1. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark Creation of a functional B cell receptor/Antibody

2. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark Germ line gene organization © 2001 by Garland Publishing

3. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark Gene organization © 2001 by Garland Publishing

4. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark The 12/23 rule of recombination recombination signal sequence (RSS) { Only combined 12 RSS to 23 RSS

5. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark Mechanism of gene rearrangement

6. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark Addition of P and N nucleotides TdT: terminal deoxynucleotidyl transferase

7. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark Questions to be addressed Can multiple D genes be inserted? –Violation of 12/23 rule Can D genes be inserted backwards? Is there a D gene preference? Is there a reading frame preference for D genes? –If yes, is it part of the gene rearrangement? Who is doing the end trimming?

8. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark P nucleotides SequencesPermutated sequences Distance from heptamer to gene end No. of seq No. with P % with PNo. of seq No. with P % with Pp-value VH gene 1144847432.716351036.3<10-5 21027484.71068656.10.091 3762537.0612365.90.245 JH gene 13246018.5350236.6<10 -5 218421.020931.40.560 321983.7250145.60.220 5’ end of D gene 151912824.7619548.7<10 -5 2343319.0347267.50.275 3474255.3454173.70.168 3’ end of D gene 16168614.0684588.50.001 22663011.3276248.70.195 346051.148591.90.241 Table 1. Manuscript 2.

9. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark How many types of D genes? Conventional D genes – Identified in 81% of sequences unmutated sequences, 64% of mutated sequences D genes with irregular RSS (DIR) Chromosome 15 OR Two D genes Inverted D genes –Long inverted D genes can not be excluded

10. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark Inverted (palindrom) D genes

11. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark D genes with irregular RSS (DIR) Very long, >180 bp Contain a family 1 D gene Found in 1% of sequences, inverted in 1.2% Some explained as family 1 gene plus N additions Median length of remaining not different from in permutated sequences

12. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark Two D genes 2 D genes found in 1% of sequences Frequency not different from permutated sequences Some explained as one long D genes with deletion Some not possible due to D genes location Median lengths of longest gene resembles normal D genes, shortest resembles permutated sequences

13. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark Chromosome 15 OR 10 OR resembling D genes on chromosome 15 High homology to conventional D genes Very few OR15 in un-mutated sequences Median length not different from hits in permutated sequences

14. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark D gene usage 27 conventional D genes, 34 known alleles

15. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark D-gene usage and JH gene + JH proximal D genes more often recombined to JH4 than JH6 and JH distal D genes more often to JH6

16. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark D gene reading frames The recombination mechanism utilises each D gene reading frame at same frequency Reading Frame StopHydrophilicHydrophobic GenePNPP P D2-2*01RIL**YQLLC (1)6.534.7GYCSSTSCYA (2)61.232.6DIVVVPAAM (3)32.232.6 D2-2*02RIL**YQLLY (1)11.346.7GYCSSTSCYT (2)55.020.0DIVVVPAAI (3)33.833.3 D2-2*03WIL**YQLLC (1)0.050.0GYCSSTSCYA (2)66.750.0DIVVVPAAM (3)33.30.0 D2-8*01RILY*WCMLY (1)2.442.9GYCTNGVCYT (2)68.328.6DIVLMVYAI (3)29.328.6 D2-8*02RILYWWCMLY (1)0.0 GYCTGGVCYT (2)88.90.0DIVLVVYAI (3)11.1100 D2-15*01RIL*WW*LLL (1)1.532.5GYCSGGSCYS (2)70.837.5DIVVVVAAT (3)27.730.0 D2-21*01SILWW*LLF (1)8.350.0AYCGGDCYS (2)58.325.0HIVVVIAI (3)33.325.0 D2-21*02SILWW*LLF (1)0.054.5AYCGGDCYS (2)78.018.2HIVVVTAI (3)22.027.3 Total-10.833.6-62.232.4-26.934.0 Tabel 4. Manuscript 2.

17. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark N nucleotide dependence on end nucleotide Position X+1 Position XATGCP-value A0.2920.1460.2920.2710.04 T0.2600.2900.2070.2430.016 G0.2040.1720.4530.1720.0004 C0.1360.2040.2310.430<0.0001 Expected0.2100.2010.2920.298- N addition is not random but dependent on end nucleotide

18. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark Trimming of gene ends  Trimming depends on the gene end and can not only be described by a simple removal of one nucleotide at a time Avg. 3.8 bp

19. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark Results regarding recombination and diversity and open questions DIR, OR15, multiple D genes and VH replacements are not used at a significant rate Inverted D genes are used rarely All D genes not used at same frequency  What determines if a D genes is used? D gene usage somewhat dependent on JH gene  Does multiple D-J recombination steps take place? All D gene reading frames used at equal rate at the recombination step  At what step in the development is the selection for the hydrophilic reading frame

20. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark Results regarding recombination and diversity and open questions (cont.) N addition not random but dependent on end nucleotide  Does nucleotide availability or the specificity of TdT determine the N addition? Trimming not random but dependent on gene and sequence  What enzyme(s) is responsible for the trimming?

21. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark The translated functional rearrangement

22. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark Numbering Schemes

23. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark Numbering Schemes  The Kabat numbering scheme is a widely adopted standard for numbering the residues in an antibody in a consistent manner. However the scheme has problems!  The Chothia numbering scheme is identical to the Kabat scheme, but places the insertions in CDR-L1 and CDR-H1 at the structurally correct positions. This means that topologically equivalent residues in these loops do get the same label (unlike the Kabat scheme).  The IMGT unique numbering for all IG and TR V-REGIONs of all species relies on the high conservation of the structure of the variable region [1-6]. This numbering, set up after aligning more than 5 000 sequences, takes into account and combines the definition of the framework (FR) and complementarity determining regions (CDR) [8], structural data from X-ray diffraction studies [9], and the characterization of the hypervariable loops [10]. http://www.bioinf.org.uk/abs/#kabatnum http://imgt.cines.fr/

24. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark Identification of CDR regions Indentifying the CDRs CDR-L1 StartApprox residue 24 Residue beforeis always C Residue afteris always W. Typically WYQ, but also, WLQ, WFQ, WYL Length10 to 17 residues CDR-L2 Startalways 16 residues after the end of CDR-L1 Residues beforegenerally IY, but also, VY, IK, IF Lengthalways 7 residues CDR-L3 Startalways 33 residues after end of CDR-L2 Residue beforeis always C Residues afteralways FGXG Length7 to 11 residues CDR-H1 StartApproximately residue 31 (always 9 after a C) (Chothia/AbM defintion starts 5 residues earlier) Residues beforealways CXXXXXXXX Residues afteralways W. Typically WV, but also WI, WA Length5 to 7 residues (Kabat definition); 7 to 9 residues (Chothia definition); 10 to 12 residues (AbM definition) CDR-H2 Startalways 15 residues after the end of Kabat/AbM definition of CDR-H1 Residues beforetypically LEWIG, but a number of variations Residues afterK[RL]IVFT[AT]SIA (where residues in square brackets are alternatives at that position) LengthKabat definition 16 to 19 residues (AbM definition and most recent Chothia definition ends 7 residues earlier; earlier Chothia definition starts 2 residues later and ends 9 earlier) CDR-H3 Startalways 33 residues after end of CDR-H2 (always 3 after a C) Residues beforealways CXX (typically CAR)