On the Similarity of Sequences from mitochondrial Conserved Sequence Blocks and the breakpoint junctions of “illegitimate translocations” found in some leukemias and lymphomas.

 

Alice C. Lichtenstein, MS

aclsnippets@gmail.com

 

Note: this installment of Snippets has been reformatted and revised (2013)

 

Abstract

 

The conserved sequence blocks (CSBs) from kinetoplastid mitchodrial minicircles, CSBs from human mitochondrial DNA and initiation sites of heavy and light strands of human mitochondrial DNA were compared to sequences found at the unusual  junctions of  patient samples or cell lines associated with Multiple Myeloma, Follicular Lymphoma, Acute leukemias involving the translocations of chromosome 4 and 11, t(14;18) of Hodgkin’s Disease and normal cells,  T cell ALL, tumor tissue from non-Hodgkin’s diffuse large cell lymphoma patient,  and mRNA for PML/RAR alpha and T cell CLL. A number of similarities were found at or near the “illegitimate translocation” join sites that might give a new perspective as to the generalized origins of the sequences involved, without, as yet,  unveiling any new mechanism for these translocations.

 

Introduction

 

Illegitmate translocations” that combine non-immunoglobulin chromosomes with immunoglobulin chromosomes are thought to be the result of VDJ recombinase creating an aberrant join mimicking a V-D-J or V-J DNA sequence.   The new chimeric sequence, which can delete a promoter, add a new constitutive promoter, transcribe for a chimeric protein causes a fundamental change in the cell that may lead to some neoplasias. There is debate about the origins of these new breakpoints because the RSS heptamer which usually brings V, D, and J genes together is not always found near the translocated junctions in the germline DNA and sometimes there are insertions or deletions which differ from the original germline sequences.

 

Rather than look at the sequences around the junctions as aberrant VDJ recombinants (which they may be), it was useful to look at them another way; through a g:u complement. A complement sequence was derived from the translocated sequence using only the bases Guanine and Uridine (G and U). For our purposes, C, T and U complement G, and A and G both complement U. To look for alignments the G:U sequence was then used as a template to look for complements that resembled CSBs etc. In the end analysis, this author substituted a pyrimidine for a pyrimidine or a purine for a purine in the original translocated sequence. 

 

As a result, at these breakpoints or joins one could see a similarity between the Conserved sequence blocks, CSB 1, or CSB 2, or CSB 3 found in the mitochondrial mini circular DNA of Leishmania, Cryptobia  and other kinetoplasts. Also, importantly, some of the breakpoints also resembled CSB 1, -2, and -3 of human mitochondrial DNA, which are the RNA primer/DNA replication sites of mitochondrial light strands. In fact, all three CSBs covered an RNA primer/DNA replication initiation site.

 

 

 

Materials and Methods

 

Mitochondrial Conserved Sequence Blocks

 

The following two sets of conserved sequence block were used. Human mitochondrial sequences were from Chang, D. and Clayton, D, (1985). "DHP-RNAprimer 3' end" was the 3' end of the RNA sequence used as a primer and "DHP 5' end" delineated where DNA synthesis began.

 

Human mitochondrial CSB1:

DHP-DNA 5' end

DHP-RNA primer 3' end

a

a

t

t

a

a

t

t

a

a

t

g

c

t

t

g

t

a

g

g

a

c

a

t

a

a

t

a

a

#210     CSB-3     #238

 

TAATTA are replicated as DNA nucleotides and ATGC are the 3' end of the RNA primer site. Replication is from 3’ to 5' as in lagging strand replication.

 

 

Human mitochondrial CSB 2:

DHP-DNA 5' end

 

DHP-RNA primer 3' end

c

c

a

a

a

c

c

c

c

c

c

c

t

c

c

c

c

c

g

c

t

#298 CSB-2     #322

 

CAAACCCCCC was the DNA sequence and TCCC is the 3' end of the RNA primer site sequence. Replication from 3’ to 5’. (note similarity of CCCCGCTTC to CSB-2s of kinetoplasts.)

 

Human mitochondrial CSB 3:

 

DHP-DNA 5’ end

DHP-RNA primer 3' end

c

t

g

c

c

a

a

a

c

c

c

c

c

a

a

a

a

a

c

a

a

a

g

#344 CSB-3     #363

 (note similarity to nonamer of RSS)

 

And CSBs found in kinetoplastid DNA minicircle sequences are shown below.;

.

 

from fig. 1 Ray, D Conserved Sequence Blocks in Kinetoplast Minicircles from Diverse Species of Trypanosomes, (1989) Molcecular and Cellular Biology, Vol 9 No.3 p. 1365

MINICIRCLE CSB's

 
CSB-1

Crithidia fasciculata

A

G

G

G

G

C

G

T

T

C

 

 

 

Trypanosoma equiperdum

A

T

G

G

G

C

G

T

G

C

 

 

 

Leishmania tarentolae

A

G

G

G

G

C

G

T

T

C

 

 

 

T. brucei

A

T

G

G

G

C

G

T

G

C

 

 

 

T. congolense

A

A

G

G

G

C

G

T

T

C

 

 

 

T. cruzi

A

G

G

G

G

C

G

T

T

C

 

 

 

T. lewisi

G

A

G

G

G

C

G

T

T

C

 

 

 

 

CSB-2

Crithidia fasciculata

T

C

C

C

G

T

T

C

 

 

 

 

 

Trypanosoma equiperdum

T

C

A

C

G

T

G

C

 

 

 

 

 

Leishmania tarentolae

C

C

C

C

G

T

T

C

 

 

 

 

 

T. brucei

T

C

C

C

G

T

G

C

 

 

 

 

 

T. congolense

T

C

C

C

G

T

A

C

 

 

 

 

 

T. congolense

C

G

C

C

G

T

A

C

 

 

 

 

 

T. cruzi

C

C

C

C

G

T

A

C

 

 

 

 

 

T. lewisi

C

C

C

C

G

T

A

T

 

 

 

 

 

 

CSB-3

Crithidia fasciculata

G

G

G

G

T

T

G

G

T

G

T

A

 

Trypanosoma equiperdum

G

G

G

G

T

T

G

G

T

G

T

A

 

Leishmania tarentolae

G

G

G

G

T

T

G

G

T

G

T

A

 

T. brucei

G

G

G

G

T

T

G

G

T

G

T

A

 

T. congolense

G

G

G

G

T

T

G

G

T

G

T

A

 

T. cruzi

G

G

G

G

T

T

G

G

T

G

T

A

 

T. lewisi

G

G

G

G

T

T

G

G

T

G

T

A

 

 

CSB-3 is universally conserved in kinetoplasts, but not CSB-1 and CSB-2. In the comparisons below, sequences used were from (Ray, D. (1989)) unless otherwise noted.

 

CSBs were compared to various leukemia and lymphoma sequences obtained from Gen Bank and the literature. In some cases a g:u complement was made to the original sequence and then a second complement to the g:u intermediate was used to determine alignments.

 

Please note that CSBs from both kinetoplasts and human mitochondria are presented in the same colors. i.e. CSB-1 is yellow, CSB-2 is light green, and CSB-3 is light blue.

 

Kinetoplastid Gap

 

The " kinetoplastid gap" is an actual "gap" in one of the strands of kinetoplastid minicircle DNA that, if filled in, would be the complement most of the CSB-3 sequence. This gap  is important in that it is conserved in all the minicircles of T. equiperdum (Ntambi, J and Englund, P. (1985)). It is tempting to assume that the kinetoplastid gap exists in other kinetoplasts too, but no papers allude to this.

 

 

 

    3'

KINETOPLASTID GAP

5'            
-G T G T   C C C C T T T-
-C A C A T A A T G T G G T T G G G G A A A-
           

CSB-3

     

 

 

 

Ori L UMS and Ori H H14 Binding Sites for the Universal Minicircle Binding Protein

 

Abu-Einell et. al. (1999),  found  two conserved minicircle DNA sequences Ori L UMS and Ori H14 that seemed to be the binding sites for a protein (UMSBP) which has been  implicated in kinetoplast minicircle replication (Itay et. al (2006)).

 

L is the lagging strand and H is the heavy strand for DNA minicircle replication. Note the overlap in sequences with CSB-1 (yellow), CSB-2 (light green) and CSB-3  (light blue), The authors report that the H strand initiates on or near a hexamer sequence which appears to be a complement of the predicted oriH sequence. (Ori=origin of replication).

 

UMSBP DNA binding sites
                             
                             
G G G G T T G G T G T A Ori L UMS
                             
      3' C C C G C A 5'   Hexamer
                             
G T A G G G G C G T T C T G  
                        Ori H 14

 

For some of the sequences listed below a "G:U" complement was used to find alignment. A complement sequence of only g and u bases was made from the original translocations and then another complement, using four bases was created from the g:u intermediate. Alignments were then determined. In essence one was substituting a pyrimidine for a pyrimidine and a purine for a purine in the original sequence

 

In the results below >>> or <<< is used to show the direction of replication.

 

The two replication start sites of human mitochondria; LSP and HSP were also compared to the original translocations via a g:u complement.

 

 

CHI Hot Spots

 

Last but not least the sequence for chi "hotspots" (GCTGGTGG) was  used for comparison. These hotspots are thought to somehow regulate or serve as a binding site for the RecBCD enzyme found in E. coli DNA recombination and repair. (Amundsen, S. (2007)).  It was included in the comparisons below because it is thought that there is a single strand generated by the enzyme which interested this author.

 

As an aside, the sequence for the complement of the kinetoplastid gap is similar to a chi "hotspot", but  not an exact match. Gray is g:u complement.

 

 

 

    3'

KINETOPLASTID GAP

5'            
-G T G T   C C C C T T T-
-C A C A T A A T G T G G T T G G G G A A A-
           

CSB-3

     
g u g u g u u g u g u u g g u u u u u u u
            g   g t g g t c g            
            CHI hotspot recomb sequence?            

 

 

 

Comparison Results

 

The first translocation sequence was from a paper by B. H. Ye et. al (1995) from tumor tissue from a non-hodgkins’s diffuse large-cell lymphoma patient. (der 14) (The bright  yellow color demarcates the join  site.) The Cryptobia sequences are from an article by Lukes, J.et. al.(1998). The human mitochondrial sequence is from Chang, D. and Clayton, D.(1985). Although labeled as from a patient with non-Hodgkin's diffuse large-cell lymphoma, the Ye article calls it a B cell lymphoma. BCL6 transcription is de-regulated by this translocation.

 

GENBANK S81106 Non-Hodgkin’s Diffuse large-cell lymphoma patient

BCL 6

IgH

g

a

g

g

t

t

g

g

t

g

a

t

g

t

g

c

t

g

t

g

c

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

g

 

g

g

t

t

g

g

t

g

a

 

 

CSB 3 L. tarentolae et.al.

 

g

 

 

 

 

 

 

 

t

 

a

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

g

 

g

g

t

t

g

g

t

g

a

 

 

CSB 3 Cryptobia helices

 

 

g

 

 

 

 

 

 

 

t

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

g

 

g

g

t

t

g

g

t

g

a

 

g

CSB 3 Cryptobia helices

 

 

g

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 If a g:u complement was used, the alignments were better

 

GENBANK S81106 Non-Hodgkin's Diffuse large-cell lymphoma patient

BCL-6

IgH

       
g a g g t t g g t g a t g t g c t g t g c        
u u u u g g u u g u u g u g u g g u g u g g:u complement
g g g g t t g g t g a                    

CSB-3 L. tarentolae  etc, OriL UMS

                  t                              
                                                 
g g g g t t g g t g a                    

CSB-3 Cryptobia helices, Ori L UMS

                  t                              
                                                 
g g g g t t g g t g a   g                

CSB-3 Cryptobia helices

                                                 
                                                 
    a a t c a g   g a t g t   c   g t a  

CSB-1 hu mitoch

    5'   a                   t         3'          
    <<RNA 3' end (primer) CSB-1 human mitochondria          
    a a t t a a t g                              
   

<<DNA5' start site

                     

CSB-1 hu mitoch

                                                 
                                                 
    g g t t g g t g a   a t           Kinetoplastid gap comp 3'-5'
    3'             t       5'                      
                                                 
                t a a t g t g   t     Kinetoplastid gap comp 5'-3'
                              g                  
                5'               3'                
                                                 
      g c t g g t g g                  

Chi "hotspot" recomb seq.

                                                 
                                                 

 

In kinetoplasts, the kinetoplastid gap complement is single stranded. (Ntambi J. and Englund, P. (1986).

 

 

Genbank U73662 Human t(4:14)(p16:q32) breakpoint junction sequence, Multiple myeloma cell line H929
4p 16.3 mu switch region Ig Heavy Chain chromosome 14q32
g t t g c c c a g a c c a a c t c a a c
u g g u g g g u u u g g u u g g g u u g
        c c c g   a c          

CSB-2 T. cruzi?

      c         t                      
        c c c g   a t          

CSB-2 T. lewisi?

      c         t                      
        c c c g   a c          

CSB-2 T. congolense?

      t         t                      
c c c g   a

Ori H hexamer

c
g   t g t t c g a a t t a a t  

CSB-1 human mitochondria

  a             t                      

RNA 3' end >>

DNA 5' >>

         
                (*)                      

 

Even though the Genbank sequence shows a join as shown above, it looks as though there is something happening three bases over, where the RNA3'/DNA5' CSB-1 bases align. (*)

 

 

Genbank U73664 Multiple myeloma cell line SKMM2

bcl-1 locus on 11.q13 gamma switch region 14q32
t g t t c c c g c a g g t g a g c a
g u g g g g g u g u u u g u u u g u
      t c c c g t a  

CSB-2 T. congolense

                    c              
      3' c c c g c a 5'

Ori H hexamer

                                   
  a t   t t c g t a a   t a a   CSB-1
g     g               t       t Hu Mitochon.

RNA 3' end >>

DNA 5' >>

   
t a c t t t t g c g g           LSP region
#401 t   g #413 Hu Mitochon
<<L strand (LSP transcription start 3'-5') LSP flanking region            
Sequences below are derived directly from the g:u complement
g u g g g g g u g u u u g u u u g u
                                   
g t a g g g g c g t t c g

Ori H H14

                      t            

 

 

 

 

Genbank U73660 Multiple Myeloma Human t(4;14)(p16;q32)
14q32 gamma 2 switch region on IgH chain 4p16.3
c a g a c c t g c c c c t t c t t t a a t g g t  
g u u u g g g u g g g g g g g g g g u u g u u g  
c a a a c c c   c c c c t c c c c c g     CSB-2 Hu Mitoch.

<<<DNA 5' end

RNA primer 3' end

             
                                                 
                t t t   t c t t t t g g c g g t  
                399     a         407             414  
                <<<LSP start site 3'-5' hu mitochondria flanking bases  
                                                 
c a a a t t t a t c t t t t                      
393           t             407                      
<<<LSP transcription (replication) start site 3-5' hu mitochondria                        
                                                 
        c t t g c c c c          

CSB-2 L. tarentolae, L. mex. amazon.

        c t t g c c c t          

CSB-2 C. fasciculata

                                                 

 

 

 

 

 

U73671 Human t(6:14) breakpoint junction sequence multiple myeloma cell line SKMM1

mu switch region 14q32.3 sequence from chromosome 6
c c t c a t g c c c t g c t g a a g c  
g g g g u g u g g g g u g g u u u u g  
                                       
      c a t g c c c c          

CSB-2 T. cruzi

      c a t g c c c t          

CSB-2 T. congolense

      c g t g c c c c          

CSB-2 T. brucei

      t a t g c c c c          

CSB-2 T. lewisi

                                       
      5' a c g c c c 3'          

Ori H Hexamer

                                       

Ori H H14

  c g t c t t g   c g g g g t  
                                       
                                       
t t t t   t a t c t t   t t g g g g t  
398                     t       c     414  

<<LSP replication start site hu mitochondria

flanking bases  
                                       
Sequences below derived from the g:u complement directly
g g g g u g u g g g g u g g u u u u g  
                  g g t g g t c     Chi sequen.
                                g      

 

 

 

Jager, U. et. Al.  (2000) Sequences from patients with follicular lymphoma showed the following: the chromosome with BCL-2 translocated to a site between a D-J sequence. The sequences below do not show the J region, just the BCL-2 -D aberrant join.

 

 

Clone 3 bcl 2 mbr+ D region additions
t c c t g c c c c a a g t a g g g a g            
g g g g u g g g g u u u g u u u u u u

g:u complement

        a c c c c a a a   g a                    
        <<Minor  LSP replic.

HSP>>

                   
                                                 
        a c c c c a a a c a a a g a a

CSB-3 human mitochondria

                      a a                        
        <<DNA 5' start tcn

<<<RNA 3' end

                     
c t c c   c c c c a a a c            

CSB-2 Human Mitochondria

        c                                        
R3' DNA 5' end >>>                        
  c t t g c c c c a a g c a  

CSB-2 L. tarentolae, L. mexicana amazonensis

                    a     386  

 

                 
  c t t g c c c t a a a   g g

a

CSB-2 C. fasciculata  (x)

                              1681                  
                                                 
                                                 
Sequences below derived from the g:u complement directly
g g g g u g g g g u u u g u u u u u u            
        c a a a   t t t a t c t t t t

LSP replication start site hu mi.

        394       t                   408            
        <<<<LSP replication start site 3'-5' human mitochondria              
  a a   c a a a   c c c a    

Minor LSP replication start site hu mitochondria

551     c         c       559                        
 

<<<minor LSP replication start site 3'-5'

                       
                                                 
          g a a a c c c g t t c

CSB-2 C. fasciculata  (x) etc,

                  t                              

(x) C. fasciculata accession GenBank M19266.1

 

Notes: The two LSP sequence alignments are derived from a direct pyrimidine: pyrimidine and purine:purine substitution from the g:u complement, and not the original cloned bcl/D region sequence. 

 

 

 

Clone 4 bcl2 mbr+ D region additions
c c t c c t g c c c t c c t a g g g c t g c c c a
g g g g g g u g g g g g g g u u u u g g u g g g u
    t t t t a   t c t t t t g g g g   c a t

LSP hu mit.

    397                         c           416      
   

<<<<LSP replication init. site 3'-5' hu mitochondria

Flanking sequence

     
                                                 
      c t t g c c c t                  

CSB-2 C. fasciculata

      c t t g c c c c                  

CSB-2 L. tarentolae

Ori H H14       g t   c t t   c g g g g   t g        
                        g           a            
                                                 
CSB-1                       g g g g c   g t t c  
                                                 
  t c t t c g c c c c c t c            

CSB-2 Hu Mitochondria

  311                       323                      
 

RNA 3' end>>

D5'                      
                                                 
                    c c c c a a a a   c a

CSB-3 Hu Mitoch

                                    a            
                    <<DNA 5' site

<<<RNA 3' end

       
                                                 
                    c c c c a a a g   c a c c c  
                    554               a       566    
                    <<minor LSP init site

HSP initiation site hu mitochondria>>>

 
                                                 
Sequences derived directly from g:u complement
g g g g g g u g g g g g g g u u u u g g u g g g u
            c a   a g a a a c c c c a a c   a a c
            565   c                     a   c     550
           

<<HSP initiation site hu mitochondria

minor LSP init site>>

                                                 
                                c t g g t g g Chi seq.
                              g                  

 

 

From the results above, it became apparent that if one uses a gu complement it is easy to see the similarity of the CSB-3 sequence and the sequence that determines the minor LSP initation site and the HSP initiation site. (from fig 5 Chang and Clayton (1985)) Not only the sequences are very similar, but the alignment of where the RNA primer ends in CSB-3 and the HSP initiation start is perfect.

 

Comparison of CSB-3 sequence and minor LSP/HSP initiation sites
g u g g u u u g g g g u u u u u g u  

g:u complement

t g c c a a a c c c c a a a a a c a  

CSB-3 human mitochondria

346

<<DNA 5' end

<<RNA primer 3' end

             
  a c c a a a c c c c a a a g a c a  

minor LSP and HSP init sites

  548                               564              
 

<<minor LSP initiation site

HSP initiation site>>>

             
                                                 

 

 

 

Clone 9 bcl2 mbr+ D region addition
t c c t g c c c t c c t t c c g c c c c a c    
g g g g u g g g g g g g g g g u g g g g u g    
                                               
c c c c   c c c t c c c c c   g c t t t g

CSB-2 hu mitoch

303                                     c

320

     

<<DNA 5' end

<<RNA 3' end

Flanking bases

   

 

                                               
t t t t a t c t t t t                    

LSP init site hu mit.

398                   408                          
<<LSP initiation site human mitochondria                            
  c t t g c c c t       c t t g c c c t  

CSB-2 C. fasciculata

t c c c g t t c       t c c c g t t c    

CSB-2 C. fasciculata

  c t t g c c c c       c t t g c c c c  

CSB-2 L. tarentolae

c c c c g t t c       c c c c g t t c    

CSB-2 L. tarentolae

                                               

 

 

Y. Gu et. al. (1992) looking at the (4:11) (q21:23) chromosome translocations in acute leukemias which they say involve VDJ recombinase also show the following:

 

 

MV4:11 (der (11))

Chromosome 11         Chromosome 4
c t t c g g c t t g g g t g t a c c a c c a t c a
g g g g u u g g g u u u g u g u g g u g g u g g u
                t g g g c g t g c    

CSB-1 T. brucei, T. equip.

              a                                  
                t g g g c g t (t) c t g

Ori H 14

                  c c c g c a        

Hexamer

                                                 
              t t   g g t g t a   t a c

T. equiperdum seq?

                                a                
              kinetoplastid gap complement? 5'-3'              
                t   a a t g t g t t g            
                              g                  
                kinetoplastid gap complement? 3'-5'            
                                                 
Sequence derived directly from g:u complement
g g g g u u g g g u u u g u g u g g u g g u g g u
g g g g t t g g                      

Ori LUMS part ????

                  c c c g c a        

Hexamer

                                                 
                            g c g g t g g

Chi sequence?

                              t                  

 

 

 

 

Note that the following has repeats of the HSP/LSP and  CSB-3 human mitochondrial sequences. Chromosome 4 has them going 5'-3' and 3'-5'

 

RS4:11 der 11
Chromosome 4 Chromosome 11
g g a a g g a g g t t t c c a a a a g g g g a g c a g a a g t c t t t g a c a c t c a a t a t a c t
u u u u u u u u u g g g g g u u u u u u u u u u g u u u u u g g g g g u u g u g g g u u g u g u g g
                                                                                                   
    a   a g a a a c c c c   a a               a c a g a a a c c c c   a a c      

Human mitochondria HSP start

      c                                      

<<HSP

minor LSP>>

                       
   

<<HSP

minor LSP>>

                                                 

Human mitochondria HSP start

                                                                                                   
            a a a c c c c a a a g a   a                                          

Human mitochondria HSP start

                                    c                                                              
            <<Minor LSP

HSP>>

                                                           
                                    a a g a a a c a a a a a c c c c   a a c g t c t

Human mitoch. CSB-3

                                                                    a     c                        
                                             

RNA 3' end >>

DNA 5' start

                   
            a a a c c c c   a a a a a   a a a g c                                  

Human mitoch. CSB-3

                                      c       a                                                    
            <<DNA5' start site  

<<RNA3' end site

                                                 
                                                                                                   
g   a g g g g   g t t t c t                   g t a g g g g c t t t c              

CSB-1 L. tarentolae (x)

  t           c                                             g                                      
g   a g g g g   g t t   c t g                 g t a g g g g c t t c t g            

Ori H H14 5'-3' (y)

  t           c                                             g                                      
                g t c t t c g g g g a   g g g                                      

Ori H H14 3'-5'

                          g           t                                                            
                                                                                                   
Sequences below derived directly from the g:u complement
u u u u u u u u u g g g g g u u u u u u u u u u g u u u u u g g g g g u u g u g g g u u g u g u g g
                                        t t t t a t c t t t g g                                    
                                                          t                                        
                                        <<LSP                                        
                            305                                                   329                  
                            c c c t c c c c c c g c t t c t g g       c c a c a g   CSB-2 (part) hu mitochon.
                            DNA start 5'

<<RNA 3' end

                                                 
                                                    c c c c a a a g a c c          

HSP Human Mitoch

                                                    <<LSP minor

HSP>>

                         
                                                                                                   
                                        t c c c g t t c                            

CSB-2 C. fasciculata

                                        c c c c g t t c                            

CSB-2 L. tarentolae

                                                                                                   
                                                                                                   

 (x) Accession:KO1980.1 Leischmania tarentolae kinetoplast minicircle Lt154

(y) Accession M19266.1 Crithidia fasciculata

 

Gupta, R.K. et. al. (1992), show the following translocations for Hodgkin’s disease that involve chromosomes 14 and 18 and lead to the deregulation of BCL-2.

 

 The nonamer sequence for the result below is from Yu, K et. al (2002)

 

Patient A

BCL-2 (Chromosome 18) N region J6 (chromosome 14)      
t c c t t c c g c g g g g g c c c c c t g g c t a c t a c t a c t a c g g t a t g g a c t c t      
g g g g g g g u g u u u u u g g g g g g u u g g u g g u g g u g g u g u u g u g u u u g g g g      
                                                                                                   
      t c t t g c g g g g a t                                                  

Ori H 14

        c t t g c g g g g a                                                    

CSB-1 C. fasciculata, L. tarentolae

        c t t g c g g g a a                                                    

CSB-1 T. congolense

        c c c g c a                                                            

Ori Hexamer

                                      t a a t   g t   g t t g                  

Kinetoplastid Gap???

                                                    g                                              
  c c c c c c a c a g a a a c c c c     a a c c a                              

Human mitoch HSP start site/minor L

  571                               555   a                                                            
 

<<<HSP initiation site

minor LSP>                                                                
                                                                                                   
              a c a a a a a c c c c     a a c c g t c                          

CSB-3 Human Mitochondria

                                      a                                                            
               

RNA 3' end>>

DNA start site>>>

                                             
                                                                                                   
              a c a a a a a c c t c c c                                        

Nonamer Vh seq 4-34 etc (Italics)

              5'                       3'                                                            
Sequences derived directly from the g:u complements
g g g g g g g u g u u u u u g g g g g g u u g g u g g u g g u g g u g u u g u g u u u g g g g      
                                g g g g t t g g t g   t a                      

Ori L UMS, CSB-3

                            a a g g g g t t g g t g   t a a t   a c a c        

Kinetoplastid gap in T. equip seq

                                        400                   409                                      
                    c c c c a a a g a a c c                                                        
                                    c                                                              
                    <<LSP minor

HSP>>

                                                       
                                      g c t g g t g g                          

Chi recombination hot spot sequence

        a a a c c c c c c t                                                    

CSB-2 Human mitochondria

      c                                                                                            
      <<DNA 5' Start site                                                                      
                                                                                                   

 

 

Patient B

BCL-2 N Region J6
g g t c t t c c t g a a a t g g a g t g g g g a c t a c t a c t a c g g t a t g g a c g t c t g g g
u u g g g g g g g u u u u g u u u u g u u u u u g g u g g u g g u g u u g u g u u u g u g g g u u u
  g t c t t c   c g g g a t g g g                                                

CSB-1 T. congo. T. lewisi, C.f.

              g       g                                                                            
  g t c t t c   c g g g a t g                                                    

Ori H H14

              g       g                                                                            
                                                        c a t c a t a a c a   a a a   a t t t

5'-3' Hu m

                                                        277                   a               294      
                                                                                                   
                                      a a g g g     g t t g   t g t a a t a c a  

Kinetoplastid Gap??? T. equiperd.

                                      394                     g                 412   or              
          c t t c g a a a   g g g g                   t t g   t g t a a t a c a  

Kinetoplastid gap?? T. equiperd

          388                       399                   400     g           409     412                    
                                                    a                                              
          c c c c a a a a c a     a c a g a a a c c   c                                            
                      g          

<<HSP

LSP minor>>                                            
          <<LSP minor

HSP>>

                                                                     
Sequences below derived directly from g:u complement
u u g g g g g g g u u u u g u u u u g u u u u u g g u g g u g g u g u u g u g u u u g u g g g u u u
                          a t t t t a t c t t t g g c g g t a   t g   c a c   t t t LSP start site hu mitoch
                                              t                             t     424                
                         

<<LSP

Flanking region                
c c a   a g a a a c c c c a     c c a                                                              
      c                     a a                                                                    

<<HSP

minor LSP>>

                                                             
                                            t t g g t   g t a a t a c   a c a c     Kinetoplastid gap? in T. equip
                                            400                   409                                  
                                                                                                   
                                            c t g g t g g                          

Chi hot  spot seq?

                                          g                                                        
                                                  g c g g t g g                    

Chi hot  spot seq?

                                                    t                                              
                                                      g g t g g c g                

Chi hot  spot seq?

                                                                t                                  
                                                                                                   

 

 

from table 3. The mbr sequence indicating the translocation breakpoint for patient A (bright yellow box)
c g g g g g c t t t c t c    
g u u u u u g g g g g g g    
t a g g g g c   t t c t   Ori H H14, CSB-1
              g         g    
c a g a a a c c c c       HSP hu mitoch.
                             

<<HSP init site

minor LSP>> site          

 

 

c

a

g

t

g

g

t

g

c

t

t

a

c

 

 

mbr breakpoint patient C 

c

a

g

t

g

g

t

g

c

t

t

a

c

 

 

mbr breakpoint cell line doHH2

t

a

g

 

g

g

c

g

t

t

c

 

 

 

 

CSB-1

 

 

  g

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 An incomplete analysis of Patient D (no J6 region shown)

 

Patient D
BCL 2 N region      
c t t t a c g t g g c c t g a a g g t a g c      
g g g g u g u g u u g g g u u u u u g u u g      
t c t t g c g   g g c t t g g g g a t g

CSB-1, ori H H14

              g             c                    
        a t g t g g   t t g g g g      

CSB-3, Ori L UMS

                                                 

 

 

 

Of interest in patients A through C, (patient C's sequence not shown) is that the J6 sequence is broken at a TAC or  ACT repeat and then translocated to the N region.

 

 

Wang, Junjie et. al (2000), in their paper titled, “The t (14;21), (q11.2;q22) chromosomal translocation associated with T-cell acute lymphoblastic leukemia activates the BHLHB1 gene” sequenced the following

 

der 21
GL 21 GL 14 (J alpha)
c t t g c g c g c g t g g c t g t c g g g a t a gc
g g g u g u g u g u g u u g g u g g u u u u g u ug
                                         

2 CSB-1s (ori H??)

c t t g c g   g   a t g g t c g t c g g g g      
            g   g               g                
                                                 
c t t g c g   g   a t g                  

Ori H H14?

            g   g                                
                  a t   g t   g t t g g g g

Ori L UMS?

                            g                    
      a c a c a c a t a a t   g t   g g   kin gap T. equiperdum?
                    409                            
c c c g c a                              

Ori H hexamer

c c c g t g c                             CSB-2 kinetoplast
                                                 
Sequences derived directly from g:u complement
g g g u g u g u g u g u u g g u g g u u u u g u ug
          c a c g t a t   g g c g g t t t t   c ta
          #420                                      
         

<<LSP start site 3'flanking region

L strand start site 3'-5'>>>

                                                 
                            a c   g c c c

Ori H hexamer

                    g c t g g t g g      

CHI hot spot seq

 

 

 

 

GenBank AB067754.1
Homo sapiens mRNA for PML/RAR alpha, partial cds
PML 15q22 exon 6 RAR alpha 17q21                
g g c t c g g a g a c a a g a g a a c t t g a c  
u u g g g u u u u u g u u u u u u u g g g u u g  
                                                 
a a c c c a a a g a c a          

Human mitochondrion HSP init site

    c                                            

<<<LSP minor

HSP>>

                         
                                                 
a a c c c a a a a a c a a a g a a

Human CSB-3 human mitochondria

    c                                            

<<DNA 5' start

<<RNA 3' end

                         
    t c c a a a a a c a g a      

Nonamer sequence  VH seq 4-34 etc

      3'               5'                          
  g c t t g g g g a t g          

Ori H H14

    t       c                                    
g g c t t g g g g a t g g g      

CSB-1 C. fasciculata, T. congolense, etc

    t       c                                    
                                                 
Sequences below derived directly from g:u complement
u u g g g u u u u u g u u u u u u u g g g u u g  
CSB-2 Human mitoch g c c c c c   t c c c c c c a a a c c    
        312                                   295    
       

RNA 3' end>>

DNA 5' end>>

     
c c a a a c c c c c   c t c c c c c g  

CSB-2 Human mitoch

295                                   312            

<<DNA 5' end

<<RNA 3' end

           
t c a a a   t t t t a t c t t t t   g g

LSP start site hu mitochon.

393                                     410          

<<LSP start site

Flanking region          
g g t t t t t a t t t t

LSP start site hu mitochon.

      410           c         396                    
     

Flanking

LSP start site>>

                   
                                                 

 

 

B cell lymphoma AY137971 Homo sapiens immunoglobulin heavy chain DH d3-3/BCL2 translocation breakpoint sequence

Immunoglobulin heavy chain DH D3-3 N region BCL 2                  
t t t t t g g a g t g g t t a t c a g g g t a g g g g c c c t c a g g g a a c a g                  
g g g g g u u u u g u u g g u g g u u u u g u u u u u g g g g g u u u u u u g u u                  
                                c a   g g t a g g g g c t t c t g g g a a a c g a

Ori H H14 C. fasciculata L. tarent

                                                      g           c       a                        
          g g g g c g   t t   c t g                                              

Ori H H14 C. fasciculata????

                                                                                                   
t t t c c g g g g t g g t t a                                                    

CSB-3 C. fasciculata (x) Ori LUMS

483                 t     g                                                                          
                                        a c a g a a a c c c c   a a a                              
                                       

<<HSP

LSP minor>>

                             
                                                  a a a c c c c a a a   g a c a                    
                                                 

<<LSP minor

HSP>>

                   
Sequences below derived directly from g:u complement
g g g g g u u u u g u u g g u g g u u u u g u u u u u g g g g g u u u u u u g u u                  
                  g c t g g t g g                                                

Chi hotspot recombination seq.

                            c a a t t t t a c t t t t g g                        

LSP Human mitochondria

                              a             t                                                      
                           

<<LSP start site

                                             
                                                                                                   
                                                                                                   

 

(x)  C. fasciculata accession Genbank M19266.1

 

 

S78526 human peripheral blood follicular lymphoma

BCL-2 JH
t g c a g t g g t g c t t a a t g a c c a a c t g g t t c g a c c c t g g g g c c a g g g c a a c c
g u g u u g u u g u g g g u u g u u g g u u g g u u g g g u u g g g g u u u u g g u u u u g u u g g
                                                                                                   
              a c a c   t a a t g g t t g g                                  

Kinet T. equiperdum? Ori L UMS

                      a         t                                                                  
                                          a t t   g c t c g   c t t c g g g g t

Ori H H14 T. equiperdum?

                                                            t       g                              
                                a a c c a a c c a a c c c a a                                      
                                                  a     c                                          
                               

<<minor LSP

HSP>>

                                     
              g t g   t t a a t a a t t a a t   g   t t                      

seq near CSB-1 human mitochondria?

              206               t                   c   226                                            
                              <<DNA 5' start site RNA 3' end site (part)                                            
                                                                                                   
                                                                                                   

 

 

 

 

One of three translocations found in a patient with T-cell chronic lymphocytic leukemia and ataxia-telangiectasia (Davey, M. et. al. (1988))

Xa50 (TCR germline)

 

Ja33

 
g c a c c c c c a c a g a a a t a a c c a g g g a g g a a a g c t t a t c t t c g g a c a g g g a  
u g u g g g g g u g u u u u u g u u g g u u u u u u u u u u u g g g u g g g g g u u u g u u u u u  
  c a c c c c c a c a g a a a c                                                

Hu mitoch. HSP start site

 
      c                       558                                                                    
 

<<<HSP

                                                                   
                      a a   g t g g t t g g g g                                

CSB-3 Ori L UMS

 
                          t                                                        
    g t c t t t a t   g a   g t g g t t g g g g                                

CSB-3 C. fasciculata

 
                          t                                                        
                                                                                   
                                    t c g g g   a g g g g   g t t c            

CSB-1 C. fasciculata, Ori H H14?

 
                                              t           c                                        
                                                                                                   
Sequences below derived directly from g:u complement
u g u g g g g g u g u u u u u g u u g g u u u u u u u u u u u g g g u g g g g g u u u g u u u u u  
  g t a g g g g c g t t c   t g                                                

Ori H H14

 
                                                                                                   
                              g c   g g t t t t c t   t t t t a a a c          

LSP start site 3'-5'

 
                              412                     a               394                              
                                         

LSP start site>>

                             
                                t c a a   t t t t   t t t t t g g              

LSP start site 5'-3'

 
                                393       a         a             410                                  
                               

<<LSP start site

                                       
                                  t c g c c c c t c c c c c c a a a c          

CSB-2 Human mitoch

 
                                        c         c                                                
                                 

RNA 3' end>>

DNA 5' start>>

                             
    c a a a g a c a c c c c c                                                  

HSP start site

 
     

HSP >>

                                                                     
                                                                                                   
                                                                                                   
                                                                                                   

 

 

 

Results:

Using a g:u intermediate it  appears that there are similar sequences found in comparisons between some kinetoplastid CSBs, human mitochondrial. DNA CSBs and the translocation breakpoints of some leukemias, myelomas and lymphomas.  These alignments also appear even with the inserted sequences ("N additions") between the germline chromosomes.  For all the sequences used above there was at least one kinteoplast CSB that aligned at or near the translocation junctions.Mostly CSB-1 or CSB-2 were found. Many of the translocations also showed alignments with the Light strand initiation sites or Heavy strand initiation site sequences

 

Given the limited samples used above,  the prevalent alignments were CSB 2-like in Multiples myelomas, and follicular lymphomas . Interestingly in the four patients with Hodgkins, the sequences around the mbr-bcl2 sequences resembled CSB 1 and CSB 1 flanking sites of kinetoplastids. Also, the sequence around the ALL and AML cell lines which had an MV4:11 and RSV4:11 were like CSB 1 and a der21 in ALL had a double sequence resembling CSB 1. The corresponding der 14 had an added region which resembled CSB 2 and the CSB 2 region where there is a persistent RNA/DNA complex in mouse mitochondrial DNA replication.. (Lee and Clayton, 1996).

 

The T cell CLL had an alignment similar to a CSB 1-like sequence at the 3’ end of a primer RNA, and the X alpha germline had a sequence similar to the CSB 2 of hu mitochondria near the 3’ end of the RNA primer.

 

Again, the fact that these sequences were similar does not imply an active causal relationship with the origins of these translocations.

 

Discussion

 

  • The homologous sequence fits were subjective and no statistical analysis was done. Although the adage that you usually find what you're looking for might be true in all the above comparisons, there were many regions for which this didn't hold true.
  • Given the diversity of the samples, it was nearly impossible to determine when and how the translocations took place.  A low, but varying level of Bcl-2 translocations occur in the normal population.
  • Although there was similarity in most instances between the kinetoplastid mitochondrial minicircle CSB’s and translocated chromosomal junctions it didn’t demonstrate a direct relationship between the two. One would have had to describe and prove a mechanism(s) that were both genetic and/or somatic given the diversity of samples.
  • Alignments were also found to the kinetoplastid gap which is a single stranded section of DNA in an otherwise doublestranded DNA minicircle that is conserved as CSB 3. (Ntambi, J and Englund, P (1985)) It  is "filled in" just before cell replication. (more on this in a later Snippets entry)
  • Using a g:u intermediate, the sequence at the L strand promoter site (LSP) of human mitochondria resembled the human mitochondrial CSB-2 sequence. Each had long stretches of pyrimidines although the LSP region had a predominance ot "T"s and CSB-2 had a predominance of "C"s. In the same fashion, CSB-3 resembled the minor LSP/HSP initiation site.

 

 

 

CSBs and Replication

 

.CSBs are related to replication in minicircle mitochondrial DNA as they are shown to be binding sites of a protein involved in replication. In a paper, Abu-Einell et. al. (1999)) the author describes two sequences where a zinc finger protein, UMSBP binds during replication at the origins of replication of C. fasciculata L and H strands. The paper describes a 14 mer and a 12 mer binding site for the protein.

 

·        Note that the 14 mer (Ori H H14) GTAGGGGCGTTCTG is similar to CSB 1 of L. tarantolae and C. fasciculata and the 12 mer (Ori L UMS) GGGGTTGGTGTA is similar to CSB 3 of  all kinetoplasts.

 

·        Of note, is the fact that 5' end of Ori L UMS resembles 5’T2G4;  the telomeric repeat templated in the RNA portion of Tetrahymena telomerase (5’-CA2C4A2); mentioned in a  paper by Maizels and Weiner, (1994) “Phylogeny from function: Evidence from the molecular fossil record that tRNA originated in replication, not translation”, PNAS vol 91, p 6729-6734

 

 

 

CSBs may be involved as primer sites for L strand synthesis in transcription as well in mitochondrial DNA replication:

 

·        Of note is a similarity to the location where the 3’ end of a “DHP RNA” primer and 5’ end of a transcript are cleaved in part of CSB 2 in the replication region around the L strand promoter. (Chang and Clayton, 1985) Since there are still debates about the D loop and R loop regions of mitochondrial DNA it is hard to interpret the length of the RNA/DNA hybrids and their occurrence under different cellular conditions. In an article  Wanrooij, et. al. (2008) the authors argue that they have demonstrated that POLRMT (mitochondrial polymerase) transcribes long strands of RNA on ds DNA, but short RNA strands on ssDNA. The authors conclude that these short primers are able to provide pol-gamma with a binding site for continued DNA synthesis.

 

 

·        What may be of importance is that each DNA minicircle in T. equiperdum has a “Kinetoplastid gap” of a conserved sequence (CSB 3 or Ori L UMS). The H strand, although replicated as one long strand is thought to be replicated as in lagging strand synthesis with an RNA primer. The primer is clipped out and the DNA is filled in when the cell replicates (Ntambi, et al. 1998). The L strand is also replicated ala lagging strand synthesis, but with the more familiar short strands. It would be useful to find out if all the kinetoplasts have a kinetoplastid gap that is single stranded. Although CSB-3 is a universal sequence among kinetoplasts no one has shown that it is part of a kinetoplastid gap in all the kinetoplasts.

 

 

 

Abberrations in Mitochondrial DNA Associated with tumorigenesis.

 

  • In a paper, Sanchez-Cespedes et. al. (2001) the authors describe a sequence in the D loop of mitochondria as “a mutational hotspot in primary tumors”. It is a region of insertions and deletions in varying tumors reported in the paper. Assuming their nucleotide numbering is the same as other published researchers, the area they are describing is almost  the same as CSB 2 in human mitochondria.  Through a g:u complement, the sequence also aligns with the LSP initiation site.

 (Gray color is g:u complement, light green is CSB-2 sequence in human mitochondria.)

 

 

Mutational "Hot Spot" in primary tumors Sequence from Sanchez-Cespedes et. al. (2001)
c a a a c c c c c c c c c t c c c c c c g c t t c
        303                                        
g u u u g g g g g g g g g g g g g g g g u g g g g
c a a a c c c c c c c     t c c c c c   g c t t c
298                                               323

<<DNA 5' start

<<RNA primer 3' end CSB-2 human mitochondria

                                                 
c a a a t t t t   t c t t t t                    
                a           408                    

<<LSP initiation site 3'-5'

                     
                                c c c c g t t c  
                                kinteoplast CSB-2  
                                                 

 

Micro RNAs

 

 Ji, Q. et. al. (2009)  is a paper that elucidates the involvement of mir-34 (under p53 control) in tumor suppression. One of the proteins regulated by mir-34 is bcl 2.

 

Assuming that the sequence of mir-34 might be similar to a promoter-like sequence region of bcl 2, a comparison was made of mir-34 with the “illegitimate translocations” of chromosomes with a bcl 2 sequence. The following was found using Genbank AY194151.1 for the mir34 homo sapiens micro RNA sequence.

 

 

 

GENBANK S78526 bcl2...JH Human Ig
BCL2 JH Human Ig
t g c a g t g g t g c t t a a t g a c c a a c t g
g u g u u g u u g u g g g u u g u u g g u u g g u
t g c a g t g   t   c t t a g c g g t t g g t t Mir 34
g                             t                  
Sequences derived directly from g:u complement
g u g u u g u u g u g g g u u g u u g g u u g g u
                              g c t g g   t g g CHI
          g c t g t g g                         CHI
                g                                

 

 

Nothing can really be concluded from the above sequence, other than the the mir-34 sequence aligns with the translocation when using a g:u intermediate. Of note is that  the Chi hot spot sequence that binds with E. coli's RecBCD helicase-nuclease ( responsible for initiating homologus recombinations and DNA repair) is found from the g:u complement. What that means with respect to the above translocation is totally unclear and no conclusions may be drawn. It is hypothesized that the recBCD protein complex does create a single stranded loop during recombination/repair.  (Amundsen, S. K. (2007))

 

For now, one can’t say anything about the involvement of these particular sequences in the actual origination of the translocations other than they appear in more than one kind of B cell and T cell neoplasia. 

 

CSB-like sequences are found in tRNAs, and RSS sequences between the heptamer and nonamer to name two. So, the sequences may reflect other functions previously used, like replication or interchanges of small circular DNA or some overlap with lagging strand synthesis like the variation between transcription and RNA primer formation in a D loop or RNA loop found in mitochondrial DNA. .

 

References

Abu-Einell et. al. (1999), Universal Minicircle Sequence binding protein, A sequence specific binding protein that recognizes the Two Replication Origins of the Kinetoplast DNA Minicircle, The Journal of Biological Chemistry, vol 274, pp. 13419-13425

 

Amundsen, Susan K., (2007), Intersubunit signaling in RecBCD enzyme, a complex protein machine regulated by Chi hot spots, Genes Dev, vol 21, 3296-3307.

 

Chang, D. and Clayton, D, (1985), Priming of human mitochondrial DNA replication occurs at the light-strand promoter, PNAS vol 82, p. 351-355.

 

Davey, et. Al., (1988), Juxtaposition of the T Cell alpha locus (14 q 11) and a region 14 q 32 of potential importance in leukomegenisis by a 14;14 translocation in a patient with T cell chronic lymphocytic leukemia and ataxia-telangiectasia, PNAS vol 85, p. 9287-91

 

Gu, Y. et. al. (1992) The (4:11) (q21:23) chromosome translocations in acute leukemias involve the VDJ recombinase, PNAS 89, p.10464

 

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 Originally posted June 4, 2010, reformatted May 2011, then Jan 2013