Supplemental Material for:

MultiPipMaker and Supporting Tools: Alignments and analysis of multiple genomic DNA sequences

Scott Schwartz, Laura Elnitski, Mei Li, Matt Weirauch, Cathy Riemer, Arian Smit, NISC Comparative Sequencing Program, Eric D. Green, Ross C. Hardison, and Webb Miller.   Submitted.

Guide to using MultiPipMaker

MultiPipMaker uses an input format like that of the PipMaker server. The user enters the number of sequences for comparison on the first page, and MultiPipMaker returns a second page for entering exactly the requested number of sequences. The user assigns a single-word name for each sequence (used to identify the sequence row in the alignment) and submits sequence files via a browser function. Additional files can be submitted describing locations of genes and exons in the first (reference) sequence, positions of interspersed repeats for masking and annotation (generated by RepeatMasker, run locally by the user or at the server, positions of color underlays for various features, and other annotations. The color underlay for the first sequence can be displayed as the default in every panel of the alignment, or custom underlay files can be submitted for each panel, provided that the coordinates are relative to the first sequence. User specified options for sequences other than the primary one include the choice of searching one or both strands, the amount of coverage desired (show all matches, chaining, and single coverage), and higher sensitivity but with a lower time limit. The user also selects the format of output files that accompany a MultiPip, including a nucleotide level alignment displayed as a PDF file and a textual alignment that is machine readable. These options are described fully at the MultiPipMaker web server and in Elnitski et al. (1).

Follow this link to see an example of MultiPipMaker, with sample input and output files.

Transient transfection and expression assays

To test the hypothesis that strongly conserved noncoding sequences in the introns of WNT2 are involved in gene regulation, we measured their enhancing or silencing effects on a heterologous promoter (the SV40 early promoter), which is active in many cell lines, including human HeLa and 293 cells. The fibroblastic HeLa cells have been used in many enhancer assays (e.g. 2), whereas the SV40 promoter is expressed at a high level without enhancers in 293 kidney epithelial cells (3), which constitutively express the adenovirus activator E1A (4). The segments of the human WNT2 gene that aligned with other mammalian species at the highest percent identity and gap-free length (over 400 bp), CNC1 and CNC2, were amplified by PCR and cloned into the parental reporter plasmid pGL3-promoter from Promega, generating test plasmids pGL3-CNC1 and pGL3-CNC2. The parental plasmid contains a modified firefly luciferase gene (5) whose transcription is driven by the SV40 early promoter, but it lacks an enhancer. The plasmid pGL3-control, which has the SV40 enhancer inserted into pGL3-promoter, served as the positive control, and the plasmid pGL3-basic, which lacks a promoter, served as a negative control. The test and control plasmids were transfected in HeLa and 293 cells using the cationic lipid Tfx-20(tm) from Promega, both in titrations of DNA (data not shown) and as triplicate transfections at 0.25 micrograms DNA per ml (6). In the latter case, the reporter constructs (expressing the firefly luciferase gene) were co-transfected with an additional control plasmid that expresses the Renilla luciferase gene. Expression from the latter plasmid controls for any variation in transfection efficiency. Cells were harvested 48 hr after transfection, and cell extracts were assayed for firefly and Renilla luciferase activity, following protocols from the manufacturer of the assay reagents (Promega). The means and standard errors of the ratios (firefly luciferase / Renilla luciferase) were computed.


1.Elnitski, L., Riemer, C., Schwartz, S., Hardison, R. and Miller, W. (2003) PipMaker: a World Wide Web server for genomic sequence alignments. Current Protocols in Bioinformatics, Wiley and Sons, Inc., New York, pp. in press.
2.Banerji, J., Rusconi, S. and Schaffner, W. (1981) Expression of a beta-globin gene is enhanced by remote SV40 DNA sequences. Cell, 27, 299-308.
3.Robbins, P.D., Rio, D.C. and Botchan, M.R. (1986) Trans activation of the simian virus 40 enhancer. Mol Cell Biol, 6, 1283-1295.
4.Imperiale, M.J., Feldman, L.T. and Nevins, J.R. (1983) Activation of gene expression by adenovirus and herpesvirus regulatory genes acting in trans and by a cis-acting adenovirus enhancer element. Cell, 35, 127-136. Wet, J.R., Wood, K.V., DeLuca, M., Helinski, D.R. and Subramani, S. (1987) Firefly luciferase gene: Structure and expression in mammalian cells. Mol Cell Biol, 7, 725-737.
6.Elnitski, L., Li, J., Noguchi, C.T., Miller, W. and Hardison, R. (2001) A negative cis-element regulates the level of enhancement of hypersensitive site 2 of the b-globin locus control region. J Biol Chem, 276, 6289-6298.