Characterization of the nm23-M2, nm23-M3 and nm23-M4 mouse genes:comparison with their human orthologs

K. Massea,1, S. Dabernata, P.-M. Bourbona, M. Laroua, L. Amreina, P. Barrauda,2, Y. Perela,M. Camaraa,3, M. Landrya,4, M.-L. Lacombeb, J.-Y. Daniela,*

aBiologie de la Differenciation et du Developpement, Universite Victor Segalen-Bordeaux2, 146 rue Leo Saignat, 33076 Bordeaux, FrancebINSERM U402, Faculte de Medecine Saint Antoine, 75012 Paris, France

Received 3 March 2002; received in revised form 24 June 2002; accepted 8 July 2002

Received by E. Boncinelli

Abstract

The nm23 gene family is thought to be involved in physiopathological processes such as growth, differentiation and cancer promotion,

progression or metastasis. We report here the mouse nm23-M3 and nm23-M4 complementary DNA sequences and the genomic cloning,

characterization and tissue expression pattern of the nm23-M2, nm23-M3 and nm23-M4 genes, in comparison with their human and rat

orthologs and with the human nm23-H1 and mouse nm23-M1 genes. The organization and structure of the members of this gene family are

remarkably similar in human and rodents. Accordingly, the striking similarities between the human and mouse nm23 genes enable the use of

mouse transgenic and knock-out models for studying the role of nucleoside diphosphate kinase isoforms in human physiopathology. q 2002

Elsevier Science B.V. All rights reserved.

Keywords: Nucleoside diphosphate kinase; Gene organization; Expression

1. Introduction

Nucleoside diphosphate (NDP) kinase was initially

described as a housekeeping enzyme which catalyzes the

transfer of a g-phosphate between nucleoside tri- and di-

phosphates, via a phosphohistidine intermediate (Lascu

and Gonin, 2000, for review). Over the past 10 years,

however, new data have rekindled interest in this protein

which exists as different isoforms encoded by a family of

highly related genes. Currently, eight human nm23 genes

have been characterized. The first four human members of

this gene family, nm23-H1, nm23-H2, nm23-H3 (or DR-

nm23) and nm23-H4, express protein products which

possess NDP kinase activity and are named respectively

NDP kinase A–D (Lacombe et al., 2000, for review).

A growing set of evidence suggests that in addition to

their basic enzymatic activity, NDP kinase isoforms might

display other functions more or less related to signal trans-

duction pathways, cell growth and differentiation, embryo-

nic development, tumor progression, metastasis and

apoptosis (see for reviews, de S. Otero, 2000; Hartsough

and Steeg, 2000; Kimura et al., 2000; Lacombe et al.,

2000). Additionally, the human NDP kinase B has been

shown to be identical to the c-myc transcription factor

PuF (Postel et al., 2000b, for review) and was recently

shown to be involved in DNA repair processes (Postel et

al., 2000a). However, the precise role and mechanism of

action of these additional functions of the NDP kinases

are not yet well understood. Moreover, it was established

by in vitro experiments that NDP kinase is a heterohexa-

meric enzyme which contains randomly associated subunits

consisting of A and B isoforms (Gilles et al., 1991).

Although the four recombinant NDP kinases can be recon-

stituted into heterohexamers in vitro (Lascu et al., 2000), no

information is presently available either on the physiologi-

Gene 296 (2002) 87–97

0378-1119/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved.

PII: S0378-1119(02)00836-3

www.elsevier.com/locate/gene

Abbreviations: dpc, days post-coitum; ES cells, embryonic stem cells;

EST, expressed sequence tag; K-pn mutation, killer of prune mutation;

NDP kinase, nucleoside diphosphate kinase; PCR, polymerase chain reac-

tion; RT, reverse transcription

* Corresponding author. Tel.: 133-5-5757-1284; fax: 133-5-5624-0643.

E-mail address: jean-yves.daniel@biocell.u-bordeaux2.fr

(J.-Y. Daniel).1 Present address: Department of Biological Sciences, University of

Warwick, Coventry, UK.2 Present address: Wallenberg Neuroscience Center, Division of Neuro-

biology, BMC A11, 221 84 Lund, Sweden.3 Present adress: Laboratoire de Biochimie Medicale, CHU Cocody,

Abidjan, Cote d’Ivoire, France.4 Present address: INSERM EPI 9914, Institut Francois Magendie, rue

Camille Saint-Saens, 33077 Bordeaux, France.

cal effects of the relative concentration of each subunit in

the complex formation, or on a possible involvement in vivo

of the C and D isoforms in the hexameric conformation of

the protein in a living organism. Therefore, it is difficult to

know whether each of the many suspected biological roles

of the different NDP kinase isoforms is performed in a

particular cell compartment either through their interactions

with cell-specific factors or through the precise composition

of the NDP kinase heterohexamers in this cell compartment.

A unique animal model for studying the relationships that

exist between the different NDP kinase isoforms and their

biological function ex vivo or in vivo could be used to

address these questions. As a first step towards developing

a mouse model, we have previously described the organiza-

tion and expression pattern of the mouse gene encoding

NDP kinase A (Dabernat et al., 1999a). We describe here

the cloning, characterization and tissue distribution pattern

of the murine nm23-M2, nm23-M3 and nm23-M4 genes. The

properties of these genes are compared to those of their

orthologs previously published in the rat and to the corre-

sponding human genes, either published or identified from

genome sequences registered in database.

2. Material and methods

2.1. Cloning of the mouse nm23-M3 and nm23-M4

complementary DNAs (cDNAs)

A search with the FASTA program (Pearson and Lipman,

1988) in the EMBL sequence data base, for mouse

expressed sequence tags sharing more than 80% nucleotide

identity with human cDNA for nm23-H3 and nm23-H4

(Venturelli et al., 1995; Milon et al., 1997) led to the iden-

tification of several clones corresponding to the putative

mouse isoforms nm23-M3 (13 clones) and nm23-M4 (10

clones). The consensus sequence corresponding to the

coding domain and to the 3 0 untranslated region of the

mouse nm23-M3 and nm23-M4 cDNAs was deduced from

the sequence alignment of these clones by using the CLUS-

TALW program (Thompson et al., 1991). Reverse transcrip-

tion-polymerase chain reaction (RT-PCR) reactions were

then carried out on mouse liver and heart total RNA by

using gene-specific primer pairs designed from each

cDNA consensus sequence, namely 5 0TGA CCA TCT

TTG CTA ACC/5 0CAA CCT CCA CGC AGA AAT for

nm23-M3 and 5 0GAG GAA GCC ATT CTA CCC/5 0TGA

CAG AGG TAG TAG GTC for nm23-M4. Finally, the

precise sequences of the mouse transcripts were established

by direct sequencing of the amplification products. Two

cDNA clones displaying the closest similarity with the

sequence of each RT-PCR product (ID: MMAA8932; AC:

g1660383 for nm23-M3 and ID:MMA59517; AC:

AA059517 for nm23-M4) were finally obtained from

cDNA clone I.M.A.G.E. the United Kingdom Human

Genome Mapping Project (Cambridge), verified by

sequence analysis and corrected for their nucleotide

mistakes. Site-directed mutagenesis was used to introduce

a G in position 228 (GenBank AF288689) in nm23-M3

sequence and a G and a C in position 59 and 149, respec-

tively in nm23-M4 sequence (GenBank AF288690). Three

rounds of PCR were necessary for each insertion. During the

first two rounds, the fragments upstream and downstream of

the insertion carried by the primers were amplified. Then

these partially overlapping PCR products were used

together as templates to amplify the entire coding sequence,

resulting in the production of a corrected sequence. Taq

DNA polymerase from Boehringer was used according to

the manufacturer’s recommendations (Boehringer

Mannheim, Germany). The corrected cDNAs were then

inserted into the cloning vector pBluescript II SK2 (Strata-

gene, La Jolla, CA, USA) for the production of specific

probes. The nm23-M3 and nm23-M4 cDNA sequences

were submitted to GenBank databases.

2.2. Cloning of the mouse nm23-M2, nm23-M3 and nm23-

M4 genes

2.2.1. Screening of the genomic library

The genes were isolated by screening an embryonic stem

cell (ES cell) genomic library constructed in the lEMBL3

vector, with 32P labeled probes corresponding to the 3 0

untranslated regions of mouse nm23-M2, nm23-M3 and

nm23-M4 cDNA. The ES library was a generous gift from

Prof. P. Chambon, (IGBMC, Ilkirch, France). Positive

clones were purified by three other subsequent screening

rounds, the first two by using the same probes and the last

one with a specific probe located in the 5 0 region of each

gene: Since in human and in rat, nm23-1 and nm23-2 genes

are located on the same chromosome and are distant by less

than 5000 bp (Shimada et al., 1993; Seifert et al., 1995), the

3 0 untranslated domain sequence of the mouse nm23-M1

gene was used as a probe for purifying nm23-M2 gene

clones containing the full length 5 0 upstream regulatory

domain. The genes nm23-M3 and nm23-M4 were character-

ized by using specific cDNA probes chosen in their 5 0 trans-

lated presequence region and were registered in a library

data base (GenBank AF288691 and GenBank AF288692,

respectively).

2.2.2. Identification of exon-intron boundaries of nm23-2,

nm23-3 and nm23-4 genes

The putative intron boundaries of the mouse genes were

positioned by reference to the organization of the mouse

nm23-M1 gene (Dabernat et al., 1999a). Primers were

designed from exon sequences adjacent to each putative

junction. By using these primers, the introns of each purified

phage were amplified by PCR, and then the regions neigh-

boring the exon-intron borders were sequenced. The follow-

ing PCR primer pairs were used to characterize the exon/

intron boundaries of the different genes. For the nm23-2

gene: 5 0GAC CCA CCG GCT TTC GGT/5 0GAG GTT

K. Masse et al. / Gene 296 (2002) 87–9788

GGC CAT GGT CCT (intron 1); 5 0CTC GAG CGT ACC

TTC ATT/5 0TCA TGT ACT TCA CCA GCC (intron 2); 5 0

CTC TGA AGA ACA CCT GAA/5 0GCC AAC TTG AAT

GCA GAA (intron 3) and 5 0TGG GAG GGG CTC AAT

GTG/5 0CAT CCT GTC AGT GGG ATG (intron 4). For the

nm23-3 gene: 5 0ATG ATC TGT CTG GTG CTG/5 0CTA

GCT TCA GTG CCA CCA (intron 1); 5 0CGT GAA CGA

GCG CAC GTT/5 0CAC GGG GCC AGA ACT CAT (intron

2); 5 0CGA AGA GCT ACT GCG GGA/5 0CCT ATG AGG

GCT CGC GAA (intron 3); and 5 0GGG ACG CCA TGC

CCG GTA/5 0TAT GAA AGG CAA CAA GGG (intron 4) –

and finally for the nm23-4 gene: 5 0ATG GGC AGC CTT

TTC GGG/5 0GCC TCT CAA AGC GTT GTA (intron 1);

5 0CAA GAG CGG ACG CTG GTT/5 0CAC CAC AGG

CCC AGA GCT (intron 2); 5 0ACC AGA AAG CAT CCT

TGC/5 0CTA TCA TGG CCC TTG AGA (intron 3) and

5 0ACT CAA CAG AGG CAG CCC/5 0GTC TCT CCT

TTG AGG CAG (intron 4).

2.3. DNA Sequencing and PCR amplifications

DNA sequence analysis was performed on double-

stranded plasmids, phages or PCR products by the Sanger

dideoxy chain termination method using a commercially

available kit (BigDye Terminator from Perkin Elmer

Corporation, Norwalk, CT, USA) and primers designed to

obtain overlapping sequences on both DNA strands. PCR

amplifications were carried out using the Goldstarw taq

DNA polymerase (Eurogentec Seraing, Belgium) according

to the manufacturer’s recommendations.

2.4. Northern blot analysis

Northern blot analysis was performed using a commercial

adult mouse RNA poly(A)1 blot (MTN Blot, Clontech, Palo

Alto, CA, USA). The nm23-M1, nm23-M2, nm23-M3 and

nm23-M4 specific probes were produced by PCR amplifica-

tion from the 3 0 untranslated region of each cDNA. The

primers used were 5 0TAG-GAC-GGT-GCC-GGT-TTT/

5 0TCT-AAT-GAA-TTC-TCT-GTT for nm23-M1, 5 0TAG-

ACA-TGA-AGA-AAC-CAG/5 0CAG-TTC-CAA-AGT-

CTT-TAT for nm23-M2, 5 0GAT GTT ATT GCA GTC

AGC/5 0TAT GAA AGG CAA CAA GGG for nm23-M3

and 5 0CTT GCA CTG CCT TCT GCA/5 0GTC TCT CCT

TTG AGG CAG for nm23-M4. A cDNA encoding the

ubiquitous ribosomal protein (36B4) was used as a control

probe to normalize RNA loading (Laborda, 1991). The

36B4 clone was kindly provided by Prof. P. Chambon.

The blots were analyzed using a phosphorimager (Molecu-

lar Dynamics, Sunnyvale, CA, USA) and quantitative analy-

sis was carried out by the ImageQuant software (Molecular

Dynamics). The relative messenger RNA (mRNA) abun-

dance of each nm23 isoform in each tissue extract was

normalized by reference to the amount of the control

36B4 mRNA and standardized to heart nm23 mRNA

contents. Probe labeling was performed as already described

(Dabernat et al., 1999b), and hybridization was done

according to the recommendation of the membrane manu-

facturer.

2.5. RNase mapping

Total RNAs from kidney tissue of adult mice were

extracted by using Trizol (GIBCO BRL) according to the

manufacturer’s protocol. Three recombinant pBS-SK2 plas-

mids were used in this study, namely pM2, pM3 and pM4.

Each of them contains the first exon of each mouse gene, as

defined from its 3 0 splicing site, flanked by its 5 0 adjacent

genomic DNA domains. The different inserts were 394, 430

and 440 bp long respectively. The radioactive sense and

antisense probes were synthesized according to the plasmid

supplier’s recommendations (Stratagene) using the T3 and

T7 RNA polymerases. Hybridizations and RNase digestions

were performed as previously described (Rio et al., 1988).

2.6. In situ hybridization

In situ hybridizations were carried out on C57BL/6 15.5

days post-coitum mouse embryos. The oligonucleotide

probes sequences were complementary to nucleotides

551–596 and 660–704 of the mouse nm23-M1 cDNA

(accession number U85511 GenBank), to nucleotides

477–522 and 524–569 of the mouse nm23-M2 cDNA

(Urano et al., 1992), to nucleotides 486–528 and 589–633

of the mouse nm23-M3 cDNA (numbering for initiation

ATG codon) and to nucleotides 599–643 and 771–815 of

the mouse nm23-M4 cDNA (numbering from initiation

ATG codon). The oligonucleotides were labeled by tailing

and were purified as previously described (Dagerlind et al.,

1992). In situ hybridization, autoradiography and control

experiments, using an excess of cold probes, were

performed as described previously (Dabernat et al.,

1999b). Tissue distribution was deduced from autoradio-

graphic films.

3. Results

3.1. Cloning of the mouse nm23-M3 and nm23-M4 cDNAs

and deduced amino-acid primary structure

The nm23-M3 cDNA (GenBank, accession number

AF288689) displays a 507-nucleotide-long translated region

encoding a putative protein of 169 amino acids (Fig. 1). The

first ATG possessed a Kozak-like sequence (cgccATGa)

and a single AATAAA polyadenylation consensus signal

was present 152 nucleotides downstream from the TAG

stop codon. The deduced primary structure of the NDP

kinase C mouse protein displayed 88% amino acid identity

with that of its human counterpart. By comparison with

human NDP kinase A, a NH2 17 amino acid hydrophobic

polypeptide extension was present in the mouse NDP kinase

C. This extension is identical to the corresponding domain

of the rat putative protein (GenBank, accession number

K. Masse et al. / Gene 296 (2002) 87–97 89

AY017337) and shares the same first 14 amino acids with

the human NDP kinase C. A Lys, which is equivalent to the

Arg 114 found in mammalian NDP kinase A and B was

present in the human and mouse NDP kinase C.

The nm23-M4 cDNA (GenBank, accession number

AF288690) contained an open reading frame of 558 nucleo-

tides for a predicted protein 186 amino acids long (Fig. 1).

The nucleotide sequence around the first ATG conforms to

the consensus Kozak sequence (caacATGg). The TGA stop

codon was followed by an untranslated domain of 292

nucleotides and the poly(A)1 tail was located 18 nucleotides

downstream from the ATTAAA polyadenylation signal. The

mouse protein sequence is 82% identical to its human coun-

terpart and displays several characteristics already described

for human NDP kinase D (Milon et al., 1997). The NH2

terminus extension of the mouse NDP kinase D was 32

amino acids long (33 amino acids in human) and shared

only 50% identical amino acids with the corresponding

human protein. However, this extension displayed a high

level of Arg, Leu and Ser (13 residues), Gly or Pro (10 resi-

dues) but only two Val, and no Lys, Asp, Glu or Ile. This

amino acid composition is characteristic of a mitochondrial

targeting sequence (von Heijne, 1986). By computer analysis

(von Heijne, 1986) and according to the numbering conven-

tion used in Fig. 1, a cleavage site may be postulated between

Pro(26) and Ser(25), of the protein primary sequence. This

site corresponds to the putative proteolytic cleavage site

between residues His(26) and Gly(25) already described

for the human protein (Milon et al., 2000). As already

found in human (Milon et al., 1997), the mouse NDP kinase

D presents a Ser(96) instead of the Pro(96) which is

constantly found in isoforms A, B and C. This substitution

is equivalent to the killer of prune (K-pn) mutation of the

Awd/NDP kinase of Drosophila (Timmons et al., 1995).

Moreover, the carboxy terminus of NDP kinase D is two

amino acids (PA) longer than that of the other mammalian

NDP kinases, which normally end with a tyrosine-glutamate

(YE) motif. Finally, neither NDP kinase C nor D showed the

concomitant presence of the three amino acids previously

described as being essential for the DNA binding of NDP

kinase B, namely Arg 34, Asn 69 and Lys 135 (Postel et

al., 2000b, for review). By contrast, all the mammalian

NDP kinases have the Lys 12, associated with the predicted

DNA repair functions of the protein (Postel et al., 2000a).

K. Masse et al. / Gene 296 (2002) 87–9790

Fig. 1. Primary sequence comparison between the NDP kinase domains of the mammal Nm23/NDP kinase proteins. The amino acid sequences of the different

Nm23/NDP kinase proteins are aligned on the Nm23-H1 sequence. The numbering above refers to the Nm23-H1 sequence. The intron positions are located

using vertical lines and are numbered in bold type. The consensus sequence in bold type represents identical residues within all the nm23 sequences while

regular type is used for the residues conserved between nm23-1 and nm23-2 isoforms. Gaps are indicated by dashes. Sequences are from human (Gilles et al.,

1991; Venturelli et al., 1995; Milon et al., 1997 and this paper), rat (Kimura et al., 1990; Shimada et al., 1993; Lutz: 2001 GenBank accession number

AYO17337) and mouse (Steeg et al., 1988; Urano et al., 1992 and this paper).

3.2. Organization of the human and mouse nm23-2, nm23-3

and nm23-4 genes

As shown in Figs. 1 and 2, the first four members of the

nm23 gene family characterized so far in human, rat and

mouse exhibit the same pattern of organization. All of them

have four introns flanked by five exons. The position of the

fourth intron of the nm23-H3 gene, as reported on the NCBI

site, is not convincingly located. It was therefore corrected

by reference to the other genes (Fig. 2) and according to the

Breathnach and Chambon (1981) law. On a multiple nucleo-

tide sequence alignment, the exon-intron borders between

introns two, three and four are strictly conserved (Figs. 1

and 2) and the position of the first intron is always the same

for one particular gene of this family, whatever the species.

Finally, the overall gene sizes are about 9000 nucleotides for

nm23-1 genes, 6000 nucleotides for nm23-2 genes, 1000

nucleotides for nm23-3 genes and 3500 nucleotides for

nm23-4 genes.

3.3. Promoter organization of human and mouse nm23-1,

nm23-2, nm23-3 and nm23-4 genes

The 1800 bp 5 0 region upstream the first exon of the four

human and mouse orthologous genes were compared for

their nucleotide distribution and were studied by computer

analysis with the Prestridge (1991) software for the occur-

rence of putative transcription factor binding sites (Fig. 3).

The promoters of the human and mouse genes do not display

consensus TATA or CAAT boxes or Inr sequences. A large

number of putative transcription activator binding sites are

shared by the four promoters, especially AP2, NF1, SP1,

TCF/LEF1 and response elements to glucocorticosteroid

receptors. These sites are clustered in overlapping domains

and are distributed throughout the analyzed gene 5 0-

upstream regions. In addition, other putative binding sites

such as AP1, C/EBP, CREB, LBP-1, MyoD, Pit1 or PuF are

found as a few units. Some of them are present in every

promoter analyzed, (LBP-1, MyoD or Pit-1) and the others

not. Surprisingly, when considering the same orthologous

genes, some putative binding sites are present in a species

but are absent in others (PuF in nm-23H2 versus nm23-M2

for instance).

3.4. Identification of the transcription initiation sites of

nm23-M2 and nm23-M3 genes

RNase protection assays were performed to determine the

transcription start sites of the mouse nm23-M2 and nm23-

M3 genes (Fig. 4): both gene transcripts were initiated from

a wide range of sites. The nm23-M2 gene displayed a major

start site giving rise to a first exon 68 nucleotides long.

However, the gene transcription was also initiated from

other minor sites. These sites were organized into two clus-

ters, giving a first exon ranging from 48 to 52 nucleotides

long on one hand, and 68–106 nucleotides long on the other.

The dominant start site of the nm23-M3 gene defined a first

exon 145 nucleotides long. Minor transcription initiation

sites were also observed leading to a first exon 121 or 167

nucleotides long. On repeated experiments, no signal was

found using the antisense M4 riboprobe so the transcription

initiation sites of the nm23-M4 gene could not be deter-

mined.

K. Masse et al. / Gene 296 (2002) 87–97 91

Fig. 2. Exon intron organization of human, rat and mouse nm23 genes. The capitals H, R and M refer to human, rat and mouse respectively. The orthologous

genes of the different species studied were assigned the same final number. Accordingly, nm23-R1 and nm23-R2 refer to the rat NDP kinase b and NDP kinase

a, respectively. The exons are represented by boxes and the introns are represented by a horizontal line. The total size, in base pairs, of the different exons and

introns is indicated in the corresponding columns. The size in base pairs of the coding sequence of each exon is bracketed.

K. Masse et al. / Gene 296 (2002) 87–9792

Fig. 3. Promoter organization of nm23-2, nm23-3 and nm23-4 genes. The 1.8 kb sequence containing the 5 0-flanking region of each gene was analysed with the

Prestige software method (Prestridge, 1991). DNA consensus binding sites for known transcription factors are localized within the promoter region by

reference to the major transcription initiation site numbered as nucleotide 1.

3.5. Tissue expression of nm23-M3 and nm23-M4 genes

The human and rat nm23 genes are known to be ubiqui-

tously expressed although the level of expression of the

different gene products is tissue-dependent (Lakso et al.,

1992). The differential expression of the nm23-M1 and

nm23-M2 transcripts has already been published (Dabernat

et al., 1999a). In the present paper, the expression pattern of

the nm23-M3 and nm23-M4 genes was studied by reference

to that of nm23-M1 and -M2 genes, using either commer-

cially available northern blots of adult tissue or in situ hybri-

dization of 15 days post coitum old embryos.

By northern blot analysis, a single signal was identified

for both nm23-M3 (0.9 kb) and nm23-M4 (1 kb) in the

different mouse tissue extracts analyzed (Fig. 5). By using

different independent northern hybridization assays, the

nm23-M1 and nm23-M2 genes appeared to be more highly

expressed than nm23-M3 and nm23-M4, and the differences

in transcript concentrations in the tissues were higher for

nm23-M1 than for nm23-M2. The four gene transcripts were

found in liver and kidney at a relatively high level compared

with the other tissues tested. As expected, the two nm23-M1

transcripts resulting from the use of different polyadenyla-

tion signals and already discussed in our previous paper

(Dabernat et al., 1999b) were found again in the commercial

adult mouse RNA poly(A)1 blot. Moreover, we confirm that

nm23-M1 mRNA is very highly expressed in the central

nervous system and that the concentration of nm23-M2

mRNA is high in heart and intermediate in skeletal muscles.

The level of nm23-M3 transcripts is moderate in heart,

brain, spleen and lung. Finally, nm23-M4 mRNA was

detected only in heart, kidney and liver tissues and at very

low levels.

In situ hybridization experiments performed in 15-day

post coitum embryos (Fig. 6) showed comparable results

in terms of mRNAs distribution. Nm23-M2 seemed to be

ubiquitously expressed while nm23-M1 was found espe-

cially in the central and peripheral nervous system, sensory

organs and thymus. The nm23-M3 expression pattern is very

close to that of nm23-M1 in the sense that the corresponding

transcripts were found in the nervous tissue and thymus. By

contrast, the expression level of nm23-M4 transcripts was

under the sensitivity threshold of the technique used.

4. Discussion

On their discovery, NDP kinases were ascribed a single

physiological function, namely the synthesis of nucleoside

triphosphate other than ATP (Parks and Agarwal, 1973, for

review). However, an increasing set of observations

suggests that these proteins are multifunctional and regulate

a fascinating variety of cellular activities including prolif-

eration, development and differentiation (see for review,

Lacombe et al., 2000, and the other papers of the same

issue). Most of these effects are not related to the protein

phosphotranspherase activity and the results obtained by the

different groups do not follow an obviously discernible

pattern. Consequently, to date there is no unifying hypoth-

esis for the physiological role of NDP kinases. Our aim was

to compare the first four NDP kinase isoforms in human rat

K. Masse et al. / Gene 296 (2002) 87–97 93

Fig. 4. RNase protection analysis of the nm23-M2 and nm23-M3 transcrip-

tion initiation sites. Total RNA (50 mg) from adult mouse kidney tissue was

incubated with riboprobes spanning the first exon and the 5 0 adjacent region

of the nm23-M2, nm23-M3 or nm23-M4 genes. These riboprobes were

retrotranscribed from the plasmids pM2, pM3 and pM4 and were 394,

430 and 440 nucleotides long, respectively. The annealed products were

digested with RNase and the protected fragments were analyzed on a dena-

turing 8% polyacrylamide gel as described previously (Rio et al., 1988).

The size of the protected fragments was assessed from the 3 0 end of the first

exon of the gene in comparison with a Sanger sequencing reaction. Lane 1:

sense pM2 riboprobe; lane 2: antisense pM2 riboprobe; pM2 sequencing

reaction; lane 3: sense pM3 riboprobe; and lane 4: antisense pM3 riboprobe.

and mouse in order to propose the mouse as a convenient

animal model for a thorough investigation of the NDP

kinase functions.

Our study clearly demonstrates that the first four human,

rat and mouse nm23 genes, share a quite similar overall

genomic organization: they all display five exons and the

splicing sites of the last four exons are rigorously conserved.

This conclusive evidence has never been reported so far, so

the DR-nm23 (Martinez et al., 1997) and the recently

described nm23-M3 (Kargul et al., 2000) genes, which

were described not to strictly follow the organization pattern

here described, need to be corrected. In addition, it may be

anticipated that the genomic organization of the nm23-R3

gene, whose cDNA sequence was recently reported

(GenBank accession number AYO17337), would conform

to our consensus structure model once characterized.

All the nm23 genes studied in this paper present a very

high identity ratio in their nucleic and amino acid

sequences: the position of the different a-helix and b-

sheet structural domains are conserved (Janin et al., 2000)

K. Masse et al. / Gene 296 (2002) 87–9794

Fig. 5. Tissue distribution of mouse nm23-M1, nm23-M2, nm23-M3 and nm23-M4 mRNAs. (A) A commercial blot containing 2 mg poly(A)1 RNAs of the

indicated mouse tissues was hybridized with labeled probes specific to either nm23-M1, nm23-M2, nm23-M3, nm23-M4 or 36B4 prepared as described in the

material and methods section. 36B4 is a housekeeping gene cDNA coding for a ubiquitous ribosomal protein (Laborda, 1991) used in this paper as a control for

the amount of loaded mRNA. The autoradiographs were analyzed using a phosphorimager (Molecular Dynamic, Sunnyvale, CA, USA). The relative levels of

the different mRNAs present in each line were quantified by ImageQuant software (Molecular Dynamics) and expressed as a percentage of the control 36B4

mRNA level. All the results were normalized to heart mRNA content. (B) nm23-M1 mRNA, longer transcript: white bar, shorter transcript: black bar; (C)

nm23-M2 mRNA; (D) nm23-M3 mRNA; and (E) nm23-M4 mRNA. H, heart; B, brain; S, spleen; Lu, lungs; Li, liver; SM, skeletal muscle; K, kidney; and T,

testis.

as well as the functional residues associated with either the

NDP kinase function (Lascu and Gonin, 2000) or with the

recently described DNA repair function (Postel et al.,

2000a). In addition, the four genes may be distributed into

two subgroups, the first containing the nm23-1 and nm23-2

genes and the second containing nm23-3 and nm23-4 genes.

The genes belonging to the same subgroup show a more

closely related sequence and share common features such

as the position of the translation start site. In each subgroup,

however, some individual properties such as the overall size

of the genes, the size of their introns or the presence of the

K-pn mutation can be considered as distinctive gene marks.

The prominent similarities found between the mammal

nm23 genes studied here strengthen the concept that the four

genes are generated by a series of duplications from a puta-

tive ancestor gene, which occurred before the divergence of

the different mammal species (Milon et al., 1997). The more

recently discovered nm23-H5, nm23-H6 and nm23-H7

genes which show an unequivocally different organization

pattern (Lacombe et al., 2000, for review) are clearly diver-

gent. They should not be considered as strictly belonging to

the same gene series and their products are likely to fulfill

quite distinct cellular functions.

The existence of four closely related but different NDP

kinase isoforms might result in a functional diversity. It is

tempting to postulate that the polypeptide extension found

in NDP kinase C and D could be associated with a protein

addressing property towards an assigned cell compartment.

Even though no known consensus polypeptide addressing

motives have been identified in NDP kinase C, the striking

sequence conservation of the protein NH2 terminus found in

human (Venturelli et al., 1995), rat (Lutz, GenBank acces-

sion number AY017337) and mouse (this paper) suggests

that the protein domain may carry an essential cellular func-

tion more or less related to cell membrane (Martinez et al.,

1997; Barraud et al., 2002) or mitochondrial targeting

(Negroni et al., 2000). In human, the mature NDP kinase

D has been found to be associated with the outer and inner

mitochondrial membranes, and the cleavage of its prese-

quence between the His 28 and the Gly 29 seems to be a

prerequisite for that protein localization (Milon et al., 2000).

Even if the 32-amino-acid NH2 terminal extension of the

mouse NDP kinase D shows only a 50% amino acid

sequence identity with its human counterpart, this protein

also presents the characteristics of mitochondrial targeting

in terms of amino acid sequence composition, (von Heijne,

1986) and shows a putative cleavage site located at the same

position. Therefore, the mouse NDP kinase D is also likely

to be a mitochondrial addressed protein. Remarkably, both

the human and mouse NDP kinase D naturally possess the

Pro96Ser substitution equivalent to the K-pn mutation of the

Drosophila Awd/NDP kinase. This mutation has been

shown to affect the folding/assembly pathway of the Droso-

phila (Lascu et al., 1992) and human (Milon et al., 2000)

NDP kinases without modifying their enzymatic activity

once folded and assembled into the mature hexameric

complex. Although not demonstrated to date, the presence

of the K-pn mutation in NDP kinase D might play a role in

its mitochondrial targeting by destabilizing the protein fold-

ing and thus making the trans-membrane transfer easier.

The occurrence of a differential expression pattern of the

nm23 genes has already been reported for the human and rat

NDP kinases (see for reviews: Hartsough and Steeg, 2000;

Kimura et al., 2000; Lacombe et al., 2000). This is clearly

documented at the mouse RNA level (Dabernat et al., 1999a

and this paper) and calls for two comments: Firstly, NDP

kinase B seems to be more ubiquitously expressed than the

isoforms A, C and D. Considering that all the four recom-

binant NDP kinases can be involved in the heterohexamer

assembly in vitro (Lascu et al., 2000), an exciting hypothesis

to be tested might be that NDP kinase B is a common

subunit present in every cell and that the three other

isoforms could locally interfere with this subunit to generate

an heterohexameric protein and perform a specific function

at a cellular or sub-cellular level, in relation to the relative

composition of the complex. Secondly, gene transcription

initiation depends on the binding of general transcription

factors to proximal DNA-binding elements that lead to an

increase in the affinity of the basal transcriptional machinery

for a particular promoter in a continuum having several

K. Masse et al. / Gene 296 (2002) 87–97 95

Fig. 6. Film autoradiograms of sagittal sections of mouse embryo at E15.5

after in situ hybridization with 35S-labeled probes complementary to nm23-

M1 (a); nm23-M2 (b); nm23-M3 (c); and nm23-M4 (d) mRNA. (a) A strong

signal corresponding to nm23-M1 mRNA is seen in nervous system and

also in the thymus and intestine. (b) nm23-M2 mRNA displays a wide

distribution throughout the whole embryo. (c) Expression pattern of

nm23-M3 is similar to that of nm23-M1 with a preferential expression in

nervous system, thymus and intestine. (d) nm23-M4 mRNA is hardly

detectable at this stage. However, a weak signal is seen in the intestine.

br: brain; th: thymus; he: heart; in: intestine; and sc: spinal cord. Bar: 5 mm,

same magnification for all.

levels of regulation. In addition, regulatory proteins,

referred to as activators, bind to specific sequences in the

vicinity of the promoter and influence the basal transcrip-

tional machinery through intermediary factors (cofactors)

that convey signals from activators to transcription proteins

(Veenstra and Wolffe, 2001, for review). Thus, it was tempt-

ing to look for the occurrence of a differential combinatorial

organization in transcription factor binding elements of the

proximal and distal domains of the four nm23 gene promo-

ters to tentatively explain their tissue specific regulation.

Disappointingly, the promoters of all the human and

mouse genes appeared to be similarly organized and no

clear-cut feature in the distribution of their putative tran-

scription factor binding sequences was found to clearly

explain the differences observed in the gene expression

patterns. Subtle dissimilarity in the promoter organization,

which cannot be revealed by our approach, may account for

the specific transcription patterns. Moreover, the actual

functional significance of those putative binding sites

remains to be established by appropriate methods (gel mobi-

lity shift, footprint and reporter gene analysis). Thus, to

unravel the mechanisms by which the spatial and temporal

expression of nm23 genes is controlled, additional studies

are required using suitable and informative models. Our

data constitute a good starting point.

Mouse nm23 genes express several transcripts which

diverge in their 5 0 or 3 0 untranslated domains and whose

relative levels may vary depending on the cells or tissues

considered. The isoforms A–C are known to have different

transcription start sites but the use of an alternative poly-

adenylation signal seems to be restricted to NDP kinase A

(Dabernat et al., 1999a and this paper; Dabernat et al.,

1999b). In a previous paper (Dabernat et al., 1999b), the

nm23-M1 longer transcript expression was found to be

prominent in mouse central nervous system but the func-

tional significance of the nm23-1 mRNA 3 0untranslated

region length is unclear to date. By contrast, the use of

different transcription start sites for the rat nm23-R2 subunit

is associated with differences in transcript translation effi-

ciencies leading to a post-transcriptional regulation of the

protein concentration (Kimura et al., 2000, for review).

Accordingly, it would be interesting to establish whether

those last results can be extended to the other NDP kinase

isoforms in the different mammal species.

Deciphering nm23-related functions in human health and

disease will require understanding of the tissue-specific

regulation of the genes, as well as the physiological proper-

ties and mechanism of action of their products. A mouse

model would be suitable for these purposes. The data

presented herein indicate that the rodent nm23 genes and

their products share very high similarities with their human

counterparts in terms of gene organization, tissue expression

and protein primary structure. Therefore, the generation of

nm23 knockout mouse models that we are developing

should not only provide insight into the evolution of the

function of these genes, but also help in understanding the

function of the composition of the different sub-units

present in a specific tissue at a given step of development

or during the onset of a cellular pathological process.

Acknowledgements

Karine Masse was supported by a grant from the Comite

Departemental des Pyrenees Atlantiques of the French

Ligue Nationale Contre le Cancer. This work was supported

by the same institution. We are grateful to Prof. Ioan Lascu

(IBGC, Bordeaux, France) for fruitful discussion. We would

also like to thank Prof. Pierre Chambon and Dr Andree

Dierich (IGBMC, Illkirch, France) for providing us with

the ES H1 cell line, Prof. Michel Darmon (Universite Victor

Segalen, Bordeaux, France) for the gift of cDNA

I.M.A.G.E. clones, Catherine Robinet and Helene Rober-

teau for their technical assistance, and Prof. Elisabeth

Oliver-Jones (Warwick, UK) for stylistic advice. The

Mouse Facility of the University of Bordeaux2 was funded

by the Conseil Regional d’Aquitaine, the French Ligue

Nationale Contre le Cancer and the French Fondation

pour la Recherche Medicale.

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