPTEMBER 2004 | VOLUME 5

total mutation rate). Not only that, but the more
frequently observed mutations were insertions,in contrast to reports based on pseudogenesthat indicated that most naturally occurring
mutations in worms are deletions.
So, with one study, Denver and colleagues
prompt the entire field to rethink the process ofmutation over time and our measurement of it.
It does not take long for this to generate controversy: in a thought-provoking News and Views
piece, Rosenberg and Hastings speculate on themechanisms at work in the Denver study. Either
previous estimates were wrong because they
only detected mutations that produce phenotypes, as Denver et al. would suggest, or the new
study uses methods that push the worms todevelop more mutations even in the absence ofdeleterious selection, maybe even through triggering stress-response pathways. Future studies
might have to bring the worms out of their poshretirement to settle the question.
Chris Gunter, Senior Editor, Nature
References and links
ORIGINAL RESEARCH PAPER Denver, D. R. et al.

High mutation rate and predominance of insertions in the
Caenorhabditis elegans nuclear genome. Nature 430,
679–682 (2004)
FURTHER READING Rosenberg, S. M. & Hastings, P. J.
Worming into genetic instability. Nature 430, 625–626 (2004)
WEB SITE

NemATOL: http://nematol.unh.edu/index.php

www.nature.com/reviews/genetics©2004 Nature Publishing GroupRESEARCH HIGHLIGHTSIN THE NEWSHUMAN GENETICS

Variations on a theme
The human genome is littered withsmall-scale genetic variants, such as
SNPs and repeat-length polymorphisms, but little is known about theway in which variants involving larger
regions contribute to genetic diversity. Two studies now reveal thatlarge deletions and duplications are
more common than was previously
thought, prompting a re-evaluationof the way we view human genetic
variation.
Large-scale copy-number polymorphisms/variants (CNPs/LCVs)— deletions or duplications of chromosomal segments — have been
identified previously from healthy
individuals, but technical limitations
have prevented an assessment of
whether these variants are common
on a genome-wide scale. In a collaborative study, Michael Wigler and colleagues developed a method called
ROMA (representational oligonucleotide microarray analysis) thatenables deletions or duplications tobe identified. This involves digesting
genomic DNA, amplifying the fragments, attaching a fluorescent label
and hybridizing them to an array ofcomplementary probes. The signal
strength of each probe indicates the
copy-number of the corresponding

genomic region, which can be compared between samples. Using an
average of 1 probe every 35 kb, Wigler
and colleagues analysed samples from
20 unrelated, healthy individuals froma range of geographical locations.
They identified a set of 76 different
CNPs, involving regions of 100 kb ormore, that varied between individuals, with an average of 11 CNP differences between each pair of subjects.
The polymorphisms included bothdeletions and duplications — most ofwhich have not been identified before— with a mean length of 465 kb.
Most regions of the genome had
CNPs, although they were noticeably
more frequent in some regions, suggesting that there might be CNP‘hotspots’.
Importantly, many of these CNPs
are in regions that contain genes, so
this type of variation might influencelevels of gene expression and lead tophenotypic differences between individuals. For example, one CNP-variant
contained three copies of the gene
PPYR1, which encodes the appetiteregulating neuropeptide Y4-receptor.
CNPs are also present in regions that
include genes implicated in nervoussystem development, leukaemia anddrug resistance. So, it is possible thatlarge-scale CNPs might underlie variation in a diverse range of phenotypes, from body weight to cancer
susceptibility.
In a second study, Charles Lee andcolleagues used a similar technique toidentify LCVs in samples from 39
healthy individuals. They identified
255 polymorphisms in the humangenome: an average of 12 CNPs foreach subject.
The authors of both papers pointout that their studies are not comprehensive, as the probes that they used
represent only a fraction of the
genome. Studies using larger sets ofprobes are planned for the future,
which should reveal the full extent towhich large-scale polymorphisms
contribute to the genetic differencesthat underlie human individuality.
Louisa Flintoft
References and links
ORIGINAL RESEARCH PAPERS

Sebat, J. et al. Large-scale copy-number
polymorphism in the human genome. Science
305, 525–528 (2004) | Iafrate, A. J. et al.
Detection of large-scale variation in the humangenome. Nature Genet. 1 August 2004
(doi:10.1038/ng1416)
WEB SITES

Michael Wigler’s laboratory:
http://www.cshl.org/public/SCIENCE/wigler.html
Charles Lee’s laboratory:
http://labmed.bwh.harvard.edu/pathology/Faculty/Charles_Lee.htm

NATURE REVIEWS | GENETICS

Autism Genome Project
The National Alliance for
Autism Research hasannounced the launch of
the largest autism study
ever conducted.
The study, which is acollaboration between
some 170 leading
geneticists worldwide,
will use Affymetrix DNA
microarrays to scan thewhole human genome
in search of genes thatare associated with
inherited risk to autism.
Approximately 50
academic and research
institutes will participate
in this public/private
research partnership,
which will cost in excessof US $2 million.
In the first phase,approximately 6,000
samples of DNA from1,500 multiplex families(2 children with autism
spectrum disorders andtheir parents) from all over
the world will be analysed.
The initial results are
expected by early 2005.
An ancestry test for dessert?
Shish, a London-based
restaurant that specializes
in Central Asian cuisine, isoffering DNA tests to its
diners to find out if they
are descended from
Genghis Khan.
“The unusual promotion
is to mark the Mongolian
government’s decision toallow citizens to havesurnames for the first timesince they were banned by
the communists in the
1920s” (The Daily Times,
Pakistan).
Although the offer wasavailable for only a week,
the test is offered by
Oxford Ancestors, for£195, as part of their Y-clan
analysis. As their web siteclaims — “there are now
around 16 million men
who have inherited theirY-chromosomes directlyfrom Genghis Khan.”“The results will taketwo months anddescendants will win afree meal for two” (BBCNews) courtesy of Shish!

Magdalena Skipper

VOLUME 5 | SEPTEMBER 2004 | 6 4 1©2004 Nature Publishing GroupRESEARCH HIGHLIGHTSWEB WATCH
The NHGRI Policy andLegislation Database• http://www.genome.gov/
LegislativeDatabase
A new web-based
searchable database for
US genetics-related
policy and legislative
documents promises tobe a valuable resource foranyone interested in thisever-expanding andimportant area.
Francis Collins, directorof the US National Human
Genome Research Institute(NHGRI), said this tool will be
useful for everybody “…fromacademic researchers
seeking to patent genetic
technologies to average
citizens trying to determine
what protections exist in theirstates against genetic
discrimination.”
Database users can findlegislation and laws from
specific states through aninteractive US map, as wellas doing more specificsearches for particular
combinations of content
type (for example, federal
legislative materials), topic(such as genetic testing andcounselling) and source (forexample, the Department of
Health and Human Services).
Keyword searching is also
possible, but only for words
in document titles, not thosein their content. Perhaps themost useful ‘value-added’feature that the databaseincorporates is a summary,in layman’s terms, of eachdocument: this enables quick
identification of relevant
documents without the needto trawl through pages oflegal jargon.
Of course, one of thebiggest limitations of thisextremely useful tool is that it
only encompasses US policy
and legislation. However, thisfocus is understandable
given that the US National
Institutes of Health fund thisresource. Moreover, theaddition of further content
categories this autumn, suchas foreign statutes and laws
and policy material frominternational organizations,will, at least partially, address
this limitation.

EPIGENETICS

Family feud
Although relatively few in number, imprinted loci havebeen of intense interest. The Gnas locus on distalmouse chromosome 2 is one of the more complex
imprinted regions yet discovered, having an antisensetranscript and alternatively spliced isoforms that show
biallelic as well as maternal- and paternal-specificexpression. The parent-of-origin-specific expression ofthese and other imprinted genes has yielded insights
into the mechanisms of gene regulation, whereas thefunctional role of imprinting has been one of biology’s
most intriguing mysteries. Two new studies on the Gnas
cluster published in Nature Genetics advance ourunderstanding on both fronts.
The Gnas locus encodes alternative transcripts thatarise from 4 different promoters, with different firstexons spliced to a common exon 2. Among these, Gnas
encodes Gsα, the α-subunit of the ubiquitousheterotrimeric G protein Gs, which couples receptors toadenylyl cyclase. Although Gnas is biallelically
expressed in most tissues, it is preferentially maternallyexpressed in the proximal tubules of the kidney and inbrown and white adipose tissue. Williamson et al. have
now identified a cis-acting element that regulates thistissue-specific aspect of Gnas imprinting — the firstsuch control region to be identified.
Deletion of a 2.3-Kb differentially methylated region(DMR) that encompasses exon 1A resulted in increased
expression of the paternal allele in the kidney and in
adipose tissue. The loss of tissue-specific silencing was
confirmed by a clever functional assay, in which micewith parathyroid hormone (PTH) resistance were
found to have increased Gsα-mediated PTH signalling,
thanks to increased GNAS production from thepaternal allele. The mechanism by which the exon 1A
DMR contributes to tissue-specific silencing of thepaternal allele remains to be determined.

Plagge et al. used a similar gene-targeting approach to
examine the function of a paternally expressed Gnas
transcript, Gnasxl, which encodes XLαs. Clues to thefunction of the various proteins encoded by the Gnas
locus have come from studies of mice with targeted
mutations. Mice that lack Gsα die shortly after birth,with maternal and paternal transmission showing
opposing effects on adipose tissue and metabolic rate.
As there is no evidence for exclusive paternal expressionof Gsα in any tissue, attention turned to the paternallyexpressed GNASXL as having a distinct functional role.
Plagge and colleagues found that mice in which the
XLαs-specific XL domain was deleted also died soonafter birth. The pups’ growth was retarded, they did not
suckle and their lipid stores in adipose tissue weredepleted. XLαs sites of expression — nuclei thatinnervate orofacial and tongue muscles, and thepituitary, pancreas and hypothalamus — are consistent
with its role in the postnatal adaptation to feeding andin energy homeostasis.
Notably, the XLαs-deficient phenotype provides
compelling support for the parental conflict theory of
imprinting, which proposes that paternal genes would
increase resource uptake from the mother by offspring,and that maternal genes would restrict nutritional
demands. Although this theory has received support
from mice with mutations in other imprinted genes, theauthors note that this is the first example in which an
imprinted locus encodes two proteins that act
antagonistically in postnatal physiology — regulating
cyclic AMP (cAMP) production in this instance. The
Gnas locus will no doubt continue to provide a uniquewindow into the role of imprinting in negotiating thecompeting demands of mothers and fathers.
Alan Packer, Senior Editor, Nature Genetics
References and links
ORIGINAL RESEARCH PAPERS Plagge, A. et al. The imprinted signaling

protein XLαs is required for postnatal adaptation to feeding.
Nature Genet. 36, 818–826 (2004) | Williamson, C.M. et al. A cis-acting
control region is required exclusively for the tissue-specific imprinting ofGnas. Nature Genet. 36, 894–899 (2004)
FURTHER READING Wilkins, J. F. & Haig, D. What good is genomic
imprinting: the function of parent-specific gene expression. Nature Rev.
Genet. 4, 359–368 (2003)

Nick Campbell

6 4 2 | SEPTEMBER 2004 | VOLUME 5

www.nature.com/reviews/genetics©2004 Nature Publishing GroupRESEARCH HIGHLIGHTS

EVO–DEVOFUNCTIONAL GENOMICS

A better bodyclock

Vertebrate face in profile

Faces are one of the most distinctive
parts of our anatomy; we use them totell people apart and to express ouropinions and emotions. But how doskeletal, muscle and nerve tissues combine to produce this most engaging
part of our body? By analysing azebrafish mutant, Justin Gage Crumpand colleagues show that precise hierarchical interactions between the threetissue types coordinate their assemblyin one particular segment of the face.
Vertebrate faces come in manyguises but they all develop from sevensegmented regions (the pharyngeal
arches), among which are intercalated
outpockets of endodermal tissue,
called ‘pouches’. Cranial neural crest
cells populate the arches and give rise
to cartilage, which the pouches support and pattern. Several mutations
disrupt the delicate process that controls the morphogenesis and differentiation of skeletal precursors. For
example, mutations in the genes that
encode integrins have shown that thisconserved family of heterodimeric
receptors are involved in signalling
and are required for the migration ofneural crest cells to the arches. Now,
studies of zebrafish that are mutant for
integrin-α5 have revealed a new link
between integrins and face patterning.
The mutant animals, which theauthors isolated in a screen for cartilage defects, lack the first pharyngeal
pouch and have defects in the cartilage
structures derived from the second
pharyngeal arch — a phenotype that is
linked to the loss of Integrin-α5 function specifically in the first pharyngeal
pouch. Embryos with cartilage defects
also have muscle and nerve defects
within the same face region. The factthat transplanted wild-type pouch
endoderm, but not crest, can rescue
the cartilage defects of a mutant showsthat the gene is required in the pouch
rather than in the cartilage. So, it islikely that the cartilage, muscle andnerve defects seen in the mutants aresecondary to the pouch defects.
Moreover, cell-fate mapping showedthat cells closer to the first pouch are
the most affected by the mutation andthat poor compaction and abnormal
death of the neural crest cells that surround the pouch probably cause thecartilage defects.
The most striking feature of theintegrin- α 5 mutant phenotype isthe hierarchical way in which it triggers face patterning: it seems that the
Integrin-α5-dependent endodermal
pouch patterns the crest, which inturn might control the developmentof neighbouring muscles and nerves.
Could this interconnected developmental mechanism have shaped theevolution of the head, by allowing thetissues of each head section to vary insize or shape in a coordinated fashion? Perhaps, although it remains tobe shown that similar hierarchicalrelationships hold true for other partsof the vertebrate head.
Tanita Casci

A new approach that uses gene-expression profiles to measureindividual body time (BT) promises to make a big difference tothe way we will administer drugs in the future.
Having access to an accurate clock or watch is pretty
important — as anybody who has been late for an exam or aninterview because of an unreliable timepiece can confirm! It canalso be important to read the correct time from the body’s
circadian clock: administering drugs at an inappropriate BT cancause side-effects, whereas at the correct BT a drug’s potency
might be maximized and its toxicity minimized.
To address the lack of clinically applicable methods of BT
detection, Hiroki Ueda and colleagues aimed to create a standard‘molecular timetable’ of gene-expression profiles. First, they
monitored genome-wide expression profiles from pooled liver
samples, taken from BALB/c mice over a 2-day period, undereither 12-hour cycles of light and dark, or constant dark
conditions. The peak expression times for the 168 genes that hadhigh circadian rhythmicity were distributed over a 24-hour
period and did not differ between the two light/dark regimes,
which indicated that they could provide a direct measure of theendogenous state of the circadian clock.
Next, the authors used a statistical approach that allowed the
BT information to be extracted from the expression profiles of
the time-indicating genes. The gradually changing expression
profiles of night-indicating and day-indicating genes can be
accurately modelled by the best-fitted cosine curve.
Their verification in independent samples indicated that thismethod could provide accurate estimates of BT. But how
clinically useful is such a timetable? In mice, at least,
the molecular timetable easily detectedrhythm disorders in four Clock –/– mutants
compared with wild-type mice.
Moreover, given that circadianrhythmicity could also be detected
in C3H mice, with the timetablethat was calibrated in BALB/cmice, it seems this method isrobust to changes in genetic
background.
So, Ueda and colleagues might
have provided a robust means ofestimating BT from a single-point
measurement: a long-held ambition
for many clinicians. Of course, it is along way from these experiments in inbred
mouse strains to a routine clinical tool forhumans, but given the number of genes in humans thatshow circadian expression patterns, there is certainly no obvious
reason why the same approach would not be applicable.
Nick Campbell

References and links
ORIGINAL RESEARCH PAPER Crump, J. G. et al.

References and links

An integrin-dependent role of pouch endoderm inhyoid cartilage development. PLoS Biol. 2, E244
(2004)ORIGINAL RESEARCH PAPER Ueda, H. R. et al. Molecular-timetable methods for

WEB SITE

WEB SITE

Charles Kimmel’s laboratory:
http://www.neuro.uoregon.edu/ionmain/htdocs/
faculty/kimmel.html

Hiroki Ueda’s laboratory: http://www.riken.go.jp/engn/r-world/research/lab/cdb/
system/Online links(Printed image adapted from one provided by Hiroki Ueda.)

detection of body time and rhythm disorders from single-time-point genome-wide
expression profiles. Proc. Natl Acad. Sci. USA 101, 11227–11232 (2004)

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