volume 10 Number 22 1982
Nucleic Acids Research
Nucleotide sequence of a segment of human DNA containing the three tRNA genes
Kenneth LRoy*, Howard Cooke+ and Richard Buckland^
Department of Microbiology, University of Alberta, Edmonton, Canada T6G 2E9, +MRC Mammalian Genome Unit, Department of Zoology, West Mains Rd., Edinburgh EH9 3JT, and ^MRC
Clinical and Population Cytogenetics Unit, Western General Hospital, Edinburgh EH4 2XU, UK
Received 2 July 1982; Revised and Accepted 14 October 1982
ABSTRACT
A 1.65 kb segment of DNA from a human-A recombinant, selected for human
tRNA genes, was subcloned in the plasmid pAT 153 s for sequence determination.
Three human tRNA genes were found; one, a tRNA'y gene which appears to s
specify a tRNA identical in sequence tolnthe previously described tRNA 3 'y
from rat liver (AAA.G); another a tRNA9 gene, the product of which would
be expected to recognize
only the codon CAG; and a third which would direct
synthesis of a tRNA Teu specific for CUA and G. Intervening sequences are
not found in any of these genes. The three tRNA genes are separated by
about 0.5 kb segments of DNA containing no apparent informational sequences.
Examination of flanking sequences shows some similarities at the 5'-ends
of the three genes, but less similarity to 5' flanking sequences of tRNA
genes in other eucaryotes. All three tRNA genes direct synthesis of appropriate sized products in a HeLa cell lysate in vitro transcription system.
INTRODUCTION
The sequences of a number of eucaryotic tRNA genes have been reported,
(1) but few of these have been determined for vertebrates. Mu'ller and
Clarkson (2) reported the sequence of the first 593 bases of the 3.18 kb
repeated unit from Xenopus laevis, which includes genes for a tRNAP he and
a tRNAty. This repeated unit also contains genes for two tRNA m e t species
(3) as well as tRNA as P, tRNA a l a , tRNA 1 * 5 and tRNA leu (Mu'ller and Clarkson,
unpublished, cited in Hofstetter e_t a_K , (4), and Gal 1 i et a_h , (5). More
recently Santos and Zasloff (6) determined the sequence of two tRNA m e t
genes of human origin, and Sekiya e_t a_[. (7) cloned and sequenced a 2 kb
segment of rat DNA containing genes for a tRNA as P, a tRNA^y and a tRNA9lu.
Although there is good evidence that tRNA genes are highly conserved
(1,8) there is less information concerning conservation of their flanking
sequences, and the occurrence of intervening sequences. Furthermore,
although evidence is accumulating which suggests little conservation of
tRNA gene arrangement in vertebrates (2,6,7,9) there is a need for more
IR L Press Limited, Oxford, Errand.
0305-1048/82/1022-73138 2.00/0
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extensive information to define the limits of this conservation.
In an investigation of human tRNA genes (R. Buckland, H. Cooke,
K.L. Roy, J. Dahlberg and E. Lund, in preparation) a human-A recombinant
(AHt 4) was isolated which had several tRNA genes. Because of uncertainties in the determination of which tRNA genes were present in this recombinant, a large portion of the human insert was subcloned in a series of
plasmids for sequence determination. We wish to describe the sequence of
1.65 kb of human ONA which contains three tRNA genes, all of which appear
to be functional.
MATERIALS AND METHODS
Enzymes and Reagents
Restriction endonucleases were purchased from Bethesda Research Laboratories, New England Biolabs and Boehringer-Mannheim. The Klenow fragment
of E.. coli DNA polymerase I was from New England Nuclear and PL Biochemicals.
All a[32p]-deoxyribonucleoside triphosphates (at >600 Ci/mmol) were
from New England Nuclear, as was the HeLa cell lysate i_n vitro transcription
system. DEAE-Sephacel was a product of Pharmacia.
Isolation of Restriction Fragments
All restriction endonuclease digestions were performed under conditions
specified by the suppliers. Fragments of interest were separated electrophoretically on 5, 6 or 7% polyacrylamide slab gels, detected by 300 nm
ultraviolet light after ethidium bromide staining, and extracted as suggested
by Maxam and Gilbert (10,11).
Preparation of Plasmids
XHt 4 was isolated from a human-A recombinant library (12) provided by
T. Maniatis, using standard procedures (R. Buckland, H. Cooke, K.L. Roy,
J. Dahlberg and E. Lund, in preparation). Briefly, plaques were screened
by the Benton and Davis (13) method using nick-translated DNA from the
plasmid pXt 210 (2) as a probe. Several positive plaques were picked,
purified, and grown in quantity. DNA was isolated from the recombinant
phage and characterized by restriction endonuclease digestion (Eco RI, Bam HI,
Bgl II, and Hind III). Southern transfers (14) were hybridized to pXt 210
DNA, and separately to tRNA labeled, after partial thermal degradation, with
Y [ P]-ATP using polynucleotide kinase.
Recombinant plasmids containing segments of the human DNA insert in
AHt 4 were prepared by ligation of restriction fragments into the cloning
vehicle pAT 153 (15), followed by transformation of E_. coli HB 101 (16)
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with the ligated DNA. The desired transformants were detected by small
scale plasmid isolation (17) after selection of amp r tet s or amp r tet r
colonies. Restriction endonuclease digestions verified the identity of
the inserted segments and, if necessary, their orientation.
End-Labeling of Restriction Fragments
All DNA fragments were labeled at their 3'-termini by using the Klenow
fragment of E. coli DNA polymerase I and appropriate a[^2p] labeled and
unlabeled deoxyribonucleoside triphosphates (2,19). Flush ends were labeled
at 25° (to allow exonuclease removal of the ultimate nucleotide) and staggered ends (with 5' extensions) were labeled at 15°. After labeling, the DNA
was precipitated by addition of ammonium acetate to 2M, followed by three
volumes of ethanol, as described by Maxam and Gilbert (11). Cleavage with an
appropriate restriction endonuclease was used in most cases to separate the
two labeled ends, except in a few cases where strand separation on polyacrylamide slab gels was successful.
DNA Sequencing
The chemical sequencing procedures of Maxam and Gilbert (10,11) were
used throughout with little modification. In scone experiments the T-specific
reaction described by Friedman and Brown (20) was included, and more recently
the T-specific reaction of Rubin and Schmid (21) has been used. Best results
were obtained when a fragment was chromatographed on DEAE-Sephacel after
elution from polyacrylamide gel. Electrophoretic separation of the endlabeled fragments after completion of the sequencing reactions was achieved
with thin (0.25, 0.4, 0.5 mm) polyacrylamide (6,7,8,12,16,20%) gels (22)
using the 8.3 M urea-100 mM tris-borate-EDTA buffer system of Maxam & Gilbert
(11). In some cases the modified electrophoretic separation technique
described by Smith and Calvo (23) was used. Visualization of radioactive
bands was either radioautographic at -20° using Kodak NS-54T film or, more
commonly, fluorographic at -90° using Kodak XR-1 film and Dupont Cronex
Lightning Plus intensifying screens (24).
RESULTS
Sequencing Strategy
Most of the human insert of XHt 4 was subcloned as a series of overlapping fragments in the plasmid pAT 153 (15). Figure 1 shows the relationship of the recombinant plasmids obtained to the insert in XHt 4.
Sequencing was conducted exclusively by the method of Maxam and Gilbert
(10,11) using only 3' terminal labeling (2,19). All regions of the human
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AHt 4
Bam HI H « . II
I
|
Kpn I
l y —»* i
Eco Rl
|
Hind HI
gin—»-i
Eco Rl Hp» I
t
|
Hind III
I
pHt 4-1
pHt 4 - 9
Figure 1. Restriction map of the tRNA gene-containing region of AHt 4.
Sites are shown for the major six-base-specific restriction endonucleases;
the segments subcloned for sequence analysis are also indicated. The locations
and orientations of the three tRNA genes are shown by short arrows.
DMA were sequenced at least twice, and on both strands over most of the
sequence. Restriction endonuclease digestion of the intact plasmids revealed
that Alu I, Dde I, Hinf I, Msp I, Rsa I, Sau 3AI and Taq I gave products
indicative of cleavage within the insert. A restriction map of the segment
shown in Fig. 1, together with the major sequencing reactions, is shown in
Fig. 2, and the complete sequence is shown in Fig. 3. The variety of
restriction sites labeled, and the use, in a number of cases, of more than
one enzyme to cut labeled fragments, gives us considerable confidence in
the sequence described. With one exception, all restriction sites predicted
by the sequence were cut by the appropriate enzyme, of those we had available
for testing. The enzymes not tested include Bbv I, Hae III, Mbo II, Mnl I
and Tha I.
The single exception was the Ava II site centered at 390. This site
overlaps an Eco RII site, and it is thus assumed that the methylation of
the C at position 392 also blocks Ava II cleavage. This would be analogous
to the case reported by Backman (25), and also similar to the tRNA^ 1 " gene
described by Miiller and Clarkson (2) where modification of Mbo I sites
blocked cleavage at overlapping Taq I sites. Protection of restriction
sites by methylation of overlapping sites seems to be fairly common (26).
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Bun HI
Eco Rl
Hind 111
Eco Rl
Hind Ul
Alu I
Clo I
Dd*
I
M«p I
FUa I
Sail 3AI
T»q I
Figure 2. End-labeling and secondary cleavage restriction sites of the tRNA
gene-containing region of xHt 4. The arrows indicate the direction and distance sequenced. The four and five-base-specific restriction endonuclease
sites shown are consistent with their use as end-labeling sites and/or secondary
cleavage sites.
tRNA Genes
A segment of cloned human DNA between nucleotides 343 and 417 appears
to be a gene for a tRNA^ s . This gene would direct the synthesis of the
product shown in Fig. 4a, with the CCA terminus (not encoded in the DNA)
included for completeness. The tRNA^ s shown in Fig. 4a has the same sequence
as the rabbit liver tRNA3-vs (27) prior to secondary modifications. The
tRNA^ s gene has been shown to function efficiently in vivo on injection into
Xenopus oocytes (28). Furthermore, in vitro site-directed mutagenesis has
been used to convert this gene (transferred from pAT 153 to the bacteriophage
M13 mp7) to a functional nonsense (UAG) suppressor gene (28).
Another segment of the insert appears to be a gene for a tRNA9'n capable
of recognizing the codon CAG. This presumed gene, between nucleotides 940
and 1013, also has the usual base pairing and invariant nucleotides, and
would be expected to direct the synthesis of the product shown in Fig. 4b.
This gene also has been shown to function upon Injection into Xenopus oocyte
nuclei (28) and can be mutated to an amber suppressor gene.
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100
GGATCCT66C G66AGCCCAG GT6TCCGGGA TCTGSGCCAC TAGSGACTGG SGA66AACCT CtCASAGAAG CCCATAGCCC GCASCG6CCC CGCGCGGCCG
CCTAGGACCG CCCTCGGGTC CACAGGCCCT AGACCCGtTG ATCCCTGACC CCTCCTTG6A SAGTCTCTTC G66TATCGGG CGTCGCCGGG 6CSCGCCGGC
200
GTTCCGGCK C6CACTGTTC CASCCTCTAC TATGGTACAG TCCCTGCGK GCAGCCTCGG CGGCfiGCTCT AAGAACGGGA GGCAGAAAAA GCTCAATCAG
CAAGGCCGCG GCGTGACAAG GTC6GAGATG ATACCATGTC AG6GACGCAG CGTCGGAGCC GCCCCCGA6A TTCTTGCCCT CCGTCTTTTT CGAGnAGTC
300
CAGCAGGCGA GCTTCACCCG CT6CTTCCAA ATCTGTGCCA AAATAnCTA TGCTGCACAG ATAAAATCCT CTGTCGGTTC IACAAGCCTG GCTTTTCCTA
GTCGTKGCT CGAAGTGS6C GACGAA6Gn TAGACAC6GT TT7ATAAGAT ACGACGTGTC TATTTTAfiGA GACACCCAAG ATGTTC66AC C6AAAAGCAT
TAGAGAAaC TCTTATAAGC AAAAAGTAAA G a a C S T G A AGAGCCC66A TAfinCASTC 6GTAEAGCAT CAGACnTTA ATCTGAS6ST CCAGGCTTCA
ATCTCnGGG AGAATATTCG TTTnCATTT CGAGAGCACT TCTC666CtT ATCGAGTCAG CCATCTCGTA GTCTGAAAAT TAGACTCaA GGTCCCAAGT
AG7CCCTGTT CGGGCGGCAT G T C H T G a T TTGGGTACCG CACTTC6CAT AAAATGCTAA CAAAAAIACT TACTASTnT TAAAOaAGG 7GATACAGAA
TCASGGACAA GCCCGCCGTA CAGAAACGAA AACCCAT6SC GT6AAGCGTA T T T T A C a n G T n n A T C A ATGATCAAAA ATnCGATCC ACTATGTCTT
600
A C n G A H T C CACAAAATAC A6CAAACGAC GGTGGAGGGT VTAAACATA CTTTAAAAa GTACAAACGC GAGCCGTATT ATCGAATnA CATTTTCTGG
TGAACTAAAG GIGTTTTATG TCGTTTGCTG CCACCTCCCA nATTTGTAT GAAAnnGT CATGTTTGCG CTCGGCATAA TAGCT7AAAT GTAAAAGACC
700
TAGCCACAn ACAAAGAAIA AAAAGCAAGC GAAAHAAn TCACGGATCT A n n A C H A CCCTAATATA CCAAAATaT ATTTTAACAT ATAATCAACA
ATCGGTGTAA TGTTTCTTAT TTTTCGTTCG CTTTAAnAA AGTGCCTAGA TAAAATGAAT GGGATTATAT GGTTTTAGAA TAAAATTGTA TATTAGTTGT
800
TTCTAAAAAT AnAATGAAA nTTAAGTCT TTTTGTTTGT ACAAAGCCTT CCTTCAAAAG AAGAIGTGTA TTTAGAAGCT GGCAATACAT CTGAATTCAT
AAGATTTTTA TVTTACTTT AAAATTCAGA AAAACAAACA TGTTTCGGAA GSAAGTTTTC TTCTACACAT AAATCTTCGA CCGTTATGTA GACTTAAGTA
WU
ATATTTCAAG TGCTCGATAA CGSTGTCTAC CATAICCAAC AGCAGACTTT GTTTTGTTTT CGnTTAAGT GAC&GGTGT6 GAAGACACGC AAAATACAAT
TATAAAtTTC ACGAGaATT GCCACAGATG GTATAGGTTG TCGTCT6AAA CAAAACAAAA GCAAAAnCA CTGCCCACAC CTTCTCTGCG TTTTATGTTA
GICCAGTAAT G1ACACAATC n6AAAGCCC CCACGAG6GA GGTTCCATGG TGTAATGGTT ASCACTCTGG ACTCTGAATC CA6CSATCCG A6TTCAAATC
CAGG7CATTA CATGTGTTAG AAC1TTCGGG GGTGCTCCa CCAAGGTACC ACAHACCAA TCGTGA<WICC TGAGACTTAG GTCGCTASGC TCAAGTTTAG
TCGGTGGAAC CTTAAAGCTT ITCTTTTAAT ATCAGCATGT TGAATATTGT T A A n G A m TCACASAGGG GATACTCCTA TGACCCAAGA TAATACGACA
AGCCACCTTG GAATTTCGAA AAOAAAAnA TAGTCGTACA ACTTATAACA ATTAACTAAA AGTGTCTCCC CTATGAGGAT ACTGGGTTCT ATTATGCTGT
1200
AAAAAAAAAA GTAAACTAGG ACACTKCAA TTATTTGTGC TTHCCTACC CGATAATTGA GTCCTATAM ACAGTGTTAA GAGAACGTAC AATAMTTAT
111111 11 IT CATnGATCC TGTGATGGTT AATAAACACG AAAAGGATGG CCTATTAACT CASGATAnG TGTCACAATT CTCnGCATG nATTTAATA
1300
TTAAATATGT TGGAAGTTGA GCTAACATTT GAAAACATGT TAAAATA£AG ATGTTATUA GAAACGAAAT AATAfiAGATG nATGAAGAA ACGAAATAH
AATTTATACA ACCTTCAACT CGATTGTAAA CTTTTGTACA ATT^TATCTC TACAATACTT CTTTGCTTTA.nATCTCTAC AATACnCTT TGCTTTAJAA
1*00
GCnCAGCAG nACCATCAG GTCTTAGCTT CTGGGATGGA TGCAGGCCTA TGATCTnCT GGAASACCAA ACACGGAAGA IGATGG7GAA ACCATTAOA
CSAAGTCGTC AAIGGIAGTC CAGAATCGAA GACCCTACCT ACGTCCGGAT ACTAGAGAGA CCTTCTGGTT TGTGCCTTCT ACTACCACn TGGTAATGCT
1S00
GGG6AATTCG TAAAATTACT I T C T G a i G I AAATAGGIGT AACaAACAG AACTGTTAAC AGAACTGTAG CACSTGCGTA ATCCACCAGA AAAACTCCAG
CCCCTTAACC ATTTTAATGA AAtACGTACA T T T A T C O a TTGSATTGTC nGACAAHG TCTTGACATC GTCCACGCAT T A t G T G G i a TTTTGAGG7C
1600
CCTGfiCAGU GTGSGAnCG AACCCACGa TCCGAAGA6A CTGSAGCaA AAICCAGCGC CTTAGACCGC TCGGCCACGC TAaTGCACG A A A A C n A a
GGACCGTCGC CACCaAACC n 6 6 t T 6 C t t A S G C H n a GACCTCGSAT TTAMTC6CG GAATCTGGCG A e a S 6 T 6 C 6 ATGGAC6T6C n n G A A T G A
GTCUCCTTG TCCTCnAAG AGACTAfiAAG TAAGGAATGS GCCAMGCIT
CAGGGGCAAC A66AG6ATTC ICTGATnTC A T T C C n A a
Figure 3. The sequence of the tRNA gene-containing region of XHt 4. The
orientation and extent of the tRNA genes is indicated by arrows. The twenty
seven base pair tandem repeat is also shown.
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G CU
GI
c8
SA
G C
CG
"
S
A
CO
S
CAGGC
c
gI
uS
u/
G UA *UGUG G '
CCGAGUUCA
I?
§§
G C
U
A
UuU
G C
U
A
CuG
* "
°. §
/C
G°
LUAG
£. UGGG uu°
«r
U
§c
.
"
u
G
G
1 vc
Figure 4. Thel n sequences of the
tRNAs encoded by XHt 4. (a) the tRNA J ,
(b) the tRNA9 , (c) the tRNA leu . In each case only the CCA terminus (not
encoded by the DMA) has been added; none of the secondary modifications are
indicated.
Between base pairs 1502 and 1585, but on the opposite DNA strand, a
tRNA^ eu gene recognizing the codons CUA and CUG is located. Its unmodified
sequence is shown in Fig. 4c, and it differs in only two positions from
the bovine liver tRNA leu (specific for CUU, C and A) reported by Pirtle et.il(29). This tRNA gene has not yet been tested for transcriptional activity
in Xenopus oocyte nuclei.
Examination of the 1.65 kb human DNA sequence using the tRNA program
of Staden (30) confirmed the observation that only three tRNA genes are
present.
Jji Vitro Transcription
Each of the tRNA genes has been tested in a HeLa cell lysate for the
ability to direct synthesis of a mature sized tRNA product. As shown in
Fig. 5 all three tRNA genes produce precursors which, on prolonged incubation,
are converted to products with electrophoretic mobilities indicative of
mature tRNAs. The products have not been analyzed for secondary modifications
Flanking Sequences
The 3 1 flanking sequence of the tRNA^ s gene contains two T-rich regions,
generally thought to be RNA polymerase III termination signals (31). Two
similar T-rich regions are found close to the 3' end of the tRNA9^ n gene.
The tRNA^ eu gene appears to have a single block of five Ts as a termination
signal. All three tRNA genes have what may be secondary terminators 30 to 75
base pairs to the 3' side, in each case a run of four or five Ts (Fig. 6 ) .
The 5' flanking sequences show only limited homology and little resem-
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7 8
IPMf-
Figure 5. In v i t r o t r a n s c r i p t i o n of three human tRNA genes. The plasmids
pAT 153 (lanes 1 - 3 ) , pHt 4-10 (tRNA9 l n gene, lanes 4 - 6 ) , pHt 4-11 (tRNA l e u gene,
lanes 9 - 1 1 ) , and pHt 4-12 (tRNATys gene, Bam HI t o Eco R I , lanes 12-14) were
incubated w i t h HeLa c e l l extract (NEN) and ct[ 32 P]-GTP f o r 30 min (lanes 1 , 4 , 9 ,
12), 1 hr (lanes 2 , 5 , 1 0 , 1 3 ) , or 2 hr (lanes 3,6,11,14) a t 30°. Each reaction
(25 p i ) was stopped by the a d d i t i o n o f 100 p i of 0.3 M NaOAc containing IS SDS
and 5 yg o f E_. c o l i c a r r i e r tRNA. Phenol-chloroform ( 1 : 1 ) e x t r a c t i o n (2x) was
f o l l o w e d by alcohol p r e c i p i t a t i o n , gel e l e c t r o p h o r e s i s (11.4% acrylamide and
0.6S bisacrylamide) as i n r e f 11 , and radioautography.
Lane 7 i s a marker of
32pCp-end labeled tRNA (tRNAgln) i s o l a t e d by h y b r i d i z a t i o n t o pHt 4-10 immob i l i z e d on n i t r o c e l l u l o s e (33). Lane 8 contains s i m i l a r tRNA i s o l a t e d by hyb r i d i z a t i o n t o immobilized pHt 4 - 1 1 .
-10
80
-I+I
90
1 30
1 i+0
Lys
AAGTAAAGCTCTCGTGAAGAGCC
GCGGCATGTCTTTGCTTTTGG •
Gin
TTGAAAGCCCCCACGAGGGAGGT
CCTTAAAGCTTTTCTTTTAAT•
• -TGATTTTCAcA
Leu
G GACAGTAAGTTTTCGTGCAGGT
82
90
100
CCAGGCTGGAGTTTTTCTGGT
165
AGTAATTTTAC
72
80
• -CTAGTTTTTAA
1?5
135
Figure 6. The f l a n k i n g regions o f the tRNA genes. The non-coding strands o f
the t h r e e tRNA genes are aligned at t h e i r 5 ' ends t o demonstrate the l i m i t e d
homology o f t h e 5' f l a n k i n g sequences ( d o t t e d u n d e r l i n i n g ) . The t e r m i n a t i o n
s i g n a l s are u n d e r l i n e d , and p o t e n t i a l secondary t e r m i n a t i o n s i g n a l s are a l s o
shown (dashed u n d e r l i n i n g ) . The segments c o r r e s p o n d i n g t o t h e mature tRNA
sequences are b r a c k e t e d .
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blance to the 5' flanking sequences of most other tRNA genes from higher
eucaryotes. All three of these human tRNA genes have a block of five
pyrimidines flanked on the 51 side by a purine-rich region. This is illustrated in Fig. 6, primarily to emphasize a tentative relationship to the
regulatory sequence described by DeFranco et^ aj[. (32).
The DNA between the tRNA genes does not appear to contain information
sufficient to code for a protein in any of the reading frames. This is
probably true also of the region to the left of the tRNA^ s gene, but one
reading frame is open from the Bam HI site to a termination codon at position
262. A feature worth noting is a perfect tandem repeat of 27 base pairs
between nucleotides 1243 and 1298, approximately midway between the tRNA9^n
gene and the tRNAl eu gene. The significance, 1f any, of this repeat is
unknown.
DISCUSSION
The tRNA gene region of a human-x recombinant has been sequenced and
shown to contain a cluster of three tRNA genes, each of which is active in
an in vitro transcription system. As in the study reported by Sekiya et a!.
(7) the genes are separated by about .4-.5 kb of non-coding DNA. This human
gene cluster is not tandemly repeated, as is the 3.18 kb Xenopus tRNA gene
cluster studied by Muller and Clarkson (2), since the original human-x
recombinant, xHt 4, has no other tRNA genes in 11 kb of human DNA (8 kb on
one side, and 3 kb on the other side of the sequenced region). Endonuclease
digests of human DNA have not yet been examined to determine if this cluster
is repeated as a dispersed unit in the human genome.
ACKNOWLEDGEMENTS
We thank Ms. Debra Mclntyre for skilled technical assistance,
Dr. D. Finnegan for the 3' end labeling procedure, and Drs. E. Lund,
J. Dahlberg and E. Southern for advice and suggestions in the early stages
of this work. K.L.R. wishes to thank Dr. E. Southern and Prof. P.M.B. Walker
for their hospitality 1n providing the opportunity to work at the Mammalian
Genome Unit. Research conducted at the University of Alberta was supported
by a grant from the Natural Sciences and Engineering Research Council of
Canada.
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