doi:10.1006/jmbi.2000.3795 available online at http://www.idealibrary.com on J. Mol. Biol.

(2000) 299, 931±939

Alu-mediated Phylogenetic Novelties in Gene

Regulation and Development
Hamdi K. Hamdi, Hitomi Nishio, Jeffrey Tavis, Rita Zielinski
and Achilles Dugaiczyk*

Department of Biochemistry
University of California
Riverside, CA 92521, USA
*Corresponding author
Differential gene expression lies at the heart of biology and is responsible
for all developmental processes, including the growth and differentiation
of cells. Perhaps even speciation could be de®ned as a change in differen-
tial gene expression over evolutionary time. The present work is a phylo-
genetic study of four Alu elements known to have gene regulatory
functions in the human. The four elements have been shown to regulate
the parathyroid hormone (PTH) gene via a negative calcium-response
element, the hematopoietic cell-speci®c FceRI-g receptor gene via a cis-act-
ing positive/negative regulatory element, the CNS-speci®c nicotinic
acetylcholine receptor a3 gene via a cis-acting positive/negative control
element, and the T-cell-speci®c CD8a gene via a complex transcriptional
regulator. The four Alu elements that impact differential gene expression
were found to be differentially distributed among seven primate species
(human, chimpanzee, gorilla, orangutan, baboon, rhesus, and macaque)
in a way that is congruent with an accepted phylogeny of these species.
The results establish a link between gene regulation and the divergence
of primates. This evolutionary variation in gene regulation also suggests
a novel experimental system to study the very complex transcriptional
regulation of gene expression, by studying side-by-side the regulation of
the same gene from two primate species that differ in the cis-acting regu-
latory elements of the gene.
# 2000 Academic Press
Keywords: Alu sequences; factor-binding sites; gene regulation; saltatory
events

Introduction structing phylogenies simply rely on identifying

There is an apparent discontinuity between the
observed similarity between African apes and
humans at the level of genes and proteins and the
much more dramatic differences at the morphologi-
cal level. Human evolution has been accompanied
by an extremely fast morphological change, yet it is
associated with the lowest rate of molecular evol-
ution. Furthermore, many of the sequence differ-
ences apparently do not affect the organisms at all,
so the challenge is to ®nd those mutations that do
make a difference. Most studies aimed at recon-
Present addresses: H. K. Hamdi, Department of
Pediatrics, University of California School of Medicine,
Los Angeles, CA 90095, USA; H. Nishio, Department of
Molecular Biology & Biochemistry, University of
California, Irvine, CA 92697, USA.
E-mail address of the corresponding author:
achilles.dugaiczyk@ucr.edu
percentage differences among a particular protein
or DNA sequence and do not focus on the func-
tional consequences of these differences. The fallacy
of phylogenetic methods based on sequence data
was recently exposed by Naylor & Brown (1998),
who concluded that models underlying phyloge-
netic inferences are not accommodating the evol-
utionary process. We also have questioned whether
the majority of point mutations have any taxonomic
relevance to the evolutionary process (Gibbs et al.,
1998; Hamdi et al., 1999).
In earlier studies, Prager & Wilson (1975) envi-
sioned that anatomical differences between primate
species more likely result from differences in the
developmentally regulated expression of their
genes rather than from a different nature of their
genes per se. Genes are ultimately under the control
of promoter elements. In fact, in the ®rst transgenic
animal, the human growth hormone gene was
expressed with dramatic growth of the transgenic

0022-2836/00/040931±9 $35.00/0 # 2000 Academic Press

932
mice by introducing a metal-responsive enhancer
element into the promoter region controlling the
expression of the human growth hormone gene
(Palmiter et al., 1992).
Following an earlier postulate that a correlation
can be found between the emergence of individual
Alu DNA repeats, and the divergence of primate
lines of evolution (Ryan & Dugaiczyk, 1989;
Minghetti & Dugaiczyk, 1993), we have more
recently shown that an unambiguous tree can be
derived for primate phylogeny, because the de novo
arising Alus provide reliable time markers of the
evolutionary process (Hamdi et al., 1999). Because
of their genomic mobility, high CpG content,
tissue-speci®c methylation (Hellmann-Blumberg
et al., 1993; Rubin et al., 1994) and their effect on
chromatin structure (Englander & Howard, 1995)
and gene expression (Norris et al., 1995; Vansant &
Reynolds, 1995; McHaf®e & Ralston, 1995; Brini
et al., 1993; Fornasari et al., 1997; Hambor et al.,
1993), Alu insertions are now extremely attractive
candidates for promoting differences in the devel-
opmental regulation of primate genes. In the pre-
sent study, we provide data on the existence of
differences in various promoter regions in the gen-
omes of primates, which establishes a link between
the timing of Alu elements' insertions in primate
phylogeny and differences in gene regulation
between the primate species.
Results
Regulatory Alus
Some Alu elements have been shown to affect
gene transcription by carrying cis-acting elements
which are responsive to hormones, calcium, tran-
scription factors and other effectors (Norris et al.,
1995; Vansant & Reynolds, 1995; McHaf®e &
Ralston, 1995; Brini et al., 1993; Fornasari et al.,
1997; Hambor et al., 1993). These Alu elements
reside in the 50 -¯anking regions or within genes
and have been experimentally shown to affect gene
expression in the human. It is these Alu elements
that are the focus of the present study. Their pre-
sence or absence in the genome of human, chim-
panzee, gorilla, and orangutan, and in three
representatives of Old World monkeys (baboon,
rhesus, and lion-tailed macaque) was investigated
by the polymerase chain reaction (PCR). The iden-
tity of the PCR products was veri®ed by DNA
sequence determination.
Calcium regulation of the parathyroid hormone
(PTH) gene
A negative calcium-response element type 2
(nCARE) is a 15 bp sequence with a 12 bp palin-
dromic core which was identi®ed approximately
3.6 kb upstream from the transcription site of the
human parathyroid hormone (PTH) gene (Okazaki
et al., 1991). The nCARE2, along with a distinct,
more downstream situated nCARE element are
Phylogenetic Novelties
responsible for downregulation of PTH transcrip-
tion in response to an increase in extracellular cal-
cium concentrations. The nCARE2 element was
subsequently identi®ed to reside within an Alu
element (McHaf®e & Ralston, 1995). A simpli®ed
diagram of this structure is shown in Figure 1(a).
Using DNA primers ¯anking the nCARE2 Alu,
we ampli®ed this genomic region and veri®ed the
identity of the PCR products by DNA sequencing.
The PCR products were sequenced in their entire
length but only sequences surrounding Alu inser-
tions are shown (Figure 1(b) and (c)). The same
Alu is found to be present in an orthologous pos-
ition in humans, great apes, and three Old World
monkey species. One could infer that the calcium
regulation of the PTH gene is also the same (or
close to) in all extant primate species. In retrospect,
the result is perhaps not unexpected, considering
that calcium regulation must be a fundamental
house-keeping process that has remained un-
changed since the beginning of primates.
Cell-specific regulation of the IgE receptor
(FceeRI-gg) gene
The g chains of the high-af®nity IgE receptor are
expressed in various hematopoietic cells where
they play a critical role in signal transduction
(Ravetch & Kinet, 1991). In the 2.5 kb sequence
upstream of the transcription start site, there is a
promoter speci®c to hematopoietic cells, and in
addition two adjacent cis-acting regulatory
elements, both of which are part of an Alu repeat
(Brini et al., 1993). The ®rst is a positive element
active in both basophilic and T-cells. The second
binds to nuclear factors, which appear to be differ-
ent in basophilic and T-cells, and acts as a negative
element in basophilic and as a positive one in T-
cells (Brini et al., 1993). Thus, this Alu repeat has
evolved to become both a positive and a negative
regulator. A diagram of the 50 -region of this gene is
shown in Figure 2(a).
Using primers ¯anking the Alu containing the
cis-acting regulatory elements, we ampli®ed the
orthologous regions from seven primate species
and veri®ed the identity of the PCR products by
DNA sequencing; the entire length of the PCR pro-
ducts was sequenced, but only the critical regions
are shown. This regulatory Alu element is present
in humans and great apes, but absent from Old
World monkey species, where only the empty tar-
get site is present (Figure 2(b) and (c)).
Structural and functional features of the
nicotinic acetylcholine receptor a 3 gene
Nicotinic acetylcholine receptors are ligand-
gated ion channels expressed at the neuromuscular
junction and in several areas of the vertebrate cen-
tral nervous system. Different structural subunits
combine to form different receptor subtypes with
distinctive pharmacological and electrophysiologi-
cal properties, and the fast excitatory postsynaptic
Phylogenetic Novelties
potential is due to the activation of nicotinic acetyl-
choline receptors that contain a3 as the essential
agonist-binding subunit (McGehee & Role, 1995).
The expression of certain subunits can be restricted
to very few brain structures (Wada et al., 1989; Zoli
et al., 1995), implying that stringent controls must
be exerted to achieve such ®ne tuning of spatial
and developmental regulation. The a3 promoter
was found to be under the control of upstream
sequences that reside with an Alu and contain both
a positive and a negative control element
(Fornasari et al., 1997). A diagram of the 50 -region
of this gene is shown in Figure 3(a).
Using primers ¯anking the upstream Alu con-
taining the cis-acting regulatory elements, we
ampli®ed the orthologous regions from seven pri-
mate species and veri®ed the identity of the PCR
products by DNA sequencing of their entire length,
although only critical regions are shown
(Figure 3(b) and (c)). This regulatory Alu element is
present in humans and great apes, but absent from
Old World monkey species, where only the empty
target site is present. Similar to the results on the g
933
Figure 1. Analysis of primate
genomic DNA for the presence of
an orthologous Alu repeat carrying
a negative calcium-response
element. (a) A diagram of the 50 -
¯anking region upstream of the
human parathyroid hormone (PTH)
gene (reconstructed from McHaf®e
& Ralston, 1995), showing the pos-
ition of the Alu repeat carrying a
negative calcium response element
(nCARE 2). (b) PCR analysis of the
orthologous Alu locus in the pri-
mates. The following primers were
used: PTH Pr-1, 50 -GAGTCAT-
CATCCATTTCCCATTTTC; PTH
Pr-2, 50 -GAAAAGCTGAACTGT-
CCTGACAGG. Samples (10 ml) of
PCR mixtures (see Materials and
Methods) were electrophoresed in a
5 % polyacrylamide gel. Molecular
mass standards (Std) are indicated
in base-pairs (bp); the remaining
lanes contain PCR products from
the various primate species.
(c) DNA sequence of regions sur-
rounding and including the ortho-
logous PTH Alu. Nucleotides
identical with the top sequence (ÿ),
deletions (
), and terminal repeats
(underlined) are indicated.
chain of the IgE receptor gene, new transcription
control elements of the nicotinic receptor gene
arose in the lineage leading to higher primates.
T-cell specific transcriptional enhancer in the
CD8aa gene
Expressed on the surface of T-cells, CD8 interacts
extracellularly with the major histocompatibility
complex (MHC) molecules (Norment et al., 1988)
and intracellularly associates with the tyrosine
kinase p56lck (Chalupny et al., 1991). Expression of
the human CD8a gene is restricted to cells of the
lymphoid lineage and it is developmentally regu-
lated during thymopoiesis (Leiden, 1992). In the
last (5th) intron of this gene, a T-cell-speci®c
enhancer was identi®ed that is partially located in
an Alu element (Hambor et al., 1993). This CD8a
intronic enhancer is a complex transcriptional regu-
lator containing seven binding sites for six tran-
scription factors: GATA-3, LyF-1 (two sites),
bHLH, TCF-1, CRE, and Ets-1. Four of these bind-
ing sites reside in the Alu element. There is actually
a full-length and a half-Alu repeat in close proxi-
934 Phylogenetic Novelties

Figure 2. Analysis of primate genomic DNA for the presence or absence of an orthologous Alu repeat carrying a
positive and negative control element of transcription. (a) A diagram of the 50 -¯anking region upstream of the human
high-af®nity IgE receptor (FceRI-g) gene (reconstructed from Brini et al., 1993), showing the position of the Alu repeat
carrying a positive and negative control element. (b) PCR analysis of the orthologous Alu locus in the primates. The
following primers were used: Fc Pr-1, 50 -TTCAATGACCAACTCCTAACCCAC; Fc Pr-2, 50 -TGCCCACATAACTTGG-
TAAACCAC. Samples (10 ml) of PCR mixtures (see Materials and Methods) were electrophoresed in a 5 % polyacryl-
amide gel. Molecular mass standards (Std) are indicated in base-pairs (bp), the remaining lanes contain PCR products
from the various primate species. DNA bands in the 500 bp region indicate the presence of the Alu repeat, those in
the 200 bp regions, its absence. Small differences in mobility of the PCR bands between the various primates are
caused primarily by variable lengths of the poly(A) strech terminating each Alu, and by small insertions/deletions in
the ¯anking regions. (c) DNA sequence of regions surrounding and including the orthologous FceRI-g Alu repeat, or
its unoccupied target site. Nucleotides identical with the top sequence (ÿ), deletions (
), and target sites and terminal
repeats (underlined) are indicated.

mity and in an opposite (tail-to-tail) orientation.
A map of this region is shown in Figure 4(a).
Using ¯anking primers, we ampli®ed by PCR
the one and a half-Alu structure, including ¯anking
regions, and sequenced it from the seven primate
species (Figure 4(b) and (c)). The full-size Alu, con-
taining the four transcription factor binding sites,
is found only in humans and great apes, while the
50 adjacent half-Alu is present in higher primates
and Old World monkeys (Figure 5). The present
sequence from the non-human primates has been
deposited in GenBank under accession numbers
AF251315; AF251316; AF251317; AF251318;
AF251319; AF251320. The corresponding human
sequence has been deposited under M27161 by
Nakayama et al. (1989).
Discussion
The de®ning event of speciation is a transition
from being to becoming, but the underlying geno-
mic event(s) of such a transition elude identi®-
Phylogenetic Novelties 935

Figure 3. Analysis of primate genomic DNA for the presence or absence of an orthologous Alu repeat carrying a
positive and negative control element of transcription. (a) A diagram of the 50 -¯anking region upstream of the human
a3 nicotinic receptor subunit gene (reconstructed from Fornasari et al., 1997), showing the position of the Alu repeat
carrying a positive and negative control element. (b) PCR analysis of the orthologous Alu locus in the primates. The
following primers were used: a3 Pr-1, 50 -GGTACCATCATCCTGCCTCGACAAT; a3 Pr-2, 50 -CGCCGTAGGAGA-
GACGCTAACACA. Samples (10 ml) of PCR mixtures (see Materials and Methods) were electrophoresed in a 5 %
polyacrylamide gel. Molecular mass standards (Std) are indicated in base-pairs (bp), the remaining lanes contain PCR
products from the various primate species. DNA bands in the 500 bp region indicate the presence of the Alu repeat,
those in the 200 bp regions, its absence. Small differences in mobility of the PCR products between the various pri-
mates are caused by variable lengths of the poly(A) strech terminating each Alu, and by small insertions/deletions in
the ¯anking regions. (c) DNA sequence of regions surrounding and including the orthologous a3 Alu repeat, or its
unoccupied target site. Nucleotides identical with the top sequence (ÿ), target sites and terminal repeats (underlined)
are indicated.

cation. Perhaps the recruitment of cis-regulatory
elements, which causes the transcription of genes
in different spatial or developmental contexts,
could belong to this category of events. We have
recently shown that a differential distribution of
Alu DNA elements in primates can be used to
reconstruct an unambiguous tree of primate phylo-
geny (Hamdi et al., 1999). In the present study, we
provide examples of differential distribution of
Alu-embedded gene regulatory elements in pri-
mate species that establish a correlation between
Alu insertion and primate evolution; a correlation
with a mechanism of speciation remains to be
established. Although the differential distribution
of the presently identi®ed Alus is not depicted on a
phylogenetic tree, it is important to note that it is
not a haphazard distribution. The four Alus carry-
ing gene regulatory elements are distributed
among seven primate species in a way that is con-
gruent with an accepted phylogeny of human and
apes, and the Old World monkey species. How
these genes are actually regulated in the non-
936 Phylogenetic Novelties

Figure 4. Analysis of primate

genomic DNA for the presence or
absence of an orthologous Alu
repeat carrying a transcriptional
regulator. (a) A diagram of the
human CD8a gene (reconstructed
from Hambor et al., 1993; and
Nakayama et al., 1989), showing
the position of the full-size and the
half-Alu repeats within the intron-5
enhancer. The tail-to-tail orientation
of the Alu repeats is indicated by
the white arrowheads. The coordi-
nates (1 and 880) for the 880 bp
enhancer are taken from Hambor
et al. (1993), and they correspond to
the coordinates 4199 and 5103
given by Nakayama et al. (1989).
The 24 bp difference in size corre-
sponds to positions 345 to 367 that
was tandemly duplicated in the
work by Nakayama et al. (1989).
We do not ®nd this tandem dupli-
cation in any of our seven primate
sequences. (b) PCR analysis of
the orthologous Alu locus in the
primates. The following primers
were used: CD8a Pr-1, 50 -GCA-
TATGTTACAGGTGATATACCAA-
GGGGA; CD8a Pr-2, 50 -GCT
TTGTATTTTAAGGCAGAACTTTA-
TTCTCAG. The region is somewhat
dif®cult to amplify with one set of
primers for the seven species. The
above primers work well with
human, chimp, gorilla and baboon
DNA. For the orangutan and maca-
que DNA, the annealing tempera-
ture needs to be decreased, or the
Pr-1 primer replaced with 50 -
GCGTATGTTATAAGTGATATAC-
CAAAGGGA (for orangutan) or
with 50 -GCATATGTTATAGGTGATATGCTAAGGGGA (for macaque). Samples (10 ml) of PCR mixtures (see
Materials and Methods) were electrophoresed in a 5 % polyacrylamide gel. Molecular mass standards (Std) are indi-
cated in base-pairs (bp), the remaining lanes contain PCR products from the various primate species. DNA bands in
the 500 bp region indicate the presence of the Alu repeat, those in the 200 bp regions, its absence. Small differences in
mobility of the PCR bands are caused primarily by differences in the poly(A) length terminating individual Alus, and
by small insertions/deletions in the ¯anking regions. (c) DNA sequence of regions surrounding and including the
orthologous full-size Alu repeat, or its unoccupied target site. Nucleotides identical with the top sequence (ÿ), del-
etions (
), and target sites and terminal repeats (underlined) are indicated.

human species, and how a differential regulation
might contribute to the mechanism of speciation,
must await additional studies.
Control element shared by great apes and Old
World monkeys
The Alu element residing upstream of the PTH
gene and controlling PTH expression in the
human, was found to be present in all the primate
species studied (Figure 1). This is an example of an
old insertion and retention of function from a time
deep in the past in primate evolution. The phyloge-
netic origin of this PTH Alu could be even older, if
it is found in contemporary New World monkeys,
which were not included in our present work.
Genetic novelties at the roots of
higher primates
The Alu elements residing upstream of the
human FceRI-g gene (Figure 2), upstream of the
nicotinic a3 gene promoter (Figure 3), and within
the CD8a gene (Figures 4 and 5) are differentially
distributed in primate species. They are present in
higher primates, but absent from the Old World
monkeys, where only an empty target site is pre-
sent at the orthologous site, and no Alu transposi-
Phylogenetic Novelties 937

Figure 5. Nucleotide sequence of the CD8a intronic enhancer in the various primates (H, human; C, chimpanzee;
G, gorilla; O, orangutan; B, baboon; R, rhesus; M, lion-tailed macaque). The T in position 1 corresponds to position 4
in CD8a Pr-1 (see Figure 4); and the position of CD8a Pr-2 is 63 to 95 nucleotides downstream of the last T in pos-
ition 600. Nucleotides identical with the top (human) sequence (ÿ); deletions (
), the target sites, terminal repeats,
and the cis-regulatory elements (underlined) and the beginning of the half-Alu and the full-size Alu (arrows) are
indicated. The two Alu repeats are in a tail-to-tail orientation, separated by a short stretch of G bases, and they are
terminated by a stretch of poly(A) of different length in the various primates. These and additional sequences have
been deposited in GenBank under accession numbers AF251315, AF251316, AF251317, AF251318, AF251319 and
AF251320.
938
tion took place. These elements represent a gain of
function in the higher primates, considering that
the ancestral state of an Alu character is always its
absence (Hamdi et al., 1999). If the newly arisen
Alus operate in cell-speci®c and developmental
gene regulation in higher primates, as they do in
humans, then the gain of these regulations was
attained at the roots to higher primates and was
carried over to all descendent lineages.
The CD8a example provides more speci®c
details about species-speci®c differences in gene
regulatory elements. In humans, the Alu insertion
contributed to the evolution of a complex tran-
scriptional regulatory element: four binding sites
for transcription factors are within it, and they
have been shown to participate in cell-speci®c gene
regulation (Hambor et al., 1993). This regulatory
Alu is present in the higher primates (Figure 4),
and its four regulatory elements are largely con-
served in the four primate species (Figure 5).
GATA-3 has two point mutation differences
between human and the other primates, and may
or may not function in the same way in the four
primate species (some differences are allowed
within a concensus sequence in cis-regulatory
elements). The sequences of the remaining three
(bHLH and two sites of LyF-1) are identical in
human, chimp, and gorilla, and differ only by one
or two nucleotides in the orangutan (Figure 5). It
seems safe to postulate that the four regulatory
elements were ``active on arrival'' at the time of
the Alu transposition, rather than slowly aquired
their function by point mutations after the Alu
transposition. This would represent a saltatory
event at the roots to higher primates, with its con-
sequences carried over to descendent lineages.
This regulatory Alu is absent from Old World
monkeys, but in its place there is an older half-Alu
in the structure of the CD8a gene. Could this half-
Alu provide the same transcriptional control
elements for the monkeys? In humans, where this
half-Alu is also present, although some 300 bp
further upstream within the intronic enhancer, no
binding sites for transcription factors were found
experimentally by Hambor et al. (1993). Presently,
we sequenced this half-Alu from the seven primate
species (Figure 5), and an inspection of this
sequence reveals no similarity to any of the bind-
ing sites identi®ed by Hambor et al. (1993) in the
full-size Alu of the human CD8a gene. It seems
safe to infer that the regulation of this gene must
be different in the monkey species. Further exper-
iments will be required to show the transcriptional
differences in the non-human primates. These are
complex experiments, considering that a large
number of cis and trans-acting factors coordinate
the process of gene transcription.
Considering that Alu elements can be speci®c to
one, or shared by two or more primate species
(Hamdi et al., 1999), and given the fact that there
are a million Alu elements in the human genome
and there have been no systematic studies to ident-
ify which of them have regulatory functions, it
Phylogenetic Novelties
must be only a matter of time before human-
speci®c Alus are found to control gene expression.
Materials and Methods
DNA
Peripheral blood (5 ml) was collected into 10 mM
EDTA and used to isolate white blood cells according to
the method of Kan et al. (1997). Genomic DNA was iso-
lated from the white blood cells using the Qiagen
QIAamp kit and the manufacturer's protocol. DNA from
the following species was used: human (Homo sapiens),
chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla),
orangutan (Pongo pygmaeus), baboon (Papio cynocephalus
anubis), rhesus monkey (Macaca mulatta), and lion-tailed
macaque (Macaca silenus). Human blood was obtained
from a local blood bank while primate genomic DNA or
blood samples were obtained from: Loma Linda Univer-
sity, CA; San Diego ZOO/CRES; University of California
Davis Primate Research Center; University of Pittsburgh
Medical Center; The Upjohn Company; Yerkes Primate
Research Center.
PCR
Ingredients for the PCR reaction (30 ml) included:
20 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2,
250 mM each dNTP, 0.2 mM each primer (described in
the Figure legends), 0.3 mg of genomic DNA, and 0.9
unit of Taq polymerase. Reactions were performed in a
Stratagene Robocycler under the following conditions:
96  C for two minutes, followed by 30 cycles of denatura-
tion (96  C for 30 seconds), annealing (50  C-68  C for 30
seconds) and elongation (62  C-68  C for 30 seconds). The
optimal annealing temperature for each set of primers
was determined experimentally by ®rst running PCR
reactions on a temperature gradient ranging from 50 to
68  C. The last cycle was followed by a ®nal elongation
step (65  C for ®ve minutes). The reactions were stopped
by the addition of 3 ml of 0.25 M EDTA at 0  C, and ali-
quots were electrophoresed on a 5 % (w/v) polyacryl-
amide (acrylamide to bis-acrylamide, 40:1, w/w) gel.
Sequencing of PCR-amplified DNA
Gel-puri®ed PCR products were either subcloned
or sequenced directly by the Maxam & Gilbert (1980) or
the ABI dye-deoxy chain terminator cycle sequencing
methodology.
Acknowledgments
We thank Lars Dugaiczyk and Michael Capriotti for
computer graphics and photography of the Figures. This
work was supported by grant CA-R-BCH-5820-H from
the University of California.
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