Supplementary online materials and methods
Gene identification and cDNAs
Four novel murine transcription units represented by the IMAGE clone 3484221, the RIKEN
clones 4933433J03 and 2010001M06, and the BMAP clone UI-R-C3-tr-h-11-0-UI and not
described in a previous work 1 were identified by the generation of a comparative analysis of ~3.3
Mb of mouse genomic sequence orthologous to the human 7q11.23 region 2. However a
comparative PipMaker analysis does not allow to precisely define the intron-exon boundaries of a
gene resulting sometimes in prediction of a putative cDNA that if translated encodes a truncated
protein. Moreover as the mouse genomic sequence was used as the reference sequence such a
comparison will mask gene duplications in the human genome. A fifth and a sixth gene
correspond to two new clusters of ESTs represented by IMAGE clone 4950109 and RIKEN clone
4930563C21, that were mapped to the mouse region syntenic to the WBS commonly deleted
region. To overcome the above-cited problems and clone these genes, we first screened the
human and mouse dbEST databases using the above reported clone sequences
(http://www.ncbi.nlm.nih.gov/BLAST/). Second, we created clusters with the matching ESTs.
These contigs were possibly extended 5’ and 3’ by rescreening of dbEST. This step was repeated
until no new matching ESTs were found 3,4. Third, we fully sequenced at least one representative
EST clone. cDNA clones were acquired from three different sources: the IMAGE and BMAP
collections (obtained from Research Genetics, http://www.resgen.com); and the NIA 15K Mouse
cDNA clone set 5, provided by The National Institute on Aging (http://www.nia.nih.gov). The
following ESTs were fully sequenced: WBSCR24: human IMAGE clone 5181751 and mouse
IMAGE clone 3484221; WBSCR25: murine IMAGE clones 602660 and 602041; WBSCR26:
murine IMAGE clones 336334 and 332395 and human IMAGE clone 5422797; WBSCR27:
murine IMAGE clones 6309628 and 4950109; TRIM50: rat BMAP clone UI-R-C3-tr-h-11-0-UI).
The ORF of Trim50 and Wbscr28 were amplified by RT-PCR from mouse testis RNA with the
following pair of primers: CCGCCGCAATTGATGGCGTGGCGGCTGACT and
CCGCCGCTCGAGCTATACCTGTCCCAGTGT and TTGTACCGCTTTCTGCTCCT and
AGGTAGAGGCGTCTCAGCAG, respectively. The three TRIM50-like human copies were
identified by blastn comparison with the public human genome and subsequent correction of
copies B and C using the Genbank entry AW964178 (MAGE EST376251). To identify the 5’end
of the TRIM50-like genes 5’Rapid Amplification of cDNA Ends was performed on polyA+ RNAs
from 12 human tissues (brain, heart, kidney, spleen, liver, colon, small intestine, muscle, lung,
stomach, testis, placenta, all BD Clontech) using the BD SMARTTM RACE cDNA amplification
kit (BD Clontech Cat. No.634914) with two different oligonucleotides hybridizing to all three
copies or specific for TRIM73 and TRIM74 (AGAGGCCACAGATGAGCTCCTGGTC
and TGGAGGAACCAGTGGAGCTGAGATG, respectively) as described 6.
References:
1.
Merla G, Ucla C, Guipponi M, Reymond A: Identification of additional transcripts in the
Williams-Beuren syndrome critical region. Hum Genet 2002; 110: 429-438.
2.
DeSilva U, Elnitski L, Idol JR et al: Generation and comparative analysis of
approximately 3.3 Mb of mouse genomic sequence orthologous to the region of human
chromosome 7q11.23 implicated in Williams syndrome. Genome Res 2002; 12: 3-15.
3.
Reymond A, Camargo AA, Deutsch S et al: Nineteen additional unpredicted transcripts
from human chromosome 21. Genomics 2002; 79: 824-832.
4.
Reymond A, Friedli M, Henrichsen CN et al: From PREDs and open reading frames to
cDNA isolation: revisiting the human chromosome 21 transcription map. Genomics 2001;
78: 46-54.
5.
Kargul GJ, Dudekula DB, Qian Y et al: Verification and initial annotation of the NIA
mouse 15K cDNA clone set. Nat Genet 2001; 28: 17-18.
6.
Denoeud F, Kapranov P, Ucla C et al: Prominent use of distal 5' transcription start sites
and discovery of a large number of additional exons in ENCODE regions. Genome Res
2007; 17: 746-759.
Supplementary online table
Table S1: Northern blot results
Gene
Genbank size
1
Northern approximate size
2000
2
Highly expressed in
Expressed in
stomach
testis
Trim50
1452
Wbscr24
1443
2000, 1800, 1600, 1400
brain, kidney, skin
Wbscr25
2047
3200, 2100
testis
Wbscr26
735
1200, 700
Wbscr27
2831
2900, 2100, 1500
933
900
Wbscr28
1
size of longer Genbank deposited transcript [bp]
2
approximate size(s) of Northern blot positive bands [bp]
small intestine, kidney, liver, testis
kidney, liver
testis
Rare transcript in
stomach, testis, thymus, liver
brain
heart
heart, skin
lung, skin, small intestine, thymus
spleen, testis
heart