volume 10 Number 9 1982
Nucleic Acids Research
Retrovirus-related sequences in human DNA: detection and cloning of sequences which hybridize
with the long terminal repeat of baboon endogenous virus
Makoto Noda , Masano Kurihara and Toshiya Takano*
Department of Microbiology, Keio University School of Medicine, Tokyo, Japan
Received 15 February 1982; Revised and Accepted 23 April 1982
ABSTRACT
Human DNA sequences which hybridized with the long terminal repeats (LTR)
of baboon type C virus M7_ were detected by non-stringent blot hybridization.
About 7 to 10 discrete bands of the LTR-related sequences were commonly
observed in the DNAs from four independent human cell lines after digestion
with either Eco RI, Hind III or Bam HI. The amounts of these sequences were
more abundant in tumor cell lines than in a non-malignant cell line. The human
sequences related to the H7_ LTR seemed to be located at relatively specific
sites on the cell IMA. The human DNA clones which hybridized with M7_ LTR were
detected in the human DNA library described by Lawn et. al. (Cell 15, 1157-1174,
1978), at a frequency of about 300 per haploid genome. Five clones were isolated which shared different extent of homology with K7 LTR and whose
restriction maps were totally different one another. The DNA structures of
two of them resembled the genome of retroviruses. These results suggest the
presence of various types of the LTR-related sequences in human DNA: some of
them might represent endogenous virus genomes of human cells.
INTRODUCTION
The eukaryotic cell DNA carries many copies of the pro-retrovirus sequences which have been accumulated during evolution (1); some of them are expressed
resulting the production of infectious retroviruses, and the others are interspersed in the cellular DNA as unexpressed proviruses.
Both of the uninte-
grated and integrated retroviral DNAs are flanked with two directly repeated
sequences of 300 to 1,200 base pairs (bp) called long terminal repeats (LTR)
(for review see ref. 2). Two structural features are generally found in the
LTR:
the initiation and termination signals for transcription, and the terminal
inverted repeats of 5 to 15 bp.
The DNA structure similar to that of the
retroviral LTR have been described in both prokaryotic and eukaryotic systems
as transposable elements (3). As expected from these structural characteristics of the LTR, the retroviral DNAs can integrate at various sites of the cell
DNA (A) and can modify the expression of certain cellular genes (5). It was
proposed that the proto-retroviral sequences might have evolved from a movable
©IRLProjJJ Umlted, 1 Falconberg Court. London W1V 5FG, U.K.
0305-1048/82/1009-28653 2.00/0
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genetic element by picking up other cellular sequences necessary for the construction of the provirus genome (6).
In human cells, the expression of endogenous proviral genes or the
production of infectious retroviruses have not been observed so far; the
translocation of the endogenous viral sequences via production and re-infection
of virions seems to be rare, if any.
Therefore, the search for the retrovirus-
like sequences in human DNAs should provide us informations on heritable
pro-retroviral DNAs in human cells.
Benveniste and Todaro (7) described that
the complementary DNA (cDNA) of the genome of a primate retrovirus, baboon
endogenous virus K7 (8), hybridized with human DNA to a small extent.
Kominami
et al. (9) reported that human DNA significantly hybridized with the 3'-terminal
region of the M7 genome.
The nucleotide sequences of this region are highly
conserved in various retroviruses (10). Recently, M. A. Martin e_t a^. (11)
have isolated retrovirua-like sequences in cloned human DNAs which carry
partial homology to a central region of the baboon type C virus genome.
They
suggested the presence of numerous copies of retrovirus-related sequences in
the human genome.
In the present study, we have detected human DNA sequences related to the
M7 LTR as discrete bands In blot hybridization.
Then, we have isolated human
DNA clones which hybridized with the MJ7 LTR, from the human gene library
described by Lawn e_t^ aL^. (12). Two independent clones, whose restriction maps
differed from each other, carried two separately located regions related to
the LTR, and weakly hybridized with other part of the M7 DNA.
The structural
array of these sequences in the two clones seemed to be colinear with that of
the Jf7 provirus.
The results suggest the presence of various types of the LTR-
like sequences in human DNA:
some of them might represent human endogenous
pro-retroviruses, and some others might be inert vestiges of proto-retroviral
sequences In human cells.
MATERIALS AND METHODS
Cell DNAs and Probes for Hybridization.
High molecular weight DNAs were
prepared from human cultured cell lines by the procedure of Hughes et^ al^. (13).
The fragments of BaEV DNA were prepared from a recombinant phage, ABEV-11, a
derivative of Charon 28 carrying the full length DNA copy of the K7_ genome (14) ,
by digestion with restriction endonucleases (Takara Shuzo Co., and New England
32
BioLabs), and purified by agarose gel electrophoresis. The
P-labeled probes
were prepared from the DNA fragments by nick translation (15) with
deoxyribonucleoside triphosphates (2,000 Ci/m mole, Amersham).
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Blot Hybridization.
The cellular DNA, 15 to 30 yg, was digested with 15-30
units of restriction enzymes in 30 to 60 yl at 37°C for 2 hrs or more under
the conditions recommended by the suppliers of the enzymes.
Complete digestion
of the DNA samples was monitored by adding a small amount of X DNA to pilot
reaction of an aliquote of each reaction mixture, and analyzing the digestion
products by gel electrophoresis.
The digested cell DNAs were resolved by
agarose gel electrophoresis according to the procedure described before (14).
The DNAs in the gel were then denatured and transfered to nitrocellulose
membrane (Schleicher & Schull) by the procedure of Southern (16). Hybridization was carried out in 1 x Denhardt's solution, 0.1Z SDS, 1M NaCl, 10 mM
EDTA, 50 mM Tris-HCl (pH 7.A), 100 yg/ml of sonicated, denatured salmon sperm
DNA, and 1-5 x 10 6 cpm/ml of
32
P-labeled probe, at 65°C for U
to 20 hrs,
according to the procedure of Honjo et a^. (17). The membrane was washed in
0.1 x SSC, 0.135 SDS at 65°C for a stringent condition, or at room temperature
for a non-stringent condition.
Dried blots were exposed to Kodak XR-1 film at
-70°C.
In restriction mapping of cloned human DNA, 0.1 to 0.5 yg of DNA were
digested with 1 to 5 units of restriction enzymes in 20 yl.
Electrophoresis
and blot hybridization were done on the digested clone DNA as described above.
Estimation of the Relative Amounts of the LTR-related Sequences. Alkalinedenatured and then neutralized cell DNA (30 yg) was spotted on a nitrocellulose
disk and immobilized by baking at 80°C for 2 hrs.
The disks were pre-treated
and hybridized at 65°C in the same way as that of blot hybridization.
Washing
of the disks was done at room temperature under the non-stringent condition
described above.
The radioactivity retained on each disk was determined with
liquid scintillation counter after drying the disks.
Library Screening of Human DNA Clones.
The human DNA library described by
Lawn et_ a\_. (12) was obtained from Tom Maniatis.
Hybridization-positive clones
were screened from the library by the plaque hybridization method of Benton and
Davis (18) using the Xho I-fragment of M7 LTR as a probe.
Hybridization and
washing were carried out under the non-stringent condition described above.
Hybridization-positive clones were purified by two cycles of the j ^ situ
hybridization.
Restriction Mapping o£ the Human DNA Clones.
The restriction maps of the clones
were constructed mainly by double digestion method with restriction enzymes.
Additionally, the partial digestion method of Smith and Birnstiel (19) was
applied to the endo-labeled phage DNA of each clone to determine the order
of the fragments.
The method of selective labeling at one cos-end of the phage
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DNA was described before (14).
RESULTS
Detection of M7-related Sequences In Human DNAs.
The subgenomic probes of the
M7 DNA were prepared from the cloned provirus, as shown in Fig. 1A.
Using the
entire K7 DNA and these fragments as probes, genomic blot analyses were conducted on the Eco RI-, Hind III- and Bam Hi-digested DNA of a human rhabdomyosarcoma cell line, A204 (20), under the non-stringent condition of
hybridization described in the methods (Fig. IB). Neither the full length nor
the fragmented K7_ probes gave any bands with Escherichla coli DNA under this
condition (data not shown).
The specificity of these probes were estimated by
including a control sample in each blot:
the Bam Hi-digested DNA of a
reconbinant phage carrying the K7 DNA (ABEV-31, see ref. 14) was electrophoresed and hybridized with each probe (Fig. IB, slots 4 ) . The Intense bands
in each of these slots were the M7_ fragments corresponding to each probe and
the other faint bands Indicated the other fragments of the M7_ DNA or the arms
of the vector phage DNA contaminated in the preparations of the probes.
From
the strong contrast of the intensity among the corresponding and contaminated
bands, these probes seemed to be satisfactorily specific and the bands
detected under these conditions should represent the specific sequences which
carried considerable extents of homology with each probe.
In consistent with earlier observation made by hybridization in liquid
phase O) , the A204 DNA gave several faint bands when it hybridized with the
entire M7 DNA as a probe (Fig. IB, blot a ) . A 9 kilobase pairs (kb) band
given by Eco RI digestion and a 18 to 20 kb band by Hind III digestion were
prominent.
By hybridization with the probe b representing the gag region of
M7, however, the A204 DNA gave rise to a smear profile of strong hybridization
(Fig. IB, blot b ) .
The probe c, that mainly corresponded to the env gene of
M7, did not give any discrete bands (Fig. IB, blot c ) . On the other hand,
many Isolated, discrete bands of various intensity ranging from 1 to 25 kb were
detected in the A204 DNA by hybridization with the probes d and e (Fig. IB,
blots d and e ) .
The probe d covered the sequence of the LTR and the adjacent
regions, while the probe e was an internal Xho I-fragment of the two tandem
LTRs (Fig. 1A). Most of the bands detected by these two probes were comlgratlng each other, especially, in the Hind III- and Bam Hi-digested A204 DNA.
Even under the stringent condition of hybridization, a few of these bands were
detectable as faint signals: e.g. 8.5 and 4 kb-Eco RI, 15 kb-Hind III, 7.5
and 6.0 kb-Bam HI fragments (data not shown).
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Therefore, the A204 DNA seemed
Nucleic Acids Research
ABEV-II . -«
•
'
*
A A
111.
probe
1 2 3 4 12 3 4 1 2 3
i.o -
Fig. 1 Detection of M7-related sequences in the human cell DNA. In B, the
DNA (20 yg/slot) from a human rhabdomyosarcoma cell line, A204, was digested
with Eco RI, Hind III or Bam HI (slots 1, 2 or 3, respectively) and resolved
by electrophoresis in 0.7% agarose gel. In the slots 4, the Bam Hi-cleaved
DNA (0.1 ng/slot) of a M7 DNA clone, ABEV-31 (1A), was applied as size markers
and hybridization controls. Each blot shown in B, a to e, was hybridized
under the non-stringent condition with the 32p- p r obe illustrated in A, a to e,
respectively. The probes were prepared from the restriction fragments of the
cloned H7_ DNA, ABEV-11 (14), as shown in A. The approximate gene location and
the sites for Eco RI (•), Bam HI (O) and Xho I (•) are shown on the map of
ABEV-11 in A.
to contain a variety of the sequences which related to the LTR of >17, at
various specific sites.
We surveyed the sequences related to the M7 LTR in the DNAs of four
different types of human cultured cells:
a human embryonic lung cell, MRC-5
(21), a human oral carcinoma cell, KB (22), a human bladder carcinoma cell,
KU-2 (23), and A204 (Fig. 2 ) .
The results were essentially similar to those
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kb
23.7-
EcoRI
Hindi
1 2 3 4
1
•?.
2
3
Bam HI
4 1
N 11
2
3 4
m ••
956.743-
*
t
2.3-
20-
Fig. 2 Human DNA
sequences related to >T7
LTR. The DNAs extracted from four independent
human cell lines, MRC-5,
A204, KB and KU-2
(slots 1, 2, 3 and 4,
respectively) were
digested with restriction enzymes indicated
at the top of the blots,
and the LTR-related
sequences were detected
by the procedure similar to that of Fig. 1.
The hybridization probe
was a 0.6 kb Xho Ifragment of W_ DNA,
which was shown as the
probe e in Fig. 1.
shown in Fig. IB, blots d and e, although only the restriction fragments of
modest sizes were detected in this particular blot, which is probably because
of the different conditions of electrophoresis and DNA transfer.
In Fig. 2,
four predominant bands of about 3, 4.4, 6 and 7.5 kb were commonly detected in
the Hind Ill-digests of these DNA samples, while three major bands ranging 3
to 7 kb were also commonly observed in the Bam Hi-digests of the human DNAs.
However, the intensity of each band was different from cell to cell.
The DNAs
from the tumor cell lines, A204, KB and KU-2, gave more intense bands than
that from the non-malignant fibroblastic cell, MRC-5.
The relative intensity
of the bands was slightly different among these human cell DNAs, especially,
in the case of KU-2.
From these results, the sequences related to the M7_ LTR seemed to be
located at specific sites on the human DNA, although the copy numbers of the
sequences detected as each band seemed to be variable, and the sequences were
also detected at additional minor sites in the DNAs of particular cell lines.
Quantities of the LTR-related Sequences in Human DNAs.
Because blotting
technique lack quantitative accuracy in the condition we used, the filter
hybridization was carried out to estimate the relative amount of the LTRrelated sequences in the total cell DNAs.
Sonicated and denatured cell DNAs
were immobilized on nitrocellulose membranes, and hybridization was done under
non-stringent condition with the radioactive probe of M7^ LTR (Fig. 3 ) . DNA of
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32,
Fig. 3 Hybridization of "P-labeled M7 LTR with human cell DNAs
immobilized on nitrocellulose disks.
The experimental procedures are
described in Materials and Methods.
The radioactivity retained on each
disks after hybridization under
non-stringent condition was plotted
against the incubation time. The
DNAs from Escherichla coli (A),
MRC-5 (O), A204 (A), KB (D) and
BEC-1 (•) were used.
"0
10
20
30
TIfE OF INCUBATION (HOURS)
the M7-infected A204 (BEC-1) rapidly hybridized and the reassociation did not
reach to the plateau in 32 hr incubation.
On the other hand, only a small
fraction of the probe hybridized with Escherichia coli DNA.
This trace
hybridization was due to either contamination of a small amount of the bacterial
DNA in the probe or non-specific hybridization.
The immobilized DNAs of the
uninfected human cells showed significant reassociation with the LTR probe,
which reached to the plateau within 10 hrs of incubation.
The DNAs from the
tumor cells, A204 and KB, hybridized with the M 7 LTR to higher extents than
the non-malignant cell, MRC-5.
This result is consistent with that of the blot
hybridization shown in Fig. 2.
Therefore, the amounts of the LTR-related
sequences in human DNA appeared to be variable.
Isolation and Characterization of Human DNA Clones Bearing the Sequences
Related to M7 LTR.
To study further the structural organization of the LTR-
related sequences in human DNA, recombinant clones were screened from the
genomic library described by Lawn ^t^ jjQ. (12) , by plaque hybridization method
(18) using
32
P-labeled M7 LTR as a probe.
clones were hybridization-positive.
Out of 2 x 1 0 5 plaques, about 80
These clones were designated as XHBL.
Five independent clones, XHBL-1, - 2 , - 3 3 , -41 and -42 were plaque-purified and
the locations of the sites for restriction endonucleases in the clone DNAs were
determined.
An example of such analyses was shown in Fig. 4.
In addition, a
partial digestion method (19) was used to determine the order of the fragments
of the clone DNAs after selective labeling at one cos end with
transcriptase
(data not shown).
P by reverse
The restriction maps of the four clones
derived as above are summarized in Fig. 5.
LTR-related Sequences in Cloned Human DNA.
The locations of the sequences
related to M 7 LTR were determined on the map of the cloned human DNAs by blot
hybridization with the LTR probe.
An autoradiogram of the blot from the gel
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A.
AHBL-1
AHBL-2
B.
AHBL-1
AHBL-2
*«B »
«B«
H .I
K B •
•• • :•«
•t
Fig. 4 Structure analysis of cloned human DNA sequences related to M7_ LTR.
The DNA of XHBL-1 or -2 was digested with Hind III (H), Xho I (X), Sal I (S),
Kpn I (K) and/or Bam HI (B). Then, the digested DNA (0.5 yg/slot) was analyzed by electrophoresis in 0.7% agarose gel (A). The LTR-related sequences were
detected by blot hybridization under the non-stringent condition using 32p_
labeled M7_ LTR as a probe (B). In the slot M, Hind Ill-digested X DNA was
applied as size markers.
of Fig. 4A is shown in Fig. 4B.
The stretch of the sequences which hybridized
with ^17 LTR was deduced from the data of the blot hybridization and the results
are summarized in Fig. 5.
In the clones XHBL-1 and -41, two sequences related
to M7 LTR were detected, which located separatedly at distances of 4 and 10 kb,
respectively.
The intensity of the bands hybridized with M]_ LTR was varied
from clone to clone, so that the heterogenlty in the nucleotide sequences of
these regions was suggested.
Retrovirus-like Structures of Human DNA Found iji the Cloned DNAs.
The LTR of
human endogenous viruses, if exist, are expected to share considerable sequence
homology with other primate retroviruses.
Some of the cloned human DNAs
obtained in this study might carry certain human endogenous proviruses.
To
test this possibility, blot hybridization of the AHBL DNAs digested with
32
restriction enzymes was done under non-stringent condition using
P-labeled
probes made of various fragments of M7 DNA, i.e., the probes e, f, g and h
shown in Fig. 6.
In the autoradiogram shown in Fig. 6, the fragments derived
from the arms of A phage were detected because each probe was contaminated
with small amounts of the vector DNA.
On the other hand, certain fragments of
the insert DNAs significantly hybridized with the probes f, g and h which repre-
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AHBL-l
l£Q_^^ | £j£_ a a i jJh^ B jQ.^J
g«g'
pol'
env
'
'
-•• -
••• -
U.9 k b
•
• — •
LTR,
AHBL-2
n
y nn F T ? T 0 . 3 ^
pol
AHBL-33
•
^^£_J^^J^^^^^^^^J
15.0 kb
LTB'
AHBL-41
^AM^&^^^__JaJT
pol
•
env--
• -
--»
- - *• •
•
—
-
•—
•
12.1 k b
•
•
XHBL-42
^^^j^^^Q^g^^^j^jg
,3.3 kb
H7 provirus
U A U A J U
8.6 kb
'LTR
Fig. 5 Structure of cloned human DNA sequences related to M7 LTR. The DMA of
five independent clones of XHBL was digested with Eco RI (T), Hind III (V) ,
Bam HI (O) , Xho I (D), Sal I (O) , Kpn I (•) and Sac I (•), and analyzed similarly as the experiments shown in Fig. 4. The order of the restriction
fragments was determined by partial digestion method described in Materials
and Methods. In the cases of AHBL-l, -2 and -41, blot hybridization analyses
were also done with the probes of the M7 DNA fragments other than the LTR, by
the experiments similar to those shown in Fig. 6. The results were summarized
and shown under the restriction map of each clone. The fragments hybridized
with the probes f, g, h and e shown in Fig. 6 are indicated by blackets named
as gag, pol, env and LTR, respectively. The size of the human DNA insert in
each clone is shown in the right-hand column. The structure of H7 provlrus
DNA (14) is also shown.
sented the parts of K7 DNA other than the LTR.
shown in Fig. 5.
The results are summarized and
Interestingly, the region related to each part of M7^ DNA were
flanked by the two separately located sequences related to the LTR in AHBL-l
and -41. In AHBL-l, we could not dissociate the regions related to gag and
LTR, but the pol-related region extended more to the right hand side than the
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H7 HNA
PROBE
I
r
12345
i
if
12345
\
r
g
..
r
12345
h
12345
fcb
Fig. 6 Detection of the sequences related to each part of M7_ DNA in the cloned human DNAs. The DNA samples of XHBL-1 and -2 (0.5 yg/slot) were digested
with r e s t r i c t i o n enzymes, resolved by electrophoresis, and identical four
pieces of blots were prepared. Each piece of blot was hybridized under the
non-stringent condition with the 32p_]_abeied M7_ DNA fragments (e to h)
prepared from XBEV-11 DNA as schematically shown at the top. Symbols for
r e s t r i c t i o n enzymes shown on the map of K7_ DNA are the same as those of Fig. 5,
except for Bgl I (•) . In the slots 1 to 3, XHBL-1 DNA digested with Eco RI,
Hind I I I or Bam HI were applied, respectively. In the slots 4 and 5, XHBL-2
DNA was doubly digested with Xho I + Sal I and with Eco RI + Bam HI, respectively, and then applied.
former two regions.
Besides, the sequence related to the env gene was i n t e r -
jacent between the regions related to the pol gene and the LTR at the right
hand side.
In XHBL-41, on the other hand, the regions related to the pol and
env genes of M£ were located at the left hand side of the right LTR-related
sequence.
The DNA of this clone did not have any sequence related to the gag
gene of M7^.
XHBL-2 had the regions related to each part of M7 DNA. However,
the regions overlapped and the structure of t h i s particular clone is not clear.
Analyses are in progress on the other several clones, which have been ascertained to carry the sequences related to K7 LTR.
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DISCUSSION
As expected from the earlier observations by Kominami e_t ill. (9), we
detected the endogenous sequences of human DNAs which weakly but significantly
hybridized with the LTR of M7 by Southern's blotting technique (16).
By diges-
tion of the human DNAs with Bam HI, Hind I I I and Eco RI, about seven to ten
discrete bands were observed in the blots hybridized with the LTR probe under
non-stringent condition.
These LTR-related sequences were not derived from
any viral contamination of the cell lines used as the cellular DNA sources in
this study, because any internal Bam HI fragments characteristic to the M7^
virus (14) were not detected in Fig. IB (a).
All the cell lines were assured
to be free from exogenous virus infection by assay of reverse transcriptase
activity (24) on the culture fluid after 100-fold concentration.
Therefore,
several classes of the endogenous sequences related to the M7 LTR seemed to be
present in human DNA.
Under the experimental condition used here, the sequence
detectable as a discrete band is expected to be either considerably r e p e t i t i v e ,
short DNA segments (not shorter than 12 bp) or longer sequences with less
homology (not less than 70Z) to the probe.
While, a preliminary experiment
suggested that most of the sequences detected by hybridization with the LTR
probe were mainly related to the U3 domain of the M^ LTR (our unpublished results) .
The U3 domain is 421 base pair-long and contains a TATA box and other
sequences responsible for transcrlptional regulation (3).
The sequences more or less related to the H7 LTR were suggested to be
located at relatively specific s i t e s , from the size distribution of each band
in the DNAs from four independent human cell l i n e s .
Although i t was difficult
to determine the exact copy number, the amounts of the LTR-related sequences
seemed to be more abundant in the three tumor c e l l s , A204, KB and KU-2, than
those in the non-malignant c e l l , MRC-5 (Fig. 3).
I t is interesting to Imagine
that the LTR-related sequences might be selectively amplified during carcinogenesis.
If the human sequences related to ff7 LTR have a transposon-llke
structure as in the case of the M7 LTR (3), they might play an Important role
in a certain process of the gene amplification in carcinogenesis (25, 26).
Further evidences, however, are necessary to prove this hypothesis.
On the other hand, A204 DNA contained a variety of sequences which strongly hybridized with the probe representing the gag region of the M7_ DNA. At
least, a part of the gag gene of M7 seemed to share partial homology with
certain repetitive sequences of the human DNA, but we have not studied further
about this possibility.
By isolating and analyzing the human DNA clones which hybridized with the
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M7 LTR, the s t r u c t u r e of the sequences r e l a t e d to the LTR were determined.
The number of the LTR-related sequences was estimated to be about 300 per
haploid genome of human c e l l s , from the frequency of the h y b r i d i z a t l o n —4
p o s i t i v e clones (4 x 10 ) In the phage l y s a t e carrying random human DNA
sequences which may cover the e n t i r e sequences of human DNA by 8 x 10
p a r t i c l e s (12).
phage
Considering t h i s copy number and v a r i a b i l i t y of the i n t e n s i t y
of several d i s c r e t e bands on the genomic b l o t in Fig. 2, i t might be p o s t u l a t ed t h a t , a t l e a s e , some of those bands represented the i n t e r n a l fragments of
some r e p e t i t i v e sequences.
A wide range of v a r i e t y in h y b r i d i z a t i o n
intensity
was observed on the autoradiograms of these clones in plaque h y b r i d i z a t i o n .
This h e t e r o g e n e i t y may represent the e x t e n t of sequence homology with the
probe, or the complexity and the copy number of the sequences homologous to
the probe, in each clone.
On the o t h e r hand, five randomly i s o l a t e d clones
had the r e s t r i c t i o n maps d i f f e r e n t
from one another, and the h y b r i d i z a t i o n
i n t e n s i t y of the r e s t r i c t i o n fragments which contained the locus r e l a t e d to
the R7_ LTR was v a r i a b l e from clone to c l o n e .
These r e s u l t s t o g e t h e r suggest
t h a t the LTR-related sequences a r e highly divergent in human DNA and may have
sequence h e t e r o g e n e i t y .
In f a c t , the human DNA fragments i n s e r t e d in each
clone did not hybridize with those of o t h e r four clones under the s t r i n g e n t
condition (data not shown).
carrying d i f f e r e n t
Thus, we have detected four independent clones
groups of LTR-llke sequences presumably diverged from a
comnon p r o g e n i t o r .
In XHBL-1 and - 4 1 , the LTR-related sequences are separated i n t o the two
s i t e s and the sequences r e l a t e d to the o t h e r p a r t s of the K7 genome a r e l o c a t ed between the two separated s i t e s .
The o r g a n i z a t i o n of these sequences
parenthesized by the two LTR-related l o c i seem to be c o l i n e a r with t h a t of the
M7 genome.
Therefore, the human DNA sequences represented by these two clones
might be endogenous p r o - r e t r o v i r u s e s , or the i n e r t v e s t i g e s of p r o v l r u s e s ,
the pseudogenes.
i.e.,
On the other hand, M. A. Martin e_t a]^. (11) i s o l a t e d a human
DNA clone which c a r r i e d the sequences homologous to a p a r t of the baboon v i r u s
genome, from the same human gene l i b r a r y t h a t we used in t h i s study.
clone i s o l a t e d by them i s d i f f e r e n t
report.
from any of the clones analyzed in t h i s
Therefore, many d i f f e r e n t types of the r e t r o v l r u s - l i k e
should be contained in human DNA.
The
sequences
As for the c l o n e , XHBL-41, the gag gene
region of the M7 genome did not h y b r i d i z e with the human DNA I n s e r t of t h i s
clone under n o n - s t r i n g e n t condition.
Therefore, t h i s clone seems to c a r r y
completely d i f f e r e n t nucleotide sequences from that of M7 in the r e g i o n .
R e t r o v i r a l p r o t e i n p30, one of the gag gene p r o d u c t s , i s known to be s p e c i e s -
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specific (27). The human provirus represented by this clone should be either
unique to the human species or specific to the animal other than the primates.
Alternatively, the gag-related region of this clone might be substituted by
unknown sequences.
XHBL-2 also hybridized with both the LTR and the other regions of the M7_
genome.
In this clone, the sequences related to the gag gene of M7 are repeat-
edly located, and the LTR-related sequence is located between the two gagrelated sequences.
The array of these sequences is apparently different from
the structure of a retroviral genome.
However, the sequences clustered in the
left hand side, which are homologous to various regions of the M7 genome,
might be a part of the endogenous provirus sequences in human DNA.
The other clones including AHBL-33 and -42 carry only one locus related
to the M7_ LTR, and the homology to the other parts of the H7 genome has not
yet been analyzed in detail.
Retroviral LTRs are known to carry consensus
sequences of certain functionally important signals, i.g., signals for transcription and translation (3). Therefore, it is possible that these clones
just represent human sequences related to one or more of such signal sites.
In this study, utilizing a primate retroviral LTR as a probe, we have
directly picked up the sequences of human DNA whose structures resemble proretrovirus genome.
The cloned human sequences obtained here are useful for
studying the behavior of such retrovirus-like genomes, and the LTR-like
sequences as well, in various cells at different stages of differentiation or
carcinogenesis.
ACKNOWLEDGMENTS This work was supported by research grants from the Japanese
Ministry of Education, Science and Culture, the Naito Foundation, and the
Waksman Foundation of Japan, and by the institutional grant.
•Present address of M. N.:
Laboratory of Tumor virus Genetics, National
Cancer Institute, Bethesda, Maryland 20205, U.S.A.
tAll the inquiries should be addressed to T. T.
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