1. Functional and comparative analysis of globin loci in pufferfish and humans
by Nynke Gillemans, Tara McMorrow, Rita Tewari, Albert W. K. Wai, Carola Burgtorf, Dubravka Drabek, Nicki Ventress, An Langeveld, Douglas Higgs, Kian Tan-Un, Frank Grosveld, and Sjaak Philipsen
Blood
Volume 101(7):
April 1, 2003
©2003 by American Society of Hematology

2. Alignments of human and Fugu α- and β-globin proteins
Alignments of human and Fugu α- and β-globin proteins.The (deduced) amino acid sequences of human (h) and Fugu (f) alpha and beta globins were aligned with the multalin program.21 The alignments are displayed with the aid of the boxshade program to illustra...
Alignments of human and Fugu α- and β-globin proteins.The (deduced) amino acid sequences of human (h) and Fugu (f) alpha and beta globins were aligned with the multalin program.21 The alignments are displayed with the aid of the boxshade program to illustrate the classification of theFugu proteins as α- or β-globins. Residues found specifically in α- or β-type globins are shown by color. The consensus line is for all 6 globins: . indicates moderately conserved residue; :, well-conserved residue; *, identical residue in all 6 globins.
Nynke Gillemans et al. Blood 2003;101:
©2003 by American Society of Hematology

3. Globin loci of the pufferfish F rubripes are located on different chromosomes.(A) Nuclei were prepared from peripheral blood of adultFugu, fixed onto poly-L-lysine–coated slides and subjected to DNA-FISH with Fugu α- (red) and αβ-cosmid (green) probes.
Globin loci of the pufferfish F rubripes are located on different chromosomes.(A) Nuclei were prepared from peripheral blood of adultFugu, fixed onto poly-L-lysine–coated slides and subjected to DNA-FISH with Fugu α- (red) and αβ-cosmid (green) probes. Four representative images, obtained by confocal scanning laser microscopy, are shown. The α- and αβ-cosmid signals are always clearly separated from each other. (B) Metaphase spreads of kidney cells isolated from colchicein-treated Fugu rubripesfingerlings were hybridized with Fugu α-cosmid (red) and αβ-cosmid (green) probes. Two chromosomes from one metaphase spread are shown. Both probes hybridize to the telomeric end of the p arm of one of the Fugu chromosomes. The chromosome with the red α-cosmid signal is much larger that the chromosome with the green αβ-cosmid signal. Original magnification × 100.
Nynke Gillemans et al. Blood 2003;101:
©2003 by American Society of Hematology

4. The αβ-cosmid contains α- and β-globin genes
The αβ-cosmid contains α- and β-globin genes.Restriction mapping and Southern hybridization were used to determine the location of the β-globin gene, and an area of approximately 35 kb surrounding the gene was sequenced.
The αβ-cosmid contains α- and β-globin genes.Restriction mapping and Southern hybridization were used to determine the location of the β-globin gene, and an area of approximately 35 kb surrounding the gene was sequenced. BLAST searches and alignments revealed the presence of 3 putative globin genes. (A) The α3-, α4-, and β-globin genes are closely linked and directed in opposite transcriptional orientations. Arrows indicate the transcription start sites. Genes are shown as boxes with exons in black. (B) Promoter area of the α3-globin gene and (C) promoter area of the β-globin gene. The beginning of exon 1 is boxed. Putative TATA boxes and an inverted CACC motif are highlighted with a gray background.
Nynke Gillemans et al. Blood 2003;101:
©2003 by American Society of Hematology

5. DNaseI hypersensitive site mapping of the F rubripes αβ-globin gene domain.(A) Features of the F rubripes αβ-globin gene domain and strategy for DNaseI hypersensitive site mapping.
DNaseI hypersensitive site mapping of the F rubripes αβ-globin gene domain.(A) Features of the F rubripes αβ-globin gene domain and strategy for DNaseI hypersensitive site mapping. Arrows indicate the transcriptional direction of the genes. Restriction sites used to cut genomic F rubripes DNA are indicated: A,SacI; E, EcoRI; S, SphI; X,XhoI. The EcoRI sites were used to isolate the fragment for transgenesis. Probes for Southern hybridization were generated by PCR amplification and are labeled 1 to 7. Repetitive sequences are denoted by gray bars: T indicates homology with a human telomeric sequence (Z ); ?, unknown repetitive element with homology to yeast tRNA-met (AL121 795,); R, areas with multiple BLAST hits in the Fugu genome; R1, homology with reverse transcriptase/rex1 transposon.23 The positions of strong erythroid-specific hypersensitive sites are indicated by black arrowheads; weak hypersensitive sites are indicated by open arrowheads. (B-C) Examples of Southern blot analysis of DNaseI hypersensitive sites in F rubripes chromatin. Nuclei were isolated from the tissues indicated and treated with increasing amounts of DNaseI. DNA was purified, digested with the appropriate restriction enzymes, and subjected to Southern blotting. In panel B, the DNA was digested withSphI, and the blot was hybridized with probe 5. Strong hypersensitive sites coinciding with the promoters of the α3- and β-globin genes are indicated by arrows. Arrowheads indicate weaker sites. In panel C, the DNA was digested with SacI, and the blot was hybridized with probe 5. No hypersensitive sites are observed in blood, kidney, or liver (not shown).
Nynke Gillemans et al. Blood 2003;101:
©2003 by American Society of Hematology

6. Genes flanking human and pufferfish globin loci
Genes flanking human and pufferfish globin loci.Schematic drawings of the globin loci in human and pufferfish genomes.
Genes flanking human and pufferfish globin loci.Schematic drawings of the globin loci in human and pufferfish genomes. The human α-globin locus serves as the reference locus and is drawn to scale; flanking genes referred to in the text are color coded. Nomenclature is according to Flint et al.10Key to the color code: magenta indicates POLR3K; green, C16orf33; light orange, C16orf8; lavender,MPG; purple, C16orf35; red, globins; and peach,AANAT. Other genes are represented by dark gray; odorant receptor genes in the human β-globin locus are represented by light gray.
Nynke Gillemans et al. Blood 2003;101:
©2003 by American Society of Hematology

7. Comparative analysis of C16orf8-related genes in pufferfish and human
Comparative analysis of C16orf8-related genes in pufferfish and human.(A) C16orf8 homologs found in the pufferfish α- and αβ-loci, and the human cytoglobin locus on chromosome 17, were aligned with the C16orf8 gene of the human α-globin locus10 and visualiz...
Comparative analysis of C16orf8-related genes in pufferfish and human.(A) C16orf8 homologs found in the pufferfish α- and αβ-loci, and the human cytoglobin locus on chromosome 17, were aligned with the C16orf8 gene of the human α-globin locus10 and visualized with the VISTA program. Because of the large size of the first intron, the alignment starts near exon 2. The graphs depict homologies between 50% and 100%. (B) Homology distance matrix of the proteins encoded by theC16orf8 homologues of pufferfish and human. FLJ22341 on human chromosome 17 has an N-terminal truncation. Therefore, the matrix was calculated using the part that all 4 proteins have in common—that is, aa 208 to 855 of FLJ22357; aa 1 to 619 of FLJ22341; aa 210 to 855 of the pufferfish α-locus homologue, and aa 212 to 855 of the pufferfish αβ-locus homologue.
Nynke Gillemans et al. Blood 2003;101:
©2003 by American Society of Hematology

8. Expression analysis of cytoglobin
Expression analysis of cytoglobin.(A) RNA was isolated from the indicated organs derived from adult mice, and expression of cytoglobin was determined by RT-PCR.
Expression analysis of cytoglobin.(A) RNA was isolated from the indicated organs derived from adult mice, and expression of cytoglobin was determined by RT-PCR. The number of PCR cycles was within the linear range of the reaction (not shown). (B) To quantitate the amplicons, the bands were scanned on a Molecular Dynamics Typhoon instrument (Sunnyvale, CA), and the relative expression levels of cytoglobin were calculated using cyclophilin A as a reference.
Nynke Gillemans et al. Blood 2003;101:
©2003 by American Society of Hematology

9. Model for the evolutionary origin of human globin loci
Model for the evolutionary origin of human globin loci.Based on phylogenetic analysis, a model for the evolution of the vertebrate globins has been proposed11 and provides the evolutionary time-scale for the model depicted here.
Model for the evolutionary origin of human globin loci.Based on phylogenetic analysis, a model for the evolution of the vertebrate globins has been proposed11 and provides the evolutionary time-scale for the model depicted here. Genes are color coded; stippled lines indicate physical lineage but uncertainty about the order of the genes, the presence of additional genes, or both. Gray bars indicate when bony fishes diverged from agnathans, amphibians diverged from bony fishes, and mammals diverged from reptiles.36
Nynke Gillemans et al. Blood 2003;101:
©2003 by American Society of Hematology