1. Three differentially expressed survivin cDNA variants encode proteins with distinct antiapoptotic functions
by Edward M. Conway, Saskia Pollefeyt, Jan Cornelissen, Inky DeBaere, Marta Steiner-Mosonyi, Kelly Ong, Mathijs Baens, Désiré Collen, and Andre C. Schuh
Blood
Volume 95(4):
February 15, 2000
©2000 by American Society of Hematology

2. FISH analysis for chromosomal localization of murinesurvivin locus
FISH analysis for chromosomal localization of murinesurvivin locus.Lymphocytes were isolated from mouse spleen and cultured as detailed in “Materials and Methods.” A biotin-labeled PAC DNA clone containing the entire survivin gene was hybridized with metaph...
FISH analysis for chromosomal localization of murinesurvivin locus.Lymphocytes were isolated from mouse spleen and cultured as detailed in “Materials and Methods.” A biotin-labeled PAC DNA clone containing the entire survivin gene was hybridized with metaphase lymphocyte chromosomes. FISH signals and the DAPI banding pattern were recorded separately, and the assignment of FISH mapping data with chromosomal bands was achieved by superimposing FISH signals with DAPI-banded chromosomes. In the left panel, dots represent the specific FISH signals detected on chromosome 11. The right panel shows the same mitotic figure, stained with DAPI to identify mouse chromosome 11.
Edward M. Conway et al. Blood 2000;95:
©2000 by American Society of Hematology

3. Comparison of human survivin and murine survivin
Comparison of human survivin and murine survivin.The predicted amino acid sequences of murine survivin140(derived from cDNA clones 5.25, 92.16, 41.1, and 42.5), survivin121 (derived from cDNA clones 7HB and 7L .15), and survivin40 (derived from cDNA clones ...
Comparison of human survivin and murine survivin.The predicted amino acid sequences of murine survivin140(derived from cDNA clones 5.25, 92.16, 41.1, and 42.5), survivin121 (derived from cDNA clones 7HB and 7L .15), and survivin40 (derived from cDNA clones 5.20, 91.1, and 5.24) are aligned with the amino acid sequence of human survivin.
Edward M. Conway et al. Blood 2000;95:
©2000 by American Society of Hematology

4. Sequence of the murine survivin gene
Sequence of the murine survivin gene.The λ phage genomic DNA clone containing all 4 exons of murinesurvivin was sequenced on both strands.
Sequence of the murine survivin gene.The λ phage genomic DNA clone containing all 4 exons of murinesurvivin was sequenced on both strands. The 4 exons, with flanking intron sequence, are shown. Each exon is numbered, and the DNA encoding exons 1 to 4 are in uppercase letters. The predicted amino acid sequence of survivin140, derived from exons 1 to 4 is also shown. Survivin121 is predicted to be derived from retention of intron 3, whereas survivin40 is derived from pre-mRNA splice removal of exon 2. Underlined DNA sequence represents the only region of murine survivin cDNA without homology to putative cDNA of human EPR-1.
Edward M. Conway et al. Blood 2000;95:
©2000 by American Society of Hematology

5. Murine survivin gene and cDNAs
Murine survivin gene and cDNAs.Exons 1 to 4 are denoted in red, light green, blue, and green, respectively.
Murine survivin gene and cDNAs.Exons 1 to 4 are denoted in red, light green, blue, and green, respectively. Intron 3 is gray, and the 3′ flanking region is pink. (A) Restriction map of murine survivin gene. The arrow indicates the ATG translational start site. Restriction enzyme sites are shown: K (Kpn1); Stu (Stu1); Spe (Spe1); B (BglII); RV (EcoRV); A (Asc1); S (Sac1); P (Pml1); Apa (Apa1); Bc (Bcl1). Location of DNA probes used for Southern blots are shown. (B) Exon organization of the 3 distinct cDNAs encoding the predicted survivin proteins. The arrow indicates the start of translation. The heavy black vertical line represents the end of the coding region. Survivin140 is derived from all 4 exons. Survivin40 is generated by removal of exon 2-derived sequences, with a resultant in-frame stop codon, and a truncated protein. Survivin121 is derived from exons 1 to 3, and a short part of intron 3, the latter being presumably retained during pre-mRNA processing.
Edward M. Conway et al. Blood 2000;95:
©2000 by American Society of Hematology

6. Tissue distribution of murine survivin mRNAs
Tissue distribution of murine survivin mRNAs.RT-PCR was performed on mRNA derived from adult tissues using primers that flanked the specific survivin mRNAs.
Tissue distribution of murine survivin mRNAs.RT-PCR was performed on mRNA derived from adult tissues using primers that flanked the specific survivin mRNAs. (A) Accumulation of mRNA for survivin140 was detected in thymus and testis (primer pair srv86 and srvas311 results in amplicon of 254 bp). Positive controls using cDNA clones 5.20 and 5.25 (see Figure 2) as target DNA, are in 2 lanes at the right side, demonstrating prominent 144 bp and 254 bp bands, respectively. (B) Survivin121 mRNA was detected in all tissues (primer pair srv86 and srvas6380 results in amplicon of 332 bp). Survivin40 mRNA using primer pairs srv86 and srvas311, or srv86 and srvas6380 (expecting amplicons of 144 bp or 222 bp, respectively) was not detectable. (C) HPRT transcripts were present in all tissues (221 bp). The right lane in panels B and C are negative controls without target DNA.
Edward M. Conway et al. Blood 2000;95:
©2000 by American Society of Hematology

7. Developmental expression of murine survivin mRNA
Developmental expression of murine survivin mRNA.RT-PCR was performed on mRNA derived from murine embryos at different developmental time points ( dpc) using oligonucleotide primers as noted in Figure 5.
Developmental expression of murine survivin mRNA.RT-PCR was performed on mRNA derived from murine embryos at different developmental time points ( dpc) using oligonucleotide primers as noted in Figure 5. (A and B) All 3 survivin mRNA species were detected, although survivin140 mRNA (254 bp) was the most prominent. (C) HPRT transcripts were present at all developmental time points (221 bp). These results are representative of experiments on 3 embryos at each time point.
Edward M. Conway et al. Blood 2000;95:
©2000 by American Society of Hematology

8. Immunoprecipitation of murine survivin from embryos. Lysates of 12
Immunoprecipitation of murine survivin from embryos.Lysates of 12.5 dpc embyros were biotinylated and immunoprecipitated with anti-survivin antibodies (lane 1) or nonspecific antibodies (lane 2).
Immunoprecipitation of murine survivin from embryos.Lysates of 12.5 dpc embyros were biotinylated and immunoprecipitated with anti-survivin antibodies (lane 1) or nonspecific antibodies (lane 2). Following separation by SDS-PAGE and transfer to nitrocellulose membranes, filters were incubated with streptavidin-HRP for detection with the ECL kit (Amersham), as detailed in “Materials and Methods.” Molecular weight markers are on the left. Bands representing survivin140 and survivin121 are visualized at approximately 16 kd and 14 kd, respectively.
Edward M. Conway et al. Blood 2000;95:
©2000 by American Society of Hematology

9. Expression of human survivin mRNA forms
Expression of human survivin mRNA forms.Prior to separation by agarose gel electrophoresis, nested RT-PCR was performed on mRNA derived from the following human cell lines and tissues: 1.
Expression of human survivin mRNA forms.Prior to separation by agarose gel electrophoresis, nested RT-PCR was performed on mRNA derived from the following human cell lines and tissues: 1. K562 leukemia cell line; 2. Jurkat T-cell line; 3. germ-cell tumor derived cell line; 4. teratocarcinoma cell line; 5. neuroblastoma cell line; 6-9. malignant tumor from patients with MALT lymphoma; 10. AS19 melanoma cell line; 11. Del leukemia cell line; 12. SW620 colon cancer cell line; 13. Hep3B hepatoma cell line; 14. Ht1080 neurofibrosarcoma cell line; 15. A431 carcinoma cell line; 16. Raji leukemia cell line. (A) Ethidium bromide-stained agarose gel of nested RT-PCR products generated with oligonucleotide primers hsrv2861 and hsrv5431. Transcripts correspond to full-length murine survivin140. (B) Southern blot using32P-labeled hsrv2888 to detect specific nested RT-PCR products generated using oligonucleotide primers hsrv2861 and hsrv5159. Several cell lines express the specific transcript corresponding to murine survivin121, whereas only the neuroblastoma cell line expresses a transcript corresponding to murine survivin40.
Edward M. Conway et al. Blood 2000;95:
©2000 by American Society of Hematology