9:1-4, 2002. First published Feb 19, 2002; doi:10.1152/physiolgenomics.00105.

2001 Physiol Genomics

J. T. Streelman and T. D. Kocher
expression and growth of salt-challenged tilapia
Microsatellite variation associated with prolactin
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brief communication
Microsatellite variation associated with prolactin
expression and growth of salt-challenged tilapia
J. T. STREELMAN AND T. D. KOCHER
Hubbard Center for Genome Studies, University of New
Hampshire, Durham, New Hampshire 03824
Received 6 November 2001; accepted in nal form 11 February 2002
Streelman, J. T., and T. D. Kocher. Microsatellite vari-
ation associated with prolactin expression and growth of
salt-challenged tilapia. Physiol Genomics 9: 14, 2002. First
published February 19, 2002; 10.1152/physiolgenomics.
00105.2001.Biologists have long argued that runs of alter-
nating purines and pyrimidines could form alternative DNA
structures, which might regulate transcription. Here, we
report that simple sequence repeat polymorphisms in the
tilapia prolactin 1 (prl 1) promoter are associated with dif-
ferences in prl 1 gene expression and the growth response of
salt-challenged shes. Individuals homozygous for long mic-
rosatellite alleles express less prl 1 in fresh water but more
prl 1 in half-seawater than shes with other genotypes. Our
work provides the rst in vivo evidence that differences in
microsatellite length among individuals may indeed affect
gene expression and that variance in expression has concom-
itant physiological consequences. These results suggest that
dinucleotide microsatellites represent an under-appreciated
source of genetic variation for regulatory evolution.
dinucleotide repeats; gene expression; promoter; evolution
TILAPIA ARE AMONG the worlds most important aquacul-
tural nshes (13). A multitude of studies have ad-
dressed means to grow bigger tilapia faster, experiment-
ing with water temperature, salinity, hybridization, and
administration of growth-promoting hormones. Notably,
tilapiine species differ in salt tolerance and growth re-
sponse in different salinities (20).
A great deal is known about the role of peptide
hormones in sh osmoregulation. Prolactin is a mem-
ber of the growth hormone (GH)/Prl gene family whose
freshwater adapting role is to increase plasma osmo-
lality by reducing gill Na

-K

-ATPase activity (15).

The tilapiine pituitary produces two forms of prolactin,
encoded by different genes (prl 1 and prl 2) that differ
in both molecular mass (24 and 20 kDa) and number of
amino acid residues (188 and 177; Ref. 25). These two
forms are roughly 70% identical at the amino acid level
and appear to have differential osmoregulatory (1) and
somatotropic (17) actions. Experiments suggest that as
shes move into saline environments, both prolactin
mRNAs and serum levels decrease; this effect is more
dramatic for prl 1 (2).
The tilapia prl 1 promoter (21) shares noncanonical
elements with that of rat. Both promoters have two
(CA/GT)
n
microsatellites interspersed among putative
binding sites for the pituitary-specic transcription
factor Pit-1 (Fig. 1). Naylor and Clark (12) demon-
strated in rat that these repeat sequences formed left-
handed Z-DNA in vitro and repressed prl 1 expression.
We reasoned that similar regulatory effects might exist
in the tilapia prl 1 promoter. Individual differences in
microsatellite length might affect prl 1 expression in
vivo and might contribute to known variance in salt
tolerance and growth at different salinities.
Despite the textbook interpretation that noncoding
simple sequence repeats evolve in a neutral fashion,
recent reports have indicated otherwise (11, 24). In
fact, the abundance and position of dinucleotide micro-
satellites in eukaryotic genomes coupled with a high
rate of mutation suggest a potential and pervasive role
in gene regulation (9). Here, we used a natural system
to evaluate the association between dinucleotide mic-
rosatellite variation, quantitative differences in gene
expression and the physiological response to contrast-
ing environments.
METHODS
We initially concentrated on the microsatellite closest to
the start of prl 1 transcription (200 bp). We crossed females
of Oreochromis mossambicus (salt-adapted) with an O. nil-
oticus (freshwater-adapted) male carrying microsatellite al-
leles that differed by 17 repeat units (CA
31
vs. CA
14
). O.
mossambicus dams were homozygous for long alleles and the
O. niloticus sire was a heterozygote. F
1
individuals were
selected for breeding such that there were three genotypes to
follow in the F
2
generation: homozygotes for short alleles
Article published online before print. See web site for date of
publication (http://physiolgenomics.physiology.org).
Address for reprint requests and other correspondence: J. T.
Streelman, Hubbard Center for Genome Studies, Univ. of New
Hampshire, 35 Colovos Road, Durham, New Hampshire 03824
(E-mail: jts3@hopper.unh.edu).
Physiol Genomics 9: 14, 2002.
First published February 19, 2002; 10.1152/physiolgenomics.00105.2001.
1094-8341/02 $5.00 Copyright 2002 the American Physiological Society 1

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(hereafter SS), homozygotes for long alleles (LL), and het-
erozygotes (SL). Seventy-ve F
2
shes from the same brood
were raised in three salinity treatments in separate 200-liter
tanks: freshwater [0 parts per thousand (ppt) NaCl], 16
ppt, and seawater (32 ppt). Two-week-old fry were acclimated
to treatments over a period of 3 days and maintained until
sh mass averaged 15 g (6 mo). Within salt treatments, all
three genotypes were grown in the same tank. Fishes were
killed, sexed, and weighed. Pituitaries were harvested and
stored at 20C in RNAlater (Ambion) until RNA extraction.
Pituitary Prl 1 gene expression was measured from F
2
individuals (cDNA preparations) using real-time PCR (8)
with B-actin as a reference. Primers and probes were de-
signed using Primer Express (version 1.5, Applied Biosys-
tems). Probe binding sites were located at intron-exon bound-
aries to prevent amplication of genomic DNA. Probes were
uorescently labeled at the 3 end. Fluorescence was moni-
tored during 40 cycles of PCR on a GeneAmp 5700 sequence
detection system (Applied Biosystems; 95C for 15 s, 55C for
30 s, and 65C for 1 min).
Critical cycle number was determined when uorescence
exceeded a threshold value set close to the background. Prl 1
relative gene expression was determined according to the
formula 2
B-actin CT Prl 1 CT
where CT is the critical cycle
number. Real-time PCR primer and probe sequences are: F
primer, cccatcaacgaactgttcga; R primer, gcatgatcaccctgcctat-
agg; probe, ctcacccaggagctggactctcacttccctc. F
2
individuals
were genotyped as described (10) or by a Sac II restriction
fragment length polymorphism (RFLP) in prl 1 cDNA, which
distinguishes parental alleles. Differences among genotypes
per treatment and treatments per genotype were evaluated
by one-way ANOVA and appropriate post hoc multiple com-
parison tests.
RESULTS AND DISCUSSION
Females were larger than males across all salinity
treatments but did not differ from males in prl 1
expression. Mean sh mass increased with increasing
salinity (P 8.4 10
06
). Expression differed among
genotypes at 0 ppt (P 0.006); SS individuals ex-
pressed approximately two times and seven times as
much prl 1 as SL and LL shes, respectively (Fig. 2A;
Table 1). Expression differed among genotypes at 16
ppt (P 0.0005) with a nearly opposite pattern to that
seen in freshwater; individuals of genotype LL ex-
pressed twice as much prl 1 as did shes of genotypes
Fig. 1. Schematic representation of the
tilapia prl 1 promoter region. Identica-
tion of putative transcription factor bind-
ing sites (e.g., Pit 1) follows Ref. 21.
Fig. 2. Prl 1 expression and sh mass
differ across salt treatments. Graphs in
AD depict means 1SE. The y-axes of
A, B, and E are normalized Prl 1 expres-
sion levels determined according to the
formula 2
B-actinCTPrl 1CT
where CT is
the critical cycle number. *P 0.05 and
**P 0.005, levels of signicance from
Tukeys post hoc multiple comparison
tests (see Table 1 for explanation).
2 MICROSATELLITES AND GENE EXPRESSION
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SS and SL (Fig. 2B, Table 1). Notably, LL individuals
were only one-half as big as those of other genotypes at
16 ppt (Fig. 2D, Table 1). There were no differences
among genotypes in either prl 1 expression or sh mass
at 32 ppt (Table 1).
We detected signicant genotype environment inter-
actions. For all genotypes, both prl 1 expression and mass
differed across salt treatments (SS, P 1.5 10
05
and
P 0.03; SL, P 3.8 10
06
and P 0.008; LL, P
0.0008 and P 0.06; for expression and mass, respec-
tively). Individuals of genotype SS and SL showed dra-
matic decreases (10 fold) in prl 1 expression from0 to 16
ppt. This response was not nearly as apparent for geno-
type LL (Fig. 2E). Each genotype grew best at a different
salinity treatment (Fig. 2F), and mean growth of geno-
types was inversely correlated with mean prl 1 expres-
sion across salinities (SS, r
2
0.957; SL, r
2
0.971; LL,
r
2
0.530). Figure 2, E and F, demonstrates divergent
norms of reaction for each genotype in response to exter-
nal salt concentration. In natural populations, these
crossover interactions are prima facie evidence for the
adaptive maintenance of genetic variation in uctuating
environments (5).
The possibility that runs of alternating purines and
pyrimidines might affect gene expression was sug-
gested nearly 20 years ago (6, 7, 14). We speculate that
repeats of varying length can induce promoter confor-
mations that differ in their ability to bind transcrip-
tional regulators. There is good evidence that tilapia
prl 1 expression is regulated in response to plasma
osmolality (18), perhaps via interactions with Pit-1.
Long prl 1 microsatellite alleles may thus act as insu-
lators, restricting access of enhancers (freshwater) and
repressors (saltwater) to Pit-1 sites.
The similarity between our results and those of Nay-
lor and Clark (12) are both unexpected and uncanny,
suggesting either the conservation of a regulatory func-
tion for CA/GT microsatellites in the prl promoter over
400 million years of vertebrate evolution or the con-
vergent acquisition of this biological role. Numerous
reports have conrmed that variably sized simple se-
quence repeats in eukaryotic promoters can elicit dif-
ferential expression when cloned into vectors (7, 12, 19,
22). Shimajiri et al. (19) indicate that the matrix met-
alloproteinase gene is downregulated by shortening of
a promoter microsatellite. Interestingly, Tae et al. (22)
report that the negative effect of a dinucleotide repeat
in the acetyl-CoA carboxylase gene is CAAT box depen-
dent and can be overcome by the presence of CAAT
enhancer binding protein. These studies suggest that
there is no general trend in the direction of effects
induced by microsatellite mutation (e.g., enhancer or
repressor) and that such effects are likely to be com-
plicated by the cellular milieu.
Because F
2
shes are not isogenic lines segregating
for different microsatellite alleles, we cannot formally
exclude the possibility that the alleles we followed are
in linkage disequilibrium with an unmeasured, causal
polymorphism. However, sequence from 1.2 kb of the
prl 1 promoter in SS vs. LL individuals revealed no
consistent substitutions in known regulatory element
binding sites. In fact, additional sequence differences
in this region are found in the second simple sequence
repeat 800 bp upstream of the start codon (Fig. 1).
Individuals of genotype SS had shorter alleles (ho-
mozygous for GT
14
) than LL shes (homozygous for
GT
39
). Taken together, both microsatellites in the prl 1
promoter of SS shes are less than half the size of the
corresponding sequence in LL individuals, a pattern of
positive covariance contrasting with data from other
bony shes (4). It is possible that the simple sequence
repeats in the prl 1 promoter physically interact with
one another to elicit transcriptional change.
The negative correlation between prl 1 expression
and sh mass is more difcult to explain. Prolactin and
GH are activated by Pit-1 in distinct pituitary cell
lineages (3), and these hormones have antagonistic
affects on sh osmoregulation (16). Our data do not
discern whether growth effects are mediated via osmo-
regulatory differences or transcriptional inhibition/in-
terconversion of GH-expressing somatotropes by Prl-
expressing lactotropes.
From an evolutionary perspective, our results run
counter to the textbook interpretation that dinucle-
otide microsatellite variation lacks functional conse-
quences. These loci are rapidly evolving and ubiquitous
components of eukaryotic genomes. Found preferen-
tially in noncoding DNA (23), dinucleotide repeats pro-
vide a cellular means to alter the amount of gene
product among individuals without changing amino
acid sequence. The association of microsatellites with a
long list of transcription factors and environmentally
Table 1. Summary of data discussed in the text
Salt Genotype Expression Signicance Mass, g Signicance
Freshwater (0 ppt) SS 14.383.56 7.751.47
SL 7.961.47 10.561.42
LL 2.750.38 P 0.05 9.941.19
16 ppt SS 0.660.08 16.692.3
SL 0.660.07 15.261.8
LL 1.370.13 P 0.005 8.770.22 P 0.05
32 ppt SS 0.220.11 15.02.38
SL 0.150.04 16.741.04
LL 0.120.04 14.321.72
Values for Expression and Mass are means 1SE, respectively. Signicance values are the results of Tukeys post hoc multiple
comparison tests. For instance, in the freshwater treatment, genotype LL exhibits signicantly less Prl 1 expression than the other two
genotypes, which do not differ signicantly from one another. SS, short alleles; LL, long alleles; SL, heterozygotes; ppt, parts per thousand.
3 MICROSATELLITES AND GENE EXPRESSION
Physiol Genomics VOL 9 www.physiolgenomics.org

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regulated genes suggests an under-appreciated role in
the ne-scale modulation of gene expression.
Research was supported by NSF/Alfred P. Sloan Foundation
Grant DBI-9803946 and USDA/NRICGP Grant 00352059267 to J. T.
Streelman.
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