Genomic cloning, chromosomal mapping, and expression analysis
Ju¨rgen Kohlhase,* Mariele Altmann,* Leticia Archangelo, Christa Dixkens, Wolfgang Engel
Institut fu¨r Humangenetik der Universita¨t Go¨ttingen, Heinrich-Du¨ker-Weg 12, D-37073 Go¨ttingen, Germany
Received: 1 June 1999 / Accepted: 26 August 1999
Abstract. Mutations of SALL1 related to spalt of Drosophila
have been found to cause Townes-Brocks syndrome, suggesting a
function of SALL1 for the development of anus, limbs, ears, and
kidneys. No function is yet known for SALL2, another human
spalt-like gene. The structure of SALL2 is different from SALL1
and all other vertebrate spalt-like genes described in mouse, Xeno-
pus, and Medaka, suggesting that SALL2-like genes might also
exist in other vertebrates. Consistent with this hypothesis, we iso-
lated and characterized a SALL2 homologous mouse gene, Msal-
2. In contrast to other vertebrate spalt-like genes both SALL2 and
Msal-2 encode only three double zinc finger domains, the most
carboxyterminal of which only distantly resembles spalt-like zinc
fingers. The evolutionary conservation of SALL2/Msal-2 suggests
that two lines of sal-like genes with presumably different functions
arose from an early evolutionary duplication of a common ancestor
gene. Msal-2 is expressed throughout embryonic development but
also in adult tissues, predominantly in brain. However, the function
of SALL2/Msal-2 still needs to be determined.
The region-specific homeotic gene spalt (sal)ofDrosophila me-
lanogaster is required for the specification of larval posterior head
and anterior tail structures during embryogenesis (Ju¨rgens 1988).
Furthermore, sal plays an important role in the embryonic devel-
opment of the larval tracheal system and the adult wing morpho-
genesis (de Celis et al. 1996; Ju¨rgens 1988; Ku¨hnlein and Schuh
1996; Lecuit et al. 1996; Nellen et al. 1996; Sturtevant et al. 1997).
sal belongs to the group of C
zinc finger transcription factors
and is characterized by a distinct molecular structure with three
double zinc finger domains distributed over the entire protein
(Ku¨hnlein et al. 1994). sal-related genes of Xenopus laevis (Xsal-
1), mouse (Msal), Medaka fish (Medaka sal), and human (SALL1,
SALL2) have been described (Hollemann et al. 1996; Kohlhase et
al. 1996; Ko¨ster et al. 1997; Ott et al. 1996). Mutations of SALL1
have been found to cause Townes-Brocks syndrome (MIM
#104780), an autosomal, dominantly inherited malformation syn-
drome, thereby suggesting an important developmental regulatory
function for SALL1 (Kohlhase et al. 1998).
The other human sal-like gene (SALL2) has a different struc-
ture compared with SALL1 and the other sal-like vertebrate genes
(Kohlhase 1996; Kohlhase et al. 1996), and the degree of similarity
between SALL2 and SALL1 is not higher than between SALL2
and Msal or Xsal-1 (Kohlhase 1996). While Msal (Ott et al. 1996),
Xsal-1 (Hollemann et al. 1996), and SALL1 encode four double
zinc finger units of the SAL-type, SALL2 encodes only three.
Also, in contrast to Msal, Xsal-1, and SALL1, SALL2 does not
contain a 3Ј intron (Kohlhase et al. 1996). The most carboxyter-
minal zinc finger domain in SALL2 consists of 51 amino acids
instead of 49 in the typical SAL double zinc finger, and it does
not contain the “SAL box”, a sequence of eight amino acids
(FTTKGNLK) characteristic for SAL zinc fingers (Ku¨hnlein et al.
1994). However, the remaining zinc finger domains, as well as the
structure of the predicted protein, are very similar to SAL, indi-
cating that SALL2 is a true member of the sal-like gene family
(Kohlhase et al. 1996). In order to investigate whether the char-
acteristic structure of SALL2 was conserved during evolution as
suggested by comparison with Msal, Xsal-1, and SALL1, we
searched for a SALL2 homologous gene in mouse. Here we report
the isolation, characterization, and chromosomal localization of
this gene (Msal-2).
Materials and methods
Genomic library screening and DNA analysis.
of a lambda FIX II mouse genomic library of the mouse strain 129 (Strata-
gene, La Jolla, Calif., USA) were hybridized with a
DNA fragment. Hybridization was carried out overnight at 60°C. Filters
were washed1×20minatroom temperature (RT) in 2 × SSPE, 0.1% SDS,
followed by2×20minat60°C in 2 × SSPE, 0.1% SDS. Hybridizing
phage clones were isolated, restriction mapped, partly subcloned into
pBluescript KS and sequenced by the dideoxy chain termination method on
an ABI 377 automated sequencer according to the Dye terminator protocol
(Applied Biosystems, Neu Isenburg). Screening procedures, DNA prepa-
ration, Southern blotting, restriction analysis, and subcloning procedures
were performed as described (Sambrook et al. 1989). Larger genomic
fragments were obtained by high stringency screening (according to stan-
dard procedures) of a computer-spotted cosmid library of the mouse strain
129ola with a
P-labeled Msal-2 fragment. Computer-spotted library fil-
ters (library no. 121) as well as cultures of isolated clones were supplied by
the Resource Center of the German Human Genome Project (RZPD; Ber-
lin, Germany). For isolation of Msal-2 exon 1, the restriction-digested
cosmid DNA was Southern blotted onto Nylonbind A membranes (Serva,
Heidelberg, Germany) and hybridized with a SALL2 exon 1 cDNA frag-
ment. Hybridization and washing conditions (reduced stringency) were as
described above for phage library screening.
Total RNA was isolated from different adult mouse-
tissues (brain, heart, liver, lung, spleen, skeletal muscle, kidney, ovary, and
testis) with Total RNA Isolation Reagent according to the manufacturer’s
instructions (Biomol, Hamburg). Approximately 20 g of total RNA of
each tissue was used for Northern blotting. Treatment of the RNA, elec-
trophoresis, and blotting procedures were performed as described (Sam-
brook et al. 1989). The filter was hybridized with a 2.4-kb BamHI genomic
DNA fragment (
P-labeled) of Msal-2, which contains the first and the
second double zinc finger domain. For testing of RNA integrity and load-
ing amount, the blot was rehybridized with a
P-labeled 1.6-kb BamHI/
BgIII fragment of human elongation factor 2 cDNA (Rapp et al. 1989).
Both hybridization steps were performed overnight at 42°C in a hybrid-
* Both authors contributed equally to this work.
Correspondence to: J. Kohlhase, email: firstname.lastname@example.org
Sequence data from this article have been deposited with the EMBL /
GenBank Databases under Accession No. AJ007396.
Mammalian Genome 11, 64–68 (2000).
© Springer-Verlag New York Inc. 2000