The genes for tumor necrosis factor (TNF-alpha) and lymphotoxin (1NF-beta) are tandemly
arranged on chromosome 17 of the mouse
S.A.Nedospasov1l2, B.Hirt1, A.N.Shakhov2, V.N.Dobrynin3, E.Kawashimar, R.S.Accolla5 and
'Swiss Institute for Experimental Cancer Research, CH-1066 Epalinges, Switzerland, 2Institute of
Molecular Biology, and 3Shemyakin Institute of Bioorganic Chemistry, USSR Academy of Sciences,
Moscow, USSR, 4Biogen S.A., Route des Acacias 46, CH-1227 Carouge/Geneva and 5Ludwig
Institute for Cancer Research, Lausanne Branch, CH-1066 Epalinges, Switzerland
Received 31 July 1986; Accepted 9 September 1986
We have isolated clones containing the gene for tumor necrosis factor
(TNF-alpha) from a mouse genomic library.
the TNF-alpha gene also hybridized to a human lymphotoxin (TNF-beta) probe.
We constructed a restriction enzyme cleavage map of a 6.4 kb region from one
of the genomic clones.
From partial sequencing data and hybridizations with
exon-specific oligonucleotide probes, we conclude that this region contains
the mouse TNF-alpha and TNF-beta genes in a tandem arrangement, that they are
separated by only about 1100 bases, and that their intron-exon structure is
very similar to that seen in man.
We probed genomic blots of DNA from
human/mouse hybrids containing single mouse chromosomes for the presence of
the mouse TNF genes.
The results show that the genes are located on mouse
chromosome 17, which also contains the major histocompatibility complex.
Therefore, both the mouse and the human TNF genes are tandemly arranged and
located on the same chromosome as the MHC.
Four out of five clones containing
Mononuclear cells of the reticuloendothelial system secrete, after
stimulation, cytokines that have a necrotic effect on tumors, and are
cytotoxic to many types of cultured transformed cells (1-6).
confusing mass of data has been recently clarified by the demonstration (7, 8)
that most if not all cytotoxic activities produced by mononuclear cells can be
attributed to the products of only two genes, which code for tumor necrosis
factor (hereafter designated as TNF-alpha) and for lymphotoxin (TNF-beta).
TNF-alpha has been shown to be identical to cachectin (9), the protein
responsible for the wasting response associated with some bacterial and
parasitic infections (10), and to the monocyte-derived cytotoxin (7).
cachectin activity of TNF-alpha seems to be due to a specific inhibition of
the expression of lipoprotein lipase in adipocytes (11, 12).
its cytotoxic activities on tumor cell lines, TNF-alpha behaves like a
cytokine for many types of normal cells:
histocompatibility antigens and of other surface markers on normal endothelial
In contrast to
it induces the expression of type I
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Volume 14 Number 19 1986
Nucleic Acids Research
production of collagenase and prostaglandin E2 (16).
reflect TNF-alpha's ability to stimulate the release of IL-1 by endothelial
TNF-alpha seems to be produced mostly, if not exclusively, by
adherent cells of the monocyte / macrophage lineage (2,
is produced by T lymphocytes (18,
it was originally detected as a secreted protein in the culture medium of
stimulated lymphocytes, there is evidence for its specific delivery to target
cells by cytotoxic T-cells and its involvement in the subsequent degradation
of the target cell DNA (20, 21).
There are many similarities in the induction and the mode of action of
the two TNFs.
In populations of peripheral blood mononuclear cells, their
synthesis is induced by bacterial endotoxins, but also by mitogens, by phorbol
esters, and by the T-cell growth factor, interleukin-2 (2, 8, 22).
cytotoxic effects that they exhibit toward tumor cell lines are quite similar
Their cytotoxicity is synergistic with that of immune interferon,
another cytokine, and it has been postulated that the in vivo effects of TNF-
alpha/-beta and immune interferon are dependent upon each other (22, 24, 25).
This hypothesis has been strengthened by the recent demonstration that immune
interferon can enhance the expression of TNF receptors in several tumor lines
Because of the obvious potential of these molecules in clinical
applications, great efforts have been made to characterize their genes and to
have them expressed to high levels in prokaryotic hosts.
brought about the cloning and sequencing of the genes and cDNAs of human TNF-
alpha and TNF-beta (28-32), of the cDNA of mouse TNF-alpha (33,34), and of the
gene and cDNA of rabbit TNF-alpha (35,36).
abundant sources of pure recombinant TNF-alpha and TNF-beta.
of both proteins from human sources revealed a significant structural
The homology also extends, albeit to a lesser degree,
region upstream of the two genes (31), suggesting similarities in the way
their expression is regulated.
Cytogenetic studies have shown that the genes
for human TNF-alpha and TNF-beta are located on the short arm of chromosome 6,
in close proximity to the major histocompatibility complex (31).
pendent analysis of a lambda clone containing the TNF-alpha gene has
established that the genes for TNF-alpha and TNF-beta are tandemly arranged in
the human genome (32, and CSHSQB,
14), enhances the growth of fibroblasts (15), and stimulates the
The latter effect may
These efforts have
As a result, there are now
as the secreted forms share 35% of their amino acid sequence
to the DNA
Nucleic Acids Research
In the present paper, we show that the genes for TNF-alpha and TNF-beta
are adjacent to each other in the mouse genome, and that their configuration
and nucleotide sequence are highly conserved between mice and humans.
Moreover, they are located on chromosome 17, which also contains the mouse
major histocompatibility complex.
MATERIALS AND METHODS
Isolation of the TNF genes from a mouse genomic library
The mouse genomic library we used was obtained from Drs L. Mori and M.
Steinmetz. It had been derived from the LH8 T-cell lymphoma, which was
isolated from Radiation Leukemia Virus-infected C57B1/6 mice and has T
suppressor cell characteristics (39).
was cloned into the BamH
Frischauf et al. (40).
106 plaques were screened by hybridization with a
synthetic oligonucleotide corresponding to nucleotides 554 to 568 in the human
TNF-alpha cDNA sequence (28).
23 out of the 25 bases of this oligonucleotide
are identical to the homologous mouse sequence.
filling in overlapping complementary oligonucleotides with 32P-labeled
nucleotides using the Klenow fragment of E. coli DNA polymerase I.
were washed in 4xSSC at 600C.
Positive plaques were replated and screened
until a homogeneous population was obtained, and recombinant lambda phage DNA
was prepared using standard techniques.
The exon-specific mTNF-alpha oligonucleotides that we used to determine
the relative positions of exons and restriction sites were located as follows
on the cDNA sequence (33): exon 1, nt 223-272; exon 2, nt 350-369; exon 3, nt
399-418; exon 4, nt 852-870.
Subcloning and restriction mapping
Selected fragments were isolated from EcoR
clones by electrophoresis in low temperature gelling agarose (41), and were
subcloned into the mWB238 or mWB239 filamentous phage vectors (42, 43).
sequencing was done using the dideoxynucleotide chain terminator method of
Maps for restriction endonuclease cleavage sites were
established by standard strategies, and verified by hybridizing selected
subclones to Southern blots.
These routine hybridizations were performed in
6xSSC at 680C, using 0.25% non-fat dry milk as a blocking agent (45).
Detection of TNF genes in mouse/human hybrid cells
The derivation and cytogenetic analysis of hybrid cells that selectively
lose mouse chromosomes has been described in detail elsewhere (46, 47).
A partial Mbo I digest of LH8 cell DNA
I site of the EMBL3 lambda phage vector described by
The probe was labeled by
I digests of the lambda
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order to detect TNF genes in these hybrids, whole DNA (5 ug) was digested with
15 units of the appropriate restriction endonuclease, separated by
N), and linked to the membrane by UV irradiation.
performed according to Church and Gilbert (48), using 32P-labeled RNA probes
synthesized in vitro with the SP6 phage RNA polymerase (49).
the polymerase, we used the following fragments cloned in the pSP6 vectors:
middle of exon 4;
the H-2Kb gene (coding for the alpha-l domain; Weiss et al.,
in agarose gels, blotted onto nylon membranes (Amersham Hybond
As templates for
a 450 bp Hinc II to EcoR
a 950 bp Pvu II fragment extending from the first intron to the
I fragment derived from exon 4;
I fragment covering the second exon of
Analysis of genomic clones
We screened a genomic library derived from the LH8 mouse T-cell lymphoma
line (39) with an oligonucleotide probe derived from the sequence of exon 4 of
the human TNF-alpha (hTNF-alpha) (28).
logous mouse sequence (33) in 23 out of 25 positions.
plaque-purified five genomic clones that hybridized to the oligonucleotide.
To verify that they actually contain the TNF-alpha gene, we probed each clone
with four different oligonucleotides whose sequence matches parts of the four
presumed exons of the mouse TNF-alpha (mTNF-alpha) cDNA.
contain a 2.8 kb EcoR
Therefore, most of the mTNF-alpha gene is contained in this EcoR
Four out of the five clones that contain the mTNF-alpha gene also
hybridized with a genomic probe derived from exon 4 of the human TNF-beta gene
These clones all shared a 1.9 kb BamH
fragment hybridizing to the hTNF-beta probe, and three out of four contained a
3.6 kb EcoR
Assuming that the human TNF-beta probe is actually specific for the
homologous mouse gene, these results indicate that the mouse TNF-alpha and
TNF-beta genes are closely linked (the genomic clones contain inserts ranging
from 10 to 25 kb).
In order to verify this hypothesis, we decided to analyze
further one of the clones, which contains 2.8 kb and 3.6 kb EcoR
that hybridize to TNF-alpha and TNF-beta probes respectively.
Structure of the mTNF-alpha gene
The 2.8 kb EcoR
the criteria mentioned above was subcloned into the mWB238 filamentous phage
This oligonucleotide matches the homo-
We identified and
All five clones
I fragment that hybridizes to all four oligonucleotides.
I to Pst I; see ref. 31).
I fragment hybridizing to the same probe.
I fragment which contains most of the mTNF-alpha gene by
Nucleic Acids Research
(tumor neaosis factor)
ml Hlnc 11EcoR I
Physical map of the mouse TNF gene cluster. The map covers the two EcoR
fragments that we subcloned and mapped in detail. The positions and structures
of the mRNAs (top) were deduced from a combination of restriction mapping,
exon-specific hybridizations, sequencing, and homology to the human genes.
Thick lines indicate untranslated regions, open boxes correspond to coding
regions. They could still be inaccurate in minor details. The arrows at the
bottom indicate the direction and extent of sequencing, with regions A, B and
C corresponding to the panels in Figure 2.
vector (42, 43).
site (33), we assumed that this would define one of the ends of the clone.
mapped the position relative to this EcoR
nuclease cleavage sites (for the Hinc II, PvuII, Nar
predicted from the cDNA sequence.
All the sites were found at positions
consistent with the map shown in Figure 1.
the Hinc II site to the EcoR
confirm that we were dealing with the mTNF-alpha gene.
complete agreement with the published cDNA sequence (33).
Further restriction sites (for Hind III, Sma I, Sac I, and Pvu II) are
located in regions presumed to be introns by analogy to the human gene.
positions of these sites relative to the presumed exons were determined by
probing relevant digests with mTNF-alpha exon-specific oligonucleotides.
analysis confirmed the relative positions of exons and restriction sites shown
in Figure 1, implying that the intron-exon distribution of the mouse gene is
very similar to that of the human gene.
and the known structure of the human gene.
exact sizes and positions of introns and exons could still be present.
Structure of the mTNF-beta gene
The 3.6 kb EcoR
subcloned into the mWB238 vector.
This fragment contains the 1.9 kb TNF-beta-
Since the mTNF-alpha cDNA sequence contains a unique EcoR
I site of other restriction endo-
I and Sac
A 450 bp fragment extending from
I site was subcloned and partially sequenced to
Our sequence is in
The mTNF-alpha gene structure shown
1 was inferred from the cDNA sequence, our restriction mapping data,
Therefore, minor errors as to the
I fragment that hybridizes to the hTNF-beta probe was
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****** ** ** -AAAAAAA ******
* *H*** ****
* ** ** ** * *
* ****** ******** ** ** ** * *** ***
* ** * *.........
*** ******** ******* ***** *****
Comparison between the nucleotide sequences of the human and mouse TNF-beta
genes. The sequences of the human gene (top) and the mouse gene (bottom) were
aligned to maximize homology, and occasional gaps (hyphens) were inserted to
maintain this optimal alignment. Asterisks mark the positions where the
nucleotides are identical in both sequences. The nucleotide numbering of the
human TNF-beta gene is according to Nedwin et al. (31). The boundary between
sequences is indicated in the areas known to be part of the coding sequence of
the human gene. Within translated regions, amino acids that differ between the
mouse and human gene are underlined. A: region between -300 and -200 relative
to the 5' end of the mRNA;
second exon, including part of the signal peptide;
in the coding sequence for the secreted protein. These regions correspond to
the arrows in Figure 1.
1 and exon 2 is marked with an arrow, and the translation of the
B: part of the first intron and beginning of the
C: part of the fourth exon,
subcloned the two internal Pvu II fragments into the mWB239 vector.
sequencing of these cloned Pvu II fragments (arrows in Figure 1) and
comparison of the sequences to the human TNF-beta gene by the matrix method of
Pustell and Kafatos (51) revealed stretches of homology.
I fragment mentioned before,
We also mapped the cleavage sites for Pvu II (see Figure 1), and
in the configuration shown in
The distribution of
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these homologies allowed a line-up of our restriction map of the mouse gene
with the known sequence of the human gene (31).
fragment length measurements in agarose gels, the distances between homologous
regions are the same in the two species.
Figure 2 shows a comparison of the stretches of mouse sequence that we
obtained with their human homologs.
Several features are worth noting:
Detectable homology between the human and mouse sequences extends at least 300
nucleotides upstream of the mRNA cap site (panel A), even though the level is
rather low (55%) and homologies are unevenly distributed.
region that we have sequenced (panel B) covers most of the first intron and
the beginning of the second exon.
Within this region, the homology becomes
stronger as one gets closer to the intron/exon boundary.
of alternating nucleotides in the intron (...TCTCTCTC...) are among the
The splice acceptor is entirely conserved, as well as the
first amino acid of the signal peptide (assuming that the initiator Met is not
(iii) Within the coding sequence for the secreted portion of the
protein (panel C), conservation is very high (75% homology at the amino acid
level in the short stretch we sequenced).
signal peptide (5 out of the 11 amino acids for which we have information are
Hybridization of digests of the mTNF-beta gene to nick-translated total
mouse DNA reveals the presence of a highly repeated element at the left end of
our map, upstream of the first Pvu II site.
probes derived from this region to Southern blots of total mouse DNA.
fragment derived from the 3'-terminal region of the mouse elastase II gene and
containing two Bl elements (a kind gift of B. Stevenson) hybridized to the
same Pvu II and BamH
strongly suggests that there is a Bl element (19) upstream of the TNF-beta
The position of the Bl element is homologous to that of an Alu sequence
found in the human TNF locus (Nedospasov et al., CSHSQB,
Linkage between the TNF-alpha and TNF-beta genes
The first indication that the genes are adjacent to each other came from
data showing that a probe derived from the mTNF-alpha 2.8 kb EcoR
in contrast to a smaller probe from the 3' terminal Hinc II - EcoR
detects a Hind III fragment and a Sma
The simplest possible explanation was that the EcoR
at the left end of TNF-alpha is also the site defining the right end of TNF-
This hypothesis was verified by hybridizing TNF-alpha and TNF-beta
Within the accuracy of
(ii) The second
This figure seems lower in the
This was confirmed by hybridizing
I fragments as nick-translated total mouse DNA.
I fragment that also hybridize to a
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Table I: Characteristics of human/mouse hybrid cells
examined containing the chromosome. No other mouse chromosomes could be
bPercentage of cells expressing the marker as detected by staining with an
H-2K specific antibody and FACS analysis.
Chromosomes detected by karyotype analysis, and percentage of metaphases
probes to DNA from the original lambda clone digested with combinations of
Pvu II, BamH I, Hind III, Sma
the results were consistent with the map presented in Figure 1.
our data show that the mouse genes for TNF-alpha and TNF-beta are extremely
close to each other.
Assuming that the two genes have equivalent structures
in mice and men (which is likely from our other data), the polyadenylation
site of mTNF-beta is only 1.1 kb away from the cap site of the mTNF-alpha mRNA
(32, and Nedospasov et al., CSHSQB,
The mouse TNF genes are located on chromosome 17
In order to establish the chromosome localization of the TNF cluster, we
performed hybridizations of probes derived from the mTNF-alpha and mTNF-beta
genes to blots of restriction endonuclease-digested DNA extracted from human,
mouse, and human-mouse hybrid cells.
The hybrid cells were derived from
fusions of a human lymphoblastoid cell line, RJ 2.2.5, with unstimulated
spleen cells from Balb/c mice (46, 47).
selectively lose mouse chromosomes rather than human ones, and can be used to
map mouse genes.
The characteristics of the human-mouse hybrids that we used
are summarized in Table I.
Figure 3 shows the results obtained when a Southern blot of total
EcoR I-digested DNA from the RJ 2.2.5 cells, the hybrids, and control mouse
samples was probed with a mTNF-beta probe.
Nedwin et al. (31), we find that the human TNF-beta gene is located on a 2.4
kb fragment, while the mouse gene is on a 3.6 kb fragment.
specific band is present only in the AD2.KD hybrid, which contains the mouse
chromosome 17, and was selected for expression of mouse H-2K surface antigens.
No mouse chromosomes other than 17 could be detected in over 40 metaphases of
the AD2.KD hybrid.
We confirmed the segregation of the TNF genes with
I, and EcoR
I and blotted onto filters.
Hybrids produced in this fashion
In agreement with the data of
The 3.6 kb mouse-
Nucleic Acids Research
Hybridization of a mTNF-beta-specific probe to EcoR
RJ 2.2.5, the human parent of the hybrids; lane b: A2.KD clone
(mouse chromosome 17); lane c: H4 hybrid line (mouse chromosome 16); lane d:
M12, mouse B cell line of Balb/c origin. See Table
I digested DNAs. Lane a:
1 hybrid line
I for a description of the
chromosome 17 by probing the same gel with a 32P-labeled RNA specific for the
mouse H-2K locus: H-2K -specific bands were seen only in the AD2.KD hybrid
(data not shown).
Hybridization of the mTNF-beta probe to a blot of BamH I-
digested DNAs revealed the expected 2.0 kb mouse-specific fragment, while the
human gene is located on a 20 kb fragment.
derived from EcoR I-digested DNAs, the 1.9 kb mouse-specific BamH
is present only in the hybrid containing chromosome 17 (data not shown).
also probed blots of mouse-human hybrid DNAs with a mTNF-alpha clone, and
obtained simil1ar resul1ts (not shown).
These results establish that the mouse TNF genes are located on
chromosome 17, which also contains the major histocompatibility complex genes.
The exact location of the TNF genes relative to the MHC is currently under
In agreement with the results
The data presented in this paper show that the structure of the mouse
genes for TNF-alpha (tumor necrosis factor, cachectin) and TNF-beta
(lymphotoxin) closely resembles that of their human counterparts (31,
Nedospasov et al., CSHSQB,
as well as their intron/exon distribution, are identical within the limits of
In addition, both genomes contain a SINE repetitive element
in similar positions (52, and Nedospasov et al., GSHSQB,
The tandem configuration of the genes,
Nucleic Acids Research
nucleotide sequence level, the mouse and human genes for TNF-beta are highly
homologous, with the highest degree of homology found within the portion
coding for the secreted form of the protein, as expected from the conservation
of function and the lack of species specificity of TNF.
from the mRNA cap site are less conserved.
TNF genes are located on the same chromosome as the major histocompatibility
The close proximity of the genes for TNF-alpha and TNF-beta reinforces
the notion that they are closely related, and most likely evolved from a gene
duplication event followed by insertion of the exons coding for the signal
peptide (which is very different between the two genes) and for the 5'
It is worth noting that even though the level of
sequence homology indicates that the two proteins may have diverged as long as
several hundred million years ago (31), they have remained very closely
Previous reports (23, 31, 38) have shown that the homologies between
the two proteins are concentrated in exon 4, which codes for 80% of the
sequence of the secreted form in both genes.
mirrored by functional homologies, since the two proteins share the same
receptor and have very similar effects on their target cells (7, 23).
The cDNA sequences of the two TNF genes clearly indicate that they are
transcribed as non-overlapping mRNAs.
its own promoter and polyadenylation signals (31).
expressed in distinct cell populations, and are induced with quite different
It would be unusual for two genes in such close physical
proximity to be regulated in a completely independent fashion.
believe that a thorough study of the regulation of the TNF genes will provide
interesting insights into the more general problem of tissue-specific
expression of related genes. It has already been noted that there is
detectable homology between the upstream, presumably regulatory regions of
TNF-alpha and TNF-beta.
This may reflect similarities in the signals used to
turn the genes on, but leaves no clues as to why they are expressed in
Since expression of the TNF genes is controlled at least in
part at the-transcriptional level (53), one might expect the determinants of
tissue specificity to be among the non-conserved features of the controlling
regions of the two genes.
The linkage of the TNF genes to the MHC is conserved between mice and
men, at least at the level of chromosomal localization.
at the present time to tie this linkage to a regulatory coordination with
Like the human genes, the mouse
This structural homology is
In addition, each gene seems to have
The two genes are
We have no evidence
Nucleic Acids Research
other genes of the MHC, but it is interesting at least as an example of
functional clustering of genes, and gives TNF-alpha and TNF-beta a firmer
place among both the effectors and the modulators of the immune response.
We thank L. Mori and M. Steinmetz (Basel Institute for Immunology) for
the mouse genomic library.
S.A.N. was partially supported by a fellowship
from the Swiss Cancer League during the course of this work.
funded in part by grants from the Swiss National Science Fund.
The work was
*To whom correspondence should be addressed
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