1. by Nancy D. Borson, Martha Q. Lacy, and Peter J. Wettstein
Altered mRNA expression of Pax5 and Blimp-1 in B cells in multiple myeloma
by Nancy D. Borson, Martha Q. Lacy, and Peter J. Wettstein
Blood
Volume 100(13):
December 15, 2002
©2002 by American Society of Hematology

2. Structure and sequence of a Pax5 mRNA isoform that was missing exons 2, 3, and 4 and was detected in the plasma cells of an MM patient in a RAP analysis.Structure and sequence of the exon junctions as observed in the plasma cells of MM patient P1.
Structure and sequence of a Pax5 mRNA isoform that was missing exons 2, 3, and 4 and was detected in the plasma cells of an MM patient in a RAP analysis.Structure and sequence of the exon junctions as observed in the plasma cells of MM patient P1. Arrows indicate a nucleotide position at which P1 appeared to be heterozygous.
Nancy D. Borson et al. Blood 2002;100:
©2002 by American Society of Hematology

3. Functional regions and genomic structure of Pax5
Functional regions and genomic structure of Pax5.(A) Locations of the paired domain (a 128 amino acid DNA-binding domain), an octapeptide homology region of unknown function, a partial homeodomain region, and a transactivating domain are indicated.
Functional regions and genomic structure of Pax5.(A) Locations of the paired domain (a 128 amino acid DNA-binding domain), an octapeptide homology region of unknown function, a partial homeodomain region, and a transactivating domain are indicated. The transactivating domain of Pax5 consists of an activator domain that is negatively regulated by a repressor domain. An alternative start site for Pax5 is indicated by the arrow. (B) An exon/intron map for the human Pax5 gene, which is located in chromosomal region 9p13. Exon sizes vary from 45 to 198 base pairs. The diagram is based on data from Celera Discovery System and Celera Genomics' associated databases and from accession no. AP002388, clone RP M6, sequenced by RIKEN Genomic Sciences Center.
Nancy D. Borson et al. Blood 2002;100:
©2002 by American Society of Hematology

4. RT-PCR products of 3′-ends of the coding region of Pax5 transcripts differ between malignant and normal B cell–enriched populations.Shown are RT-PCR products that were generated with a pair of primers designed to amplify a 585 bp product in the 3′-end of th...
RT-PCR products of 3′-ends of the coding region of Pax5 transcripts differ between malignant and normal B cell–enriched populations.Shown are RT-PCR products that were generated with a pair of primers designed to amplify a 585 bp product in the 3′-end of the coding region of Pax5. All templates were mRNA derived from B cell–enriched populations. P indicates MM patient; H, healthy donor. (Lane M) A 100-bp ladder (Boehringer Mannheim). Products of 300 bp or less in this RT-PCR were not readily identifiable. (Lane 1) A no-template control. (Lane 2) An unidentified product for P8. (Lane 3) The uppermost product observed for P3 (arrow) is a doublet composed of a 498-bp product and a 483-bp product that were identified as Pax5 isoforms missing exons 9 and 8, respectively. A product at 396 bp is an isoform missing Pax5 exons 7 and 8 in tandem. (Lanes 4 and 5) Products identified for H3 and H1 include the expected product of 585 bp, a product at 396 bp that is missing exons 7 and 8, and a product at 500 bp that is a mixed hybrid of one strand of the 585-bp and one strand of the 396-bp product.
Nancy D. Borson et al. Blood 2002;100:
©2002 by American Society of Hematology

5. Differences and similarities are observed in Pax5 isoform usage in malignant and normal B-cell–enriched populations.(A) RT-PCR products of 3′-ends of the coding region of Pax5 transcripts differ between malignant and normal B cell–enriched populations.
Differences and similarities are observed in Pax5 isoform usage in malignant and normal B-cell–enriched populations.(A) RT-PCR products of 3′-ends of the coding region of Pax5 transcripts differ between malignant and normal B cell–enriched populations. (B) Alternative Pax5 d/e is expressed in both malignant and normal B cell–enriched populations. Shown in panel A are RT-PCR products that were generated with a pair of primers designed to amplify a 754-bp product in the 3′-end of the coding region of Pax5. All templates were mRNA derived from B cell–enriched populations. P indicates MM patient; H, healthy donor. (Lane M) A 100-bp ladder (Boehringer Mannheim). (Lane 1) A no-template control. (Lanes 2-7) Patients P9, P15, and P16 had not yet received any chemotherapy at the time of the BM harvest; patients P7, P11, and P14 had received treatment with chemotherapy at the time of the BM harvest (Table 2). The expected 754-bp product is faintly observed in 2 treated patients (P7 and P11) but in none of the untreated patients. (Lanes 8-10) The expected 754-bp product is expressed in all 3 healthy subjects. All other products observed with this primer pair were nonspecific. (B) (Lane 1) A no-template control. (Lanes 2-10) All MM patients, treated and untreated, and all healthy subjects express the alternative Pax5d/e isoform.
Nancy D. Borson et al. Blood 2002;100:
©2002 by American Society of Hematology

6. Alternative 3′-end sequence (human Pax5d and Pax5e)
Alternative 3′-end sequence (human Pax5d and Pax5e).Unique nucleotide sequence that follows Pax5 exon 5 in an isoform with an alternative 3′-end and the putative translation of this sequence.
Alternative 3′-end sequence (human Pax5d and Pax5e).Unique nucleotide sequence that follows Pax5 exon 5 in an isoform with an alternative 3′-end and the putative translation of this sequence. Exons 6 to 10 of Pax5 are replaced with this alternative sequence. These data were generated through use of the Celera Discovery System and Celera Genomics' associated databases.
Nancy D. Borson et al. Blood 2002;100:
©2002 by American Society of Hematology

7. Compilation of Pax5 isoforms detected in untreated MM patients, treated MM patients, and healthy subjects.P indicates patient; MM, multiple myeloma patient; H, healthy subject.
Compilation of Pax5 isoforms detected in untreated MM patients, treated MM patients, and healthy subjects.P indicates patient; MM, multiple myeloma patient; H, healthy subject. Boxes represent detection of the Pax5 isoforms listed at left of diagram; hatched boxes for the Pax5 full-length isoform indicate that very low expression was detected in semiquantitative analyses. No attempts were made to semiquantitate any other isoforms, which were expressed at variable levels. (A) Isoforms detected by RT-PCR in B-cell populations from 5 healthy subjects, 6 untreated MM patients, and 7 treated MM patients. The isoforms shown for P11 and for P13 to P16 are also the isoforms that were observed in the CD22+ B-cell populations for these patients with one exception: The isoform missing exons 7-8 was not observed in the CD22+ cells of P14. (B) Isoforms detected by RT-PCR in plasma cells from 4 healthy subjects, 1 untreated MM patient, and 3 treated MM patients.
Nancy D. Borson et al. Blood 2002;100:
©2002 by American Society of Hematology

8. Structure and sequences of most commonly occurring Pax5 mRNA isoforms
Structure and sequences of most commonly occurring Pax5 mRNA isoforms.(A) Sequence of the exon junction observed in the Pax5 isoform from which exon 8 has been deleted.
Structure and sequences of most commonly occurring Pax5 mRNA isoforms.(A) Sequence of the exon junction observed in the Pax5 isoform from which exon 8 has been deleted. (B) Sequence of the exon junction observed in the Pax5 isoform from which exon 9 has been deleted. The base change shown in exon 8 was observed in P6. (C) Sequence of the exon junction observed in the Pax5 isoform from which exons 7-8 have been deleted. This isoform has been detected in the B cells of all healthy subjects. (D) Sequence of the exon junction observed in the Pax5 isoform from which exons 7-9 have been deleted. (E) Sequence of the isoform detected in the plasma cells of P17 that has an insert between exon 8 and exon 9.
Nancy D. Borson et al. Blood 2002;100:
©2002 by American Society of Hematology

9. Blimp-1 is prematurely expressed in B-cell populations in MM
Blimp-1 is prematurely expressed in B-cell populations in MM.Semiquantitative gene-specific RT-PCRs were performed to determine relative levels of expression of Blimp-1.
Blimp-1 is prematurely expressed in B-cell populations in MM.Semiquantitative gene-specific RT-PCRs were performed to determine relative levels of expression of Blimp-1. Shown is an example of one experiment. P indicates MM patient; H, healthy donor. All upper lanes show Blimp-1 expression; all lower lanes show β-actin expression. (Lane M) A 100 bp ladder (Boehringer Mannheim). (Lane 1) A no-template control. (Lanes 2-6) Blimp-1 products (upper lanes) and β-actin products (lower lanes) generated using mRNA templates extracted from B cells from P10, P6, and P3 and from H12 and H13, respectively. (Lanes 7-10) Blimp-1 products (upper lanes) and β-actin products (lower lanes) generated using mRNA templates extracted from plasma cells from P10 and P3 and from H12 and H13, respectively.
Nancy D. Borson et al. Blood 2002;100:
©2002 by American Society of Hematology

10. Blimp-1 is expressed in CD22+ and CD22− B cells, and its expression is inversely correlated to Pax5 expression in the same cells.P indicates MM patient.
Blimp-1 is expressed in CD22+ and CD22− B cells, and its expression is inversely correlated to Pax5 expression in the same cells.P indicates MM patient. Superscripts “+” and “−” designate CD22+ and CD22− B cells, respectively. (Lane M) A 100 bp ladder (Boehringer Mannheim). (Lane 1) A no-template control. (A) Full-length Pax5 expression in CD22+ and CD22− B-cell populations from 4 patients. (B) Accompanying Blimp-1 expression in the same cell samples as panel A. (C) A β-actin control.
Nancy D. Borson et al. Blood 2002;100:
©2002 by American Society of Hematology

11. A1 expression correlates inversely to Blimp-1 expression in B cells and plasma cells from MM patients and healthy donors.Semiquantitative gene-specific RT-PCRs were performed to determine relative levels of expression of A1 in B cells and plasma cells from ...
A1 expression correlates inversely to Blimp-1 expression in B cells and plasma cells from MM patients and healthy donors.Semiquantitative gene-specific RT-PCRs were performed to determine relative levels of expression of A1 in B cells and plasma cells from both malignant and healthy subjects. P indicates MM patient; H, healthy donor. (A) (Lane M) A 100 bp ladder (Boehringer Mannheim). (Lane 1) A no-template control. (Lanes 2-9) A1 products (upper lanes) and β-actin products (lower lanes) generated using mRNA templates extracted from percursor B cells from P2, P6, P3, P5, H1, H4, H3, and H9, respectively. (B) (Lane M) A 100-bp ladder (Boehringer Mannheim). (Lane 1) A no-template control. (Lanes 2-5) A1 products (upper lanes) and β-actin products (lower lanes) generated using mRNA templates extracted from plasma cells from P3, P10, H12, and H13, respectively.
Nancy D. Borson et al. Blood 2002;100:
©2002 by American Society of Hematology