To explore the structural basis for regulation of human serotonin tran
sporter (hSERT) gene expression, we used primer extension and 5' rapid
amplification of cDNA ends (5'RACE) techniques to estimate levels of
and identify 5'-noncoding elements of hSERT mRNAs and genomic cloning
to place these elements within the overall map of the hSERT gene. Prim
er extension on JAR cell mRNA suggested the presence of significant hS
ERT mRNA sequence upstream of the 5' end of our cloned hSERT cDNA. Usi
ng 5'RACE and reverse transcription-PCR (RT-PCR) methodologies, we clo
ned these sequences from brain and placenta and found this material to
be composed of alternatively spliced exons using a previously reporte
d noncoding exon (1A) and a novel 97-bp noncoding exon (1B). RT-PCR of
JAR cell mRNA blotted with exon-specific oligonucleotides revealed bo
th exons 1A and 1B to be regulated in a cholera toxin-dependent manner
. To clarify the structure of the hSERT gene including exon 1B, we iso
lated and characterized genomic hSERT clones from Lambda Fix II and P1
artificial chromosome libraries. In agreement with previous findings,
a single hSERT gene was identified that accounts for hybridizing band
s on genomic Southern blots and was found to utilize 13 exons to encod
e the transporter's coding sequences along with the two noncoding 5' e
xons. Exon 1B was identified similar to 14 kb downstream of exon 1A in
the hSERT gene and 737 bp upstream of exon 2, where the initiation si
te for translation is located. Exon 1B is surrounded by several elemen
ts potentially suitable for regulating serotonin transporter gene expr
ession in vivo, including consensus sites for transcription factors AP
-1, AP-2, CREB/ATF, and NF-kappa B. These data reveal additional compl
exity in hSERT gene structure and expression that may be relevant to r
egulated and compromised transporter expression in vivo.