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All of the sequences used for our alignments are available through GenBank as either unannotated or annotated BAC sequence, complete cDNA sequence, or complete gene sequence.

The original P450 multiple sequence alignment used in Paquette et al. contains 134 full-length Arabidopsis cytochrome P450 sequences, with intron phases marked (see below about phase markings). It was generated using ClustalW from GCG10 for UNIX. The following parameters were used for this alignment:



Gap Opening Penalty	10.00
Gap Extension Penalty	0.20
Delay divergent sequences	30%
DNA Transitions Weight	0.50
Protein weight matrix	BLOSUM series
DNA weight matrix	IUB
Use negative matrix	OFF


The most current P450 alignment contains 246 full-length amino-acid sequences. It was generated using ClustalX 1.83 for Windows, with the following parameters:



Pairwise Alignment parameters
Pairwise Alignments	Slow-accurate
Gap Opening Penalty	12.50
Gap Extension Penalty	0.50
Protein weight matrix	Gonnet 250
DNA weight matrix	IUB
Multiple Alignment parameters
Gap Opening Penalty	12.50
Gap Extension Penalty	0.50
Delay divergent sequences	30%
DNA Transitions Weight	0.50
Protein weight matrix	Gonnet series
DNA weight matrix	IUB
Use negative matrix	OFF



(The version presented here does not have the intron positions marked.)

The alignment was considered optimal if key regions of the P450s aligned properly. These regions were the heme-binding domain, the PERF domain, and the K-helix (KETLR). The universally-conserved cystein of the heme-binding domain was considered particularly important.

The UGT alignment used to produce Figure 4 in Paquette, Møller, and Bak was made using the following parameters:



Pairwise Alignment parameters
Pairwise Alignments	Slow-accurate
Gap Opening Penalty	10.00
Gap Extension Penalty	0.08
Protein weight matrix	Gonnet 250
DNA weight matrix	IUB
Multiple Alignment parameters
Gap Opening Penalty	10.00
Gap Extension Penalty	0.20
Delay divergent sequences	35%
DNA Transitions Weight	0.50
Protein weight matrix	BLOSUM series
DNA weight matrix	IUB
Use negative matrix	OFF



The alignment was considered optimal if the PSPG and UGT-defining sequence motif aligned properly.

Intron phases are marked on the alignment with regard to the codon in question as follows:

[ - phase 1
] - phase 2
| - phase 0

A sequence such as KET[LR means that the threonine codon has a splice site after the first base. KET]LR means the splice site lies after the second base of the threonine codon, and KET|LR means the splice site lies directly between the threonine and lysine codons.

Introns were mapped using a combination of the Technical University of Denmark's NetPlantGene prediction server and general sequence conservation among families and subfamilies. In cases where intron splice-site prediction and family sequence conservation were opposed, conservation of similarity to other family and/or sub-family members took precedence.

The original P450 multiple sequence alignment was made using ClustalW for the GCG Wisconsin package, version 10. Shading was done using Boxshade for GCG version 10. The PDF file was made using Adobe Acrobat Distiller version 4, with Microsoft Word 97.

Current aligmments are made with ClustalX 1.83 for Windows 32 environment, and shaded using a customized Boxshade program that can take up to 300 sequences. Current alignment PDF files are generated with Adobe Acrobat Distiller version 5-7 through Microsoft Word 2000 and 2003. All PDFs should be compatible with Adobe Acrobat Reader versions 4 to present.

All intron splice site prediction was done using the NetPlantGene prediction server.

Intron-exon organization and phylogeny in a large superfamily, the paralogous cytochrome P450 genes of Arabidopsis thaliana.
  Paquette SM, Bak S, Feyereisen R.
  DNA and Cell Biology (2000) May 19:307-17

On the Origin of Family 1 Plant Glycosyltransferases.   Paquette SM, Lindberg Møller B, Bak S.
  Phytochemistry (2003) 62:399-413

There is an intron position that is correctly marked in the pdf that is missing in Figure 2 of the paper. This intron position is for CYP711A1, at the 586/587 mark on the PDF alignment. The updated file was uploaded on August 27th, 2000.

A-type P450 intron map
Phase 0 introns: |; Phase 1 introns: [; Phase 2 introns: ]. P450s lacking an intronmap are intronless. Bootstrap values are out of 1000 trials. *: The genomic sequence for CYP84A4 is currently not unavailable.

Non-A type P450 intron map
Phase 0 introns: |; Phase 1 introns: [; Phase 2 introns: ]. P450 groups thought to comprise individual clades are colored. P450s lacking an intronmap are intronless. Bootstrap values are out of 1000 trials.

UGT intron map
Phase 0 introns: |; Phase 1 introns: [; Phase 2 introns: ]. UGTs lacking an intronmap are intronless. A BLOSUM protein weight matrix series was used to align the sequences and the tree was analyzed with 1000 bootstrap trials.

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