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A new type of EcoRI polymorphism of the human ribosomal DNA repeating unit revealed by analysis of cloned DNA fragments
109
Gene, 27 (1984) 109-113 Elsevier GENE 942
A new type of EcoRI polymorphism of the human ribosomal DNA repeating unit revealed by analysis of cloned DNA fragments (Human liver DNA; genomic library; cDNA; 45s rRNA; 1 Charon vectors)
Monica Mattes*, S.-A. Tsai Lai, Julio Montoya and Giuseppe Attardi Division of Biology, Calgomia Institute of Technology, Pasadena, CA 91125 (U.S.A.)
Tel. (213)-356-4930
(Received August 2&h, 1983) (Revision received November 6th, 1983) (Accepted November 9th, 1983)
SUMMARY
Several clones of rDNA have been isolated from an adult human liver DNA Charon 4A library by using cDNA probes synthesized from human 18s and 28s rRNA. The insert of one recombinant Charon 4A clone contained, besides the already known 5.7-kb EcoRI fragment of rDNA, comprising the major portion of the 18s rRNA gene and all the external transcribed spacer (ETS), a previously unidentified EcoRI fragment of rDNA of 8.5 kb in size. DNA transfer hybridization experiments utilizing EcoRI digests of the human DNA used to construct the library and of another human DNA showed the presence of the 8.5-kb EcoRI fragment in a minority of the rDNA repeats on the 5’-end side of the 5.7-kb fragment, thus defining a hitherto unidentified type of EcoRI polymorphism of these repeats.
INTRODUCTION
A limited length and sequence heterogeneity has been reported among the various rDNA units of the human nuclear genome in the same individual. In particular, three forms of polymorphism have been detected in the human rDNA family by restriction enzyme analysis of genomic DNA. The EcoRI polymorphism involves the presence in the NTS of some of the repeats of a site at approx. 5.7 kb 5’ to the * Present address: Istituto di Genetica, Universita di Pavia, 27100 Pavia (Italy) Tel. 382-303852. Abbreviations: bp, base pairs; cDNA, DNA complementary to RNA; ETS, external transcribed spacer; ITS, internal transcribed spacer; kb, kilobase pairs; NTS, nontranscribed spacer; rDNA, ribosomal DNA; SDS, sodium dodecyl sulfate. 0378-l 119/84/$03.00 0 1984 Elsevier Science Publishers
EcoRI site in the 18s rRNA gene (site 3* in Fig. 1; Arnheim and Southern, 1977; Wilson et al., 1978; Wellauer and Dawid, 1979). The Hind11 polymorphism involves the presence of a Hind11 site in the 3 ’ -end proximal third of the 28 S rRNA gene of some of the repeats (Amheim et al., 1980). The NTS length polymorphism involves a variation in length in a region within 4 kb of the 3 ‘-end of the 28s rRNA gene; this variation results in the presence of four length classes (Krystal and Arnheim, 1978; Arnheim et al., 1980; Schmickel et al., 1980). In the present work, the cloning and characterization of segments of human rDNA isolated from a human liver DNA library has led to the detection of a new type of EcoRI polymorphism, involving the presence in a relatively small portion of the repeats of a hitherto unrecognized EcoRI site at approx.
14.2 kb 5’ to the EcoRI site in the 18s rRNA gene
4.6 kb;
(site 2* in Fig. 1).
contained
the
insert
of another
a 5.7-kb fragment,
8.5-kb fragment;
Sucrose from
gradient-purified
HeLa
DNase
cells
(Boehringer,
were
28s treated
100 pg/ml,
SDS-pronase-phenol-extracted, ed, and used as templates
and, in addition,
phages
experiments
and
18s rRNAs
mic oligo(dT)-cellulose
non-bound
RNase-free
cells and 32P-labeled
nick-translated
30 min
at 4”C),
for reverse transcriptase
RNA trans-
utilizing total cytoplas-
with
ethanol-precipitat-
an
1Hr3, AHr4, AHr5 and
J_Hrll were chosen for further analysis. fer hybridization
also
and a 7.5-kb fragment.
The recombinant AND METHODS
iHr5,
finally, the insert of AHr3 consisted
of a 7.1-kb fragment MATERIALS
clone,
RNA from HeLa probes derived
from whole IHr4
or IHr5
showed
a
clear hybridization
of both probes to 18s rRNA,
in
agreement
phage DNA
with the plaque-screening
results.
Similar
together with calf thymus DNA random primers (Morandi et al., 1982). A library was constructed from a partial EcoRI digest of adult human liver DNA using lambda Charon 4A as a vector, essentially as described in published procedures (Maniatis et al., 1978; Lawn et al., 1978). Fragments in the size range 13 to 20 kb were selected by sucrose gradient centrifugation. Screening of the recombinant clones was carried out
experiments using whole nick-translated /1Hr3 DNA as a probe revealed a strong hybridization of the latter to 28s rRNA, again in confirmation of the plaque-screening results, and a weak hybridization to 18s rRNA; a hybridization of similar intensity to 18s and 28s rRNA was on the contrary observed with a probe derived from the whole iLHrl1 DNA. On the basis of these observations, it seemed very likely that the 5.7-kb fragment of the insert of /ZHr4,
by a published procedure (Benton and Davis, using 18s and 28s cDNA probes.
1977)
J.Hr5 and 1Hrll was involved in hybridization with 18s rRNA, while the 7.1-kb fragment of the inserts
Transfer onto nitrocellulose paper of HeLa cell cytoplasmic oligo(dT)-cellulose non-bound RNA, after electrophoresis through an agarose-CH,HgOH or an agarose-formaldehyde gel, and of EcoRI-
of JHr3 and AHrl 1 was responsible for their hybridization with 28s rRNA and, probably, to a small extent, with 18s rRNA. These results allowed the identification of the 5.7-kb and 7.1-kb fragments cloned in Charon 4A with those previously mapped in the human rDNA (Wellauer and Dawid, 1979; Fig. 1).
digested HeLa or VA,-B cell DNA, after electrophoresis through an agarose gel, was carried out by standard techniques. Cloned rDNA fragments, 32Plabeled by nick translation, or 32P-labeled cDNA synthesized from purified 28s or 18s rRNA were used as probes.
To analyze the distribution of the cloned DNA sequences in the genome, DNA transfer hybridization experiments were performed using the adult liver DNA utilized for the construction
RESULTS
AND
DISCUSSION
Approx. 7 x lo4 plaques from the human liver genomic library were screened separately with 32Plabeled 28s and 18s cDNAs. Two and five clones were found to give a positive signal with the 18s and 28s cDNA probes, respectively. An electrophoretic analysis of EcoRI digests of these recombinant phage DNAs revealed that the inserts of four recombinant clones (AHr8, IHr9,lHrll and IHr12) consisted of a 5.7-kb and a 7.1-kb fragment. The insert of another recombinant clone (AHr4) contained a 5.7-kb fragment and two smaller fragments of 5.1 kb and
of the library and,
in some experiments specified below, also DNA from the human cell line VA,-B. With the 18s cDNA probe, a strong band corresponding to a size of 5.7 kb and two fainter ones corresponding to sizes of 7.1 and 17.8 kb were observed (Fig. 2A). The strong 5.7-kb band and the weak 17.8-kb band were also seen when the 5.7-kb fragment of the insert of IHr 11 was used as a probe (Fig. 2B). The 7.1-kb fragment of the same insert hybridized, as expected, to a genomic fragment of the same size (Fig. 2B; the faint 5.7-kb and 17.8-kb bands reflect a low level of contamination of the 7.1-kb fragment by the 5.7-kb fragment). When the total DNA of 1Hr5 was used as a probe, four labeled bands corresponding to sizes of 5.7,8.5,
111 H 1 kb XHr8, XHrll,
XHr9 XHr12 >
5.7
4
t
7.1
7.5 ,_-------1
4
1
XHr5
8.5
+c----3-----4.6 +
XHr4
1
--
-8
’
2”
5.1
4 1
j
!.? 3”
45s
1
(
L
, k
XHr3
1 1 4
l
I I
5
!
u-
NTS
NTS ETS
ITS
Fig. 1. The cloned fragments of human rDNA isolated in the present work are aligned with the EcoRI restriction map of the rDNA repeating unit, as determined by Wellauer and Dawid (1979) and in the present work. Small downwards arrows indicate the positions ofthe EcoRI restriction sites. Asterisked numbers indicate sites not present in all repeating units. The numbers over the cloned fragments indicate their size in kb; the dashed segments represent cloned fragments not belonging to rDNA. The mapping position of the “45s RNA”, with the 5’ ends of the major (slightly shorter) and minor (slightly longer; dotted line) transcripts recently identified (Financsek et al., 1982; Miestield and Arnheim, 1983; M. Mottes, J. Montoya and G. Attardi, unpublished observations), is shown above the bottom line.
12.1 and 17.8 kb were observed, with the 5.7-kb and 12.1-kb bands being considerably stronger than the other two (Fig. 2A). The 8.5-kb fragment of the insert of LHr5 hybridized to a fragment of the same size and, in addition, to the two fragments of approx. 12.1 and approx. 17.8 kb (Fig. 2B). The hybridization pattern of the 8.5kb fragment to VA,-B DNA was similar to that obtained with liver DNA, except that the 17.8-kb band was considerably stronger, instead of weaker, than the 12.1-kb band (Fig. 2B). From a comparison of the hybridization patterns obtained with total LHr5 DNA or with the 8.5-kb fragment as a probe one can infer that the 5.7-kb fragment of IHr5 DNA hybridized to a genomic fragment of the same size and, presumably also, weakly, to a 17.8-kb fragment, like the 5.7-kb fragment of JHrl 1 (Fig. 2B). When the total 1Hr4 DNA was used as a probe, it gave the same hybridization pattern (Fig. 2A) observed with the 5.7-kb insert of LHrl 1. By contrast, no obvious hybridization was observed to fragments of a size corresponding to those of the two smaller fragments of the insert (5.1 kb and 4.6 kb); this observation strongly suggests that the latter two fragments are not related to the rDNA, and that they became joined to the 5.7-kb fragment during the cloning. When the whole JHr3 DNA was used as a probe, an intense band corresponding to a fragment with a
size of approx. 7.1 kb was observed in a genomic blot of liver DNA. On the basis of the results obtained with the 7.1-kb fragment of the insert of AHrll, it was expected that the 7.1-kb fragment of the insert of AHr3 should hybridize with a genomic fragment of the same size. However, the resolution was not such as to allow a decision as to whether hybridization of the 7.5-kb fragment of the same insert also contributed to the formation of the approx. 7.1-kb band in the genomic blot. Further RNA and DNA transfer hybridization experiments, utilizing separated 7.1-kb and 7.5-kb fragments as probes, indicated that the 7.5-kb cloned DNA fragment is unrelated to the rDNA, and presumably became joined to the 7.1-kb fragment during the cloning (unpublished data). On the basis of the results described above, the inserts of the recombinant phage DNAs analyzed here have been aligned as shown in Fig. 1. One can see that the physical map of these inserts agrees well with the previously determined map of human rDNA (Wellauer and Dawid, 1979), except for the presence in the cloned sequences of an additional EcoRI site (site 2*) located in NTS at approx. 14.2 kb 5’ to the EcoRI site in the 18s rRNA gene. The new type of EcoRI polymorphism detected here appears to involve only a relatively small fraction of rDNA units in the human liver DNA analyzed. From densitometric measurements performed on the autoradiograms it can be estimated
-17.8 -12.1 -8.5
-5.7
of 32P-labeled
Fig. 2. Hybridization liver DNA
or VA,-B
nick-translated digest
probes
of bacteriophage
(B) ‘2P-labeled
DNA derived
derived
4A DNA
to bands
represent
paper.
3’-end
labeled
probes derived from the 8.5kb
or from the 7.1-kb or the 5.7-kb insert fragment lanes and lane marked
from total recombinant
to nitrocellulose
from total IHr4 (lane 4) of iHr5
Charon
nick-translated
probes
transferred
of lHrl1
phage
(A) 32P-labeled
DNAs or inserts cDNA
synthesized
(lane 5) DNA were hybridized
with E. coli DNA insert fragment
(lanes 11,
polymerase
ofiHr5
from
to ,X&RI-digested 18s rRNA
to adult human I and
[r-3ZP]dATP
human
or “P-labeled
liver DNA. Ch, EcoRI and
[r-72P]dTTP
(lanes 5, 5), or from the whole ,lHr3 DNA (lane 3)
I and 11,,) were hybridized to adult human liver DNA (all unmarked
L) or to VA,-B DNA (VA,). i,: 3’-end labeled Hind111 digest ofbacteriophage
the sizes of the fragments
thereof
i, DNA. The numbers
corresponding
in kb.
that only about 13 % of the rDNA units exhibits the EcoRI site 2* (Fig. 1). Furthermore, the results of hybridization with genomic blots strongly suggest that, in the rDNA copies where this extra EcoRI site is present, site 3* is also present; whereas, in the rDNA copies where site 2* is absent, site 3* may or may not be present. This distribution of the EcoRI sites defines three types of EcoRI polymorphism (Fig. 3): type 1, exhibiting EcoRI site 3* but not site 2*, type 2, exhibiting neither of the two sites, and type 3, exhibiting both. The distribution of EcoRI
sites detected here also accounts for the failure to detect the EcoRI site 2* in previous studies in which the 8.5kb probe was not available. As to the nature of the event which led to the appearance of a new EcoRI site at position 2*, our results cannot distinguish between a mutational event at this position and a deletion of a segment of approx. 3.6 kb between site 1 and site 3*: such a deletion would result in a movement of site 1 closer to site 3* to position 2* (Fig. 3). In the human liver DNA investigated here, type 1
113 REFERENCES
i 18s
Type
28s
I
”
i
I z’ 1’ “1 ;
I
i
II
1
Type2
or 3*
4
5
Type
3
Fig. 3. Diagrammatic representation of the three types ofEcoR1 polymorphism of the human rDNA repeating units which have been detected in previous and the present work.
is present in about 58 % of the repeating units, type 2 in approx. 29%, and type 3, in approx. 13%. In a previous analysis of the distribution of polymorphisms in the rDNA derived from 20 pooled placental DNA samples, it was found that 80% of the rDNA repeating units (including both types 1 and 3 defined above) exhibited the EcoRI site 3*, and 20 % (corresponding to the present type 2) lacked this site (Krystal et al., 1981). Therefore, the liver DNA sample analyzed here approaches the frequency of the EcoRI site 3* in the repeating units which was reported in the above mentioned study. In another human DNA sample analyzed here (VA,-B), which also exhibits the EcoRI site 2* in a small fraction of the rDNA copies (approx. 7x), the proportion of repeating units lacking site 3* appears on the contrary to be considerably higher (approx. 64%).
ACKNOWLEDGEMENTS
This work was supported by National Institutes of Health grant GM1 1726 to G.A. and an EMBO Fellowship to M.M. M.M. wishes to express her gratitude to Dr. Vittorio Sgaramella for hospitality in his laboratory and to the Istituto Farmacologico Serono-Roma for research support during part of the work reported here. The technical assistance of Ms. Arger Drew is gratefully acknowledged.
Arnheim, N. and Southern, E.: Heterogeneity of the ribosomal genes in mice and men. Cell 11 (1977) 363-370. Arnheim, N., Krystal, M., Schmickel, R., Wilson, G., Ryder, 0. and Zimmer, E.: Genetic exchanges among ribosomal genes on non-homologous chromosomes in man and the great apes. Proc. Natl. Acad. Sci. USA 77 (1980) 7323-7327. Benton, W.D. and Davis, R.W.: Screening Igt recombinant clones by hybridization to single plaques in situ. Science 196 (1977) 180-182. Financsek, I., Mizumoto, K., Mishima, Y. and Muramatsu, M.: Human ribosomal RNA gene: nucleotide sequence of the transcription initiation region and comparison of three mammalian genes. Proc. Natl. Acad. Sci. USA 79 (1982) 3092-3096. Krystal, M. and Arnheim, N.: Length heterogeneity in a region of the human ribosomal gene spacer is not accompanied by extensive population polymorphism. J. Mol. Biol. 126 (1978) 91-104. Krystal, M., D’Eustachio, P., Ruddle, F.H. and Arnheim, N.: Human nucleolus organizers on nonhomologous chromosomes can share the same ribosomal gene variants. Proc. Natl. Acad. Sci. USA 78 (1981) 5744-5748. Lawn, R.M., Fritsch, E.F., Parker, R.C., Blake, G. and Maniatis, T.: The isolation and characterization of linked 5- and I-globin genes from a cloned library of human DNA. Cell 15 (1978) 1157-l 174. Maniatis, T., Hardison, R.C., Lacy, E., Lauer, J., O’Connell, C., Quon, D., Sim, G.K. and Efstradiatis, A.: The isolation of structural genes from libraries of eukaryotic DNA. Cell 15 (1978) 687-701. Maniatis, T., Fritsch, E.F. and Sambrook, J.: Molecular Cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982. Miesfleld, R. and Amheim, N.: Identification of the in vivo and in vitro origin of transcription in human rDNA. Nucl. Acids Res. 10 (1982) 3933-3949. Morandi, C., Masters, J., Mottes, M. and Attardi, G.: Multiple forms of human dihydrofohc acid reductase messenger RNA. Cloning and expression in Escherichiu coli of their DNA coding sequence. J. Mol. Biol. 156 (1982) 583-607. Ponttn, J., Jensen, F. and Koprowski, H.: Morphological and virological investigation of human tissue cultures transformed with SV40. J. Cell. Comp. Physiol. 61 (1963) 145-154. Schmickel, R., Waterson, J., Knoller, M., Szura, L. and Wilson, G.D.: HeLa cell identification by analysis of ribosomal DNA segment patterns generated by endonuclease restriction. Am. J. Hum. Genet. 32 (1980) 890-897. Wellauer, P. and Dawid, I.: Isolation and sequence organization of human ribosomal DNA. J. Mol. Biol. 128 (1979) 289-303. Wilson, G.D., Hollar, B.A., Waterson, J.R. and Schmickel, R.D.: Molecular analysis of cloned human 18s ribosomal DNA segments. Proc. Natl. Acad. Sci. USA 75 (1978) 5367-5371. Communicated by A.D. Riggs.
Gene, 27 (1984) 109-113 Elsevier GENE 942
A new type of EcoRI polymorphism of the human ribosomal DNA repeating unit revealed by analysis of cloned DNA fragments (Human liver DNA; genomic library; cDNA; 45s rRNA; 1 Charon vectors)
Monica Mattes*, S.-A. Tsai Lai, Julio Montoya and Giuseppe Attardi Division of Biology, Calgomia Institute of Technology, Pasadena, CA 91125 (U.S.A.)
Tel. (213)-356-4930
(Received August 2&h, 1983) (Revision received November 6th, 1983) (Accepted November 9th, 1983)
SUMMARY
Several clones of rDNA have been isolated from an adult human liver DNA Charon 4A library by using cDNA probes synthesized from human 18s and 28s rRNA. The insert of one recombinant Charon 4A clone contained, besides the already known 5.7-kb EcoRI fragment of rDNA, comprising the major portion of the 18s rRNA gene and all the external transcribed spacer (ETS), a previously unidentified EcoRI fragment of rDNA of 8.5 kb in size. DNA transfer hybridization experiments utilizing EcoRI digests of the human DNA used to construct the library and of another human DNA showed the presence of the 8.5-kb EcoRI fragment in a minority of the rDNA repeats on the 5’-end side of the 5.7-kb fragment, thus defining a hitherto unidentified type of EcoRI polymorphism of these repeats.
INTRODUCTION
A limited length and sequence heterogeneity has been reported among the various rDNA units of the human nuclear genome in the same individual. In particular, three forms of polymorphism have been detected in the human rDNA family by restriction enzyme analysis of genomic DNA. The EcoRI polymorphism involves the presence in the NTS of some of the repeats of a site at approx. 5.7 kb 5’ to the * Present address: Istituto di Genetica, Universita di Pavia, 27100 Pavia (Italy) Tel. 382-303852. Abbreviations: bp, base pairs; cDNA, DNA complementary to RNA; ETS, external transcribed spacer; ITS, internal transcribed spacer; kb, kilobase pairs; NTS, nontranscribed spacer; rDNA, ribosomal DNA; SDS, sodium dodecyl sulfate. 0378-l 119/84/$03.00 0 1984 Elsevier Science Publishers
EcoRI site in the 18s rRNA gene (site 3* in Fig. 1; Arnheim and Southern, 1977; Wilson et al., 1978; Wellauer and Dawid, 1979). The Hind11 polymorphism involves the presence of a Hind11 site in the 3 ’ -end proximal third of the 28 S rRNA gene of some of the repeats (Amheim et al., 1980). The NTS length polymorphism involves a variation in length in a region within 4 kb of the 3 ‘-end of the 28s rRNA gene; this variation results in the presence of four length classes (Krystal and Arnheim, 1978; Arnheim et al., 1980; Schmickel et al., 1980). In the present work, the cloning and characterization of segments of human rDNA isolated from a human liver DNA library has led to the detection of a new type of EcoRI polymorphism, involving the presence in a relatively small portion of the repeats of a hitherto unrecognized EcoRI site at approx.
14.2 kb 5’ to the EcoRI site in the 18s rRNA gene
4.6 kb;
(site 2* in Fig. 1).
contained
the
insert
of another
a 5.7-kb fragment,
8.5-kb fragment;
Sucrose from
gradient-purified
HeLa
DNase
cells
(Boehringer,
were
28s treated
100 pg/ml,
SDS-pronase-phenol-extracted, ed, and used as templates
and, in addition,
phages
experiments
and
18s rRNAs
mic oligo(dT)-cellulose
non-bound
RNase-free
cells and 32P-labeled
nick-translated
30 min
at 4”C),
for reverse transcriptase
RNA trans-
utilizing total cytoplas-
with
ethanol-precipitat-
an
1Hr3, AHr4, AHr5 and
J_Hrll were chosen for further analysis. fer hybridization
also
and a 7.5-kb fragment.
The recombinant AND METHODS
iHr5,
finally, the insert of AHr3 consisted
of a 7.1-kb fragment MATERIALS
clone,
RNA from HeLa probes derived
from whole IHr4
or IHr5
showed
a
clear hybridization
of both probes to 18s rRNA,
in
agreement
phage DNA
with the plaque-screening
results.
Similar
together with calf thymus DNA random primers (Morandi et al., 1982). A library was constructed from a partial EcoRI digest of adult human liver DNA using lambda Charon 4A as a vector, essentially as described in published procedures (Maniatis et al., 1978; Lawn et al., 1978). Fragments in the size range 13 to 20 kb were selected by sucrose gradient centrifugation. Screening of the recombinant clones was carried out
experiments using whole nick-translated /1Hr3 DNA as a probe revealed a strong hybridization of the latter to 28s rRNA, again in confirmation of the plaque-screening results, and a weak hybridization to 18s rRNA; a hybridization of similar intensity to 18s and 28s rRNA was on the contrary observed with a probe derived from the whole iLHrl1 DNA. On the basis of these observations, it seemed very likely that the 5.7-kb fragment of the insert of /ZHr4,
by a published procedure (Benton and Davis, using 18s and 28s cDNA probes.
1977)
J.Hr5 and 1Hrll was involved in hybridization with 18s rRNA, while the 7.1-kb fragment of the inserts
Transfer onto nitrocellulose paper of HeLa cell cytoplasmic oligo(dT)-cellulose non-bound RNA, after electrophoresis through an agarose-CH,HgOH or an agarose-formaldehyde gel, and of EcoRI-
of JHr3 and AHrl 1 was responsible for their hybridization with 28s rRNA and, probably, to a small extent, with 18s rRNA. These results allowed the identification of the 5.7-kb and 7.1-kb fragments cloned in Charon 4A with those previously mapped in the human rDNA (Wellauer and Dawid, 1979; Fig. 1).
digested HeLa or VA,-B cell DNA, after electrophoresis through an agarose gel, was carried out by standard techniques. Cloned rDNA fragments, 32Plabeled by nick translation, or 32P-labeled cDNA synthesized from purified 28s or 18s rRNA were used as probes.
To analyze the distribution of the cloned DNA sequences in the genome, DNA transfer hybridization experiments were performed using the adult liver DNA utilized for the construction
RESULTS
AND
DISCUSSION
Approx. 7 x lo4 plaques from the human liver genomic library were screened separately with 32Plabeled 28s and 18s cDNAs. Two and five clones were found to give a positive signal with the 18s and 28s cDNA probes, respectively. An electrophoretic analysis of EcoRI digests of these recombinant phage DNAs revealed that the inserts of four recombinant clones (AHr8, IHr9,lHrll and IHr12) consisted of a 5.7-kb and a 7.1-kb fragment. The insert of another recombinant clone (AHr4) contained a 5.7-kb fragment and two smaller fragments of 5.1 kb and
of the library and,
in some experiments specified below, also DNA from the human cell line VA,-B. With the 18s cDNA probe, a strong band corresponding to a size of 5.7 kb and two fainter ones corresponding to sizes of 7.1 and 17.8 kb were observed (Fig. 2A). The strong 5.7-kb band and the weak 17.8-kb band were also seen when the 5.7-kb fragment of the insert of IHr 11 was used as a probe (Fig. 2B). The 7.1-kb fragment of the same insert hybridized, as expected, to a genomic fragment of the same size (Fig. 2B; the faint 5.7-kb and 17.8-kb bands reflect a low level of contamination of the 7.1-kb fragment by the 5.7-kb fragment). When the total DNA of 1Hr5 was used as a probe, four labeled bands corresponding to sizes of 5.7,8.5,
111 H 1 kb XHr8, XHrll,
XHr9 XHr12 >
5.7
4
t
7.1
7.5 ,_-------1
4
1
XHr5
8.5
+c----3-----4.6 +
XHr4
1
--
-8
’
2”
5.1
4 1
j
!.? 3”
45s
1
(
L
, k
XHr3
1 1 4
l
I I
5
!
u-
NTS
NTS ETS
ITS
Fig. 1. The cloned fragments of human rDNA isolated in the present work are aligned with the EcoRI restriction map of the rDNA repeating unit, as determined by Wellauer and Dawid (1979) and in the present work. Small downwards arrows indicate the positions ofthe EcoRI restriction sites. Asterisked numbers indicate sites not present in all repeating units. The numbers over the cloned fragments indicate their size in kb; the dashed segments represent cloned fragments not belonging to rDNA. The mapping position of the “45s RNA”, with the 5’ ends of the major (slightly shorter) and minor (slightly longer; dotted line) transcripts recently identified (Financsek et al., 1982; Miestield and Arnheim, 1983; M. Mottes, J. Montoya and G. Attardi, unpublished observations), is shown above the bottom line.
12.1 and 17.8 kb were observed, with the 5.7-kb and 12.1-kb bands being considerably stronger than the other two (Fig. 2A). The 8.5-kb fragment of the insert of LHr5 hybridized to a fragment of the same size and, in addition, to the two fragments of approx. 12.1 and approx. 17.8 kb (Fig. 2B). The hybridization pattern of the 8.5kb fragment to VA,-B DNA was similar to that obtained with liver DNA, except that the 17.8-kb band was considerably stronger, instead of weaker, than the 12.1-kb band (Fig. 2B). From a comparison of the hybridization patterns obtained with total LHr5 DNA or with the 8.5-kb fragment as a probe one can infer that the 5.7-kb fragment of IHr5 DNA hybridized to a genomic fragment of the same size and, presumably also, weakly, to a 17.8-kb fragment, like the 5.7-kb fragment of JHrl 1 (Fig. 2B). When the total 1Hr4 DNA was used as a probe, it gave the same hybridization pattern (Fig. 2A) observed with the 5.7-kb insert of LHrl 1. By contrast, no obvious hybridization was observed to fragments of a size corresponding to those of the two smaller fragments of the insert (5.1 kb and 4.6 kb); this observation strongly suggests that the latter two fragments are not related to the rDNA, and that they became joined to the 5.7-kb fragment during the cloning. When the whole JHr3 DNA was used as a probe, an intense band corresponding to a fragment with a
size of approx. 7.1 kb was observed in a genomic blot of liver DNA. On the basis of the results obtained with the 7.1-kb fragment of the insert of AHrll, it was expected that the 7.1-kb fragment of the insert of AHr3 should hybridize with a genomic fragment of the same size. However, the resolution was not such as to allow a decision as to whether hybridization of the 7.5-kb fragment of the same insert also contributed to the formation of the approx. 7.1-kb band in the genomic blot. Further RNA and DNA transfer hybridization experiments, utilizing separated 7.1-kb and 7.5-kb fragments as probes, indicated that the 7.5-kb cloned DNA fragment is unrelated to the rDNA, and presumably became joined to the 7.1-kb fragment during the cloning (unpublished data). On the basis of the results described above, the inserts of the recombinant phage DNAs analyzed here have been aligned as shown in Fig. 1. One can see that the physical map of these inserts agrees well with the previously determined map of human rDNA (Wellauer and Dawid, 1979), except for the presence in the cloned sequences of an additional EcoRI site (site 2*) located in NTS at approx. 14.2 kb 5’ to the EcoRI site in the 18s rRNA gene. The new type of EcoRI polymorphism detected here appears to involve only a relatively small fraction of rDNA units in the human liver DNA analyzed. From densitometric measurements performed on the autoradiograms it can be estimated
-17.8 -12.1 -8.5
-5.7
of 32P-labeled
Fig. 2. Hybridization liver DNA
or VA,-B
nick-translated digest
probes
of bacteriophage
(B) ‘2P-labeled
DNA derived
derived
4A DNA
to bands
represent
paper.
3’-end
labeled
probes derived from the 8.5kb
or from the 7.1-kb or the 5.7-kb insert fragment lanes and lane marked
from total recombinant
to nitrocellulose
from total IHr4 (lane 4) of iHr5
Charon
nick-translated
probes
transferred
of lHrl1
phage
(A) 32P-labeled
DNAs or inserts cDNA
synthesized
(lane 5) DNA were hybridized
with E. coli DNA insert fragment
(lanes 11,
polymerase
ofiHr5
from
to ,X&RI-digested 18s rRNA
to adult human I and
[r-3ZP]dATP
human
or “P-labeled
liver DNA. Ch, EcoRI and
[r-72P]dTTP
(lanes 5, 5), or from the whole ,lHr3 DNA (lane 3)
I and 11,,) were hybridized to adult human liver DNA (all unmarked
L) or to VA,-B DNA (VA,). i,: 3’-end labeled Hind111 digest ofbacteriophage
the sizes of the fragments
thereof
i, DNA. The numbers
corresponding
in kb.
that only about 13 % of the rDNA units exhibits the EcoRI site 2* (Fig. 1). Furthermore, the results of hybridization with genomic blots strongly suggest that, in the rDNA copies where this extra EcoRI site is present, site 3* is also present; whereas, in the rDNA copies where site 2* is absent, site 3* may or may not be present. This distribution of the EcoRI sites defines three types of EcoRI polymorphism (Fig. 3): type 1, exhibiting EcoRI site 3* but not site 2*, type 2, exhibiting neither of the two sites, and type 3, exhibiting both. The distribution of EcoRI
sites detected here also accounts for the failure to detect the EcoRI site 2* in previous studies in which the 8.5kb probe was not available. As to the nature of the event which led to the appearance of a new EcoRI site at position 2*, our results cannot distinguish between a mutational event at this position and a deletion of a segment of approx. 3.6 kb between site 1 and site 3*: such a deletion would result in a movement of site 1 closer to site 3* to position 2* (Fig. 3). In the human liver DNA investigated here, type 1
113 REFERENCES
i 18s
Type
28s
I
”
i
I z’ 1’ “1 ;
I
i
II
1
Type2
or 3*
4
5
Type
3
Fig. 3. Diagrammatic representation of the three types ofEcoR1 polymorphism of the human rDNA repeating units which have been detected in previous and the present work.
is present in about 58 % of the repeating units, type 2 in approx. 29%, and type 3, in approx. 13%. In a previous analysis of the distribution of polymorphisms in the rDNA derived from 20 pooled placental DNA samples, it was found that 80% of the rDNA repeating units (including both types 1 and 3 defined above) exhibited the EcoRI site 3*, and 20 % (corresponding to the present type 2) lacked this site (Krystal et al., 1981). Therefore, the liver DNA sample analyzed here approaches the frequency of the EcoRI site 3* in the repeating units which was reported in the above mentioned study. In another human DNA sample analyzed here (VA,-B), which also exhibits the EcoRI site 2* in a small fraction of the rDNA copies (approx. 7x), the proportion of repeating units lacking site 3* appears on the contrary to be considerably higher (approx. 64%).
ACKNOWLEDGEMENTS
This work was supported by National Institutes of Health grant GM1 1726 to G.A. and an EMBO Fellowship to M.M. M.M. wishes to express her gratitude to Dr. Vittorio Sgaramella for hospitality in his laboratory and to the Istituto Farmacologico Serono-Roma for research support during part of the work reported here. The technical assistance of Ms. Arger Drew is gratefully acknowledged.
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