Conserved synteny screening
We used the following list of mouse sequence tags from [GenBank:NC_000078.5]: a 20 bp tandem repeat (114493451-114493793), an alpha locus portion (114496952-114497890) and seven enhancers, that is HS1.2 (114483000-114483159), HS3.A (114470545-114470747), HS3.B (114492057-114492261), HS4 (114466607-114466729), and HS5, HS6 and HS7. Those last three were localized by their primers sequences, listed in previous paper.
These sequences were aligned by BLAT (BLAST-Like Alignment Tool, http://genome.ucsc.edu/cgi-bin/hgBlat) versus the 2007 release of the Mus musculus (mouse) genome, to identify the limits of the mouse 3'RR contig (chr12:114,459,657-114,497,890 in mmu9 draft; size 38234 bp)(Figure 3). This region was used to investigate the Amniota species of which the sequenced genome was available in the "Comparative Genomics" group of tracks at the UCSC mouse genome browser site http://genome.ucsc.edu/cgi-bin/hgGateway?db=mm9. So the list of the species we checked was in the order: rat (Rattus norvegicus, rn4), Guinea pig (Cavia porcellus, cavPor3), man (Homo sapiens, hg19), chimpanzee (Pan troglodytes, panTro2), orangutan (Pongo abelii, ponAbe2), macaque (Macaca mulatta, rheMac2), marmoset (Callithrix jacchus, calJac3), dog (Canis familiaris, canFam2), panda (Ailuropoda melanoleuca, ailMel1), horse (Equus caballus, equCab2), cow (Bos taurus, bosTau4), rabbit (Oryctolagus cuniculus, oryCun2), elephant (Loxodonta africana, loxAfr3), opossum (Monodelphis domestica, monDom5), platypus (Ornithorhynchus anatinus, ornAna1), lizard (Anolis carolinensis, anoCar1), chicken (Gallus gallus, galGal3). We reported in parenthesis the scientific name and the genome draft code.
Inspecting the homologous human region in the UCSC human genome browser http://genome.ucsc.edu/cgi-bin/hgTracks?db=hg18, we searched the tracks related to the Neanderthal genome  for the presence of HS3, HS1.2 and HS4.
Database searches for enhancers
We searched for clones with the mouse (see previous section) and human version of the Alpha portion, repeat and enhancers (Alpha, GenBank:NC_000014:106053245-106054732, 1488 bp; repeat, GenBank:NC_000014:106048991-106049802, 812 bp; HS3, GenBank:NC_000014:106048351-106048676, 326 bp; HS1,2, GenBank:NC_000014:106041545-106042009, 465 bp; HS4, GenBank:NC_000014:106032614-106032974, 361 bp), using the nucleotide version of BLAST (Basic Local Alignment Search Tool, http://blast.ncbi.nlm.nih.gov/Blast.cgi). The asked databases were from the "Others" group (i.e. limiting results to non-human and non-mouse records), further specifying in the Taxonomy field to exclude "Homo (taxid:9605)". The three checked databases were: refseq_genomic (fully-sequenced genome entries from NCBI's Reference Sequence project); wgs (whole genome shotgun sequence); htgs (high throughput genomic sequences). To allow a better matching among so divergent species, especially among their 3'end tails, we chose a non-standard set of parameters: Match/Mismatch Scores 1/-1; Existence/Extension Gap Costs 0/2. Then we chose from the BLAST output the relevant sequences (see Table 1 for the list of species with sequences similar to the human queries) by empirically setting the "Expect" threshold-value to 1e-05.
Dot plot analysis
We identified 2 genomic contigs of Homo sapiens (3'RR1, [GenBank:NC_000014.8:106152458-106175002], and 3'RR2, [GenBank:NC_000014.8:106032614-106054732]), and one contig of Mus musculus [GenBank:NC_000078.5:114466607-114497890], Oryctolagus cuniculus [GenBank:AY386698.1:7160-21816] and Canis lupus familiaris [GenBank:AC187024.23:146314-165755]. Finally, we assembled a contig of Ailuropoda melanoleuca, sewing the scaffold3005_4 [GenBank:ACTA01092430.1:1574-6589] and the scaffold3005_5 [GenBank:ACTA01100430.1:1-7968] by a stretch of 600 N. We performed a series of pairwise comparisons among these six genomic contigs, by use of two dot plot analysis softwares: Gepard http://mips.gsf.de/services/analysis/gepard for the human versus human dot plots (Figure 4), and Blast2seq http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi for the human versus others species dot plots (Figure 5). Graphics were edited using Adobe Illustrator.
Database hunting for HS1.2 polymorphisms
The search for HS1.2 polymorphisms was refined using the specialized alignment tool Trace-Archive BLAST http://blast.ncbi.nlm.nih.gov/Blast.cgi?BLAST_SPEC=TraceArchive&BLAST_PROGRAMS=megaBlast&PAGE_TYPE=BlastSearch. The Trace Archive http://www.ncbi.nlm.nih.gov/Traces/trace.cgi is a repository of sequencing data from gel/capillary platforms, partitioned by genome and sequencing methods. There is a daily-growing amount of available reads from thousands of WGS projects. We performed a BLAST search on every available database referring to species identified in the previous analysis for the presence of this enhancer and to the 24 primates available in the Archive (Aotus nancymaae, Ateles geoffroyi, Callicebus moloch, Callithrix jacchus, Cercopithecus aethiops, Colobus guereza, Eulemur macaco, Gorilla gorilla, Homo sapiens, Hylobates concolor, Lemur catta, Macaca fuscata, Macaca mulatta, Microcebus murinus, Nomascus leucogenys, Otolemur garnettii, Pan paniscus, Pan troglodytes, Papio anubis, Papio cynocephalus, Papio hamadryas, Pongo pygmaeus abelii, Saimiri boliviensis, Tarsius syrichta). The sequences detected were selected for HS1.2 completeness and multi-aligned by use of ClustalW (http://www.ebi.ac.uk/Tools/clustalw2/index.html) and MAFFT (http://align.bmr.kyushu-u.ac.jp/mafft/software/) and by use of Seaview http://pbil.univ-lyon1.fr/software/seaview.html to manual revise the ClustalW/MAFFT outputs. See Figure 6 for a graphical representation of the detected HS1.2 variants.
Transfac analysis for transcription factors
The search for the transcription factor consensus was performed on the variant sequences of HS1.2 by the software AliBaba2.1 http://www.gene-regulation.com/pub/programs/alibaba2/index.html. Additional file 1 lists the transcription factors detected at least in ten loci. The full list can be inspected as Additional file 2.
Sequences of IgH constant alpha genes and of the enhancers HS3, HS1.2, HS4 retrieved after BLAST search, were used for the phylogenetic analysis (Figure 7; accession numbers and limits reported in Additional file 3). Multiple alignments of the sequences were obtained with Opal and the results were manually inspected. The best-fitting substitution model was selected using ModelGenerator , under the Akaike information criterion (AIC1), as implemented in MultiPhyl online. The following models were integrated in the phylogenetic analysis: GTR + I + G for C-alpha; HKY + I for HS3 and HS4; HKY + G for HS1.2.
An unrooted tree was constructed using the maximum likelihood method applied to nucleotides, as implemented in Garli version 0.96 http://www.bio.utexas.edu/faculty/antisense/garli/Garli.html, with bootstrap percentages obtained as a consensus after 100 replicates.