Euglena gracilis Chloroplast TransferRNA Transcription Units
NUCLEOTIDE SEQUENCE ANALYSIS OF A tRNA'l'"'-tRNA""-tRNA""tRNA'l'~'~-tRNA'''1i-tRNA'''~
GENE
CLUSTER*
(Received for publication,June 7, 1982)
Margaret J. Hollingsworth and Richard B. Hallickf
From the Departmentof Chemistry, University of Colorado, Boulder, Colorado80309
A tRNA coding locus in the Barn-Sal 9 region of
Euglena gracilis Pringsheim strain 2 chloroplast DNA
was chosen for detailed study. This DNA contains the
previously mapped tRNA coding sequences of the adjacent Euglena chloroplast EcoRI products of EcoV and
EcoH (Orozco, E. M., Jr., and Hallick, R. B. (1982) J.
Biol. C h e n 257,3258-3264). The 3.2-kilobase pair BarnSal 9 fragment was cloned into the BarnHI and Sari cut
plasmid vector pBR322, resulting in the recombined
plasmid pPG76. The tRNA coding locus was mapped to
a region of Bum-Sal 9 that contains portions of both
EcoH and EcoV. The DNA sequence of 1-kilobase pair
Bum-Sal 9, containing the entire tRNA coding locus,
was determined. A cluster of six tRNA genes wasfound.
The gene organization is as follows, where bp is base
pair: tRNAXA-64 bp spacer-tRNAFk-14 bp spacertRNA&,-4bp spacer-tRNAz$-27 bp spacer-tRNAEbc-6
bp spacer-tRNAg!&. The tRNAF& is believed to be an
elongator tRNA. The first four genes are within EcoV.
The EcoRI cleavage site that separates EcoV and EcoH
is in the tRNAG1"
g me. The tRNA"IYgene is in EcoH.
This is the largest known chloroplast tRNA gene cluster.
a large polycistronic tRNA cluster (1). Barn-Sal 9 contains
EcoV and a part of both EcoG and EcoH. It haspreviously
been shown that both EcoV and the portionof EcoH internal
to Barn-Sal 9 hybridized '"P-labeled Euglena chloroplast
tRNAs (1-3). In this study, a restriction endonuclease cleavage map of Barn-Sal 9 was constructed, the tRNA coding
locus was identified,and the DNA sequence of approximately
1 kb' containing the entire coding locus was determined via
the Maxam-Gilbert method (12). Six tRNA genes were identified. The organization of the clusteras determined from the
DNA sequence data is tRNA:;VA-64 bp spacer-tRN@&-14
bpspacer-tRNAy.\l-4
bp spacer-tRNA::?i-27 bpspacerrrr,c-6
bp spacer-tRNA;&. This is the largest known
tRNA(;1Ll
chloroplast tRNA gene cluster. All of the genes have the same
polarity with respect to each other, to the rRNA transcription
units, and to the previously characterized tRNA'"'-tRNA*""tRNAArggene cluster of EcoG.
EXPERIMENTAL PROCEDURES'
RESULTS AND DISCUSSION
Mapping-The location of the 3.2-kb BamHI-SalI digestion product of Euglena chloroplast DNA designated BarnSal 9 (or "BS9") has been previously reported (1, 2, 13). With
reference to the EcoRI map of this genome (1, 2, 14), BS9
The chloroplast DNAof Euglena gracilis hasbeen shown contains all the EcoV, as well as portions of both EcoG and
to have at least nine loci which hybridize with chloroplast
EcoH. A detailed restriction endonuclease map for this DNA
tRNAs (1-3). At least some of these loci contain multiple
was determined (Fig. 1). The enzymes used wereEcoRI, AuaI,
clustered tRNA genes that may be part of polycistronic tranAuaII, A M , MspI, HhaI, HaeIII, TaqI, Hinfl, and
DdeI.
scriptionunits (1). In Euglena, two tRNA loci have been Maps were deduced mainlyby standard methodsof single and
characterized. One, containing the tRNA"' and tRNAA1", is
double digestion. When ambiguities were possible, specific
found between the 16 and 23 S ribosomal genes (4,5). A larger
fragments were isolated for redigestion (4).In addition, 31 of
cluster in EcoG contains the four genes tRNA""", tRNAArg,
the 42 cleavage sites shown in Fig. 1 were confirmed by DNA
tRNAA"', and tRNA""' (6). Among higher plants, maize chlosequence analysis. There is one possible anomaly in the reroplast DNA hasa locus betweenthe 16 and 23 S rRNA genes
striction endonuclease map. According to the nucleotide secontainingsplit tRNA"' andtRNAA'" genes (7). However,
quence data, thereis an internal Tag1 sitein the 257-bp TayI
both the chloroplast tRNAVa1 and the tRNAH'"
genes of maize
fragment (Fig. IC) that is not cut by TaqI in enzyme digestion
are found as isolatedgenes (8, 9), as are the tRNA'"' and
reactions. The reason for this uncut TaqI site is
unknown. All
tRNAA""of tobacco chloroplast DNA (10, 11).
other sites predicted from the
nucleotide sequence data have
The Barn-Sal 9 fragment of chloroplast DNA was chosen
been confirmed during the mapping study.
for further study because it was a good candidate for having
The nucleotide sequence of the right-hand 367 bp of BS9
* This work was supported by Grant GM28463 from the National
Institutes of Health. This is paper 111 in a series Euglena gracilis
Chloroplast Transfer RNA Transcription Units. Papers I and I1 are
Orozco, E. M., Jr., and Hallick, R. B. (1982) J. Biol. Chem. 257,32583264; 3265-3275. The costs of publication of this article were defrayed
in part by the payment of page charges. This article must therefore
he hereby marked "advertisement" in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact.
Recipient of National Institutes of Health Research Career Development Award KO4 GM00372. To whom correspondence should
be addressed at, Department of Chemistry, University of Colorado,
Campus Box 215, Boulder, Colorado 80309.
*
I The abbreviations used are: kb, kilobase pair; hp, base pair; R, a
purine nucleoside; Y, a pyrimidine nucleoside; DTT, dithiothreitol.
' Portions of this paper (including "Experimental Procedures"and
a portion of "Results and Discussion") are presented in miniprint at
the end of thispaper.Miniprint is easily read with the aid of a
standard magnifying glass. Full size photocopies are available from
the Journal of Biological Chemistry, 9650 Rockvilk Pike, Bethesda,
MD20814.RequestDocument
82 "1489,
cite the authors and
include a check or money order for $1.60 per set of photocopies. Full
size photocopiesare also included in the microfilm edition of the
Journal that is available from Waverly Press.
12795
12796
E. gracilis Chloroplast tRNA Gene Cluster
bp spacer-tRNA&A':1-4 bp spacer-tRNAz;"A -27 bpspacertRNARk-6 bp spacer-tRNARgc.
None of the genes code forthe 3'-CCA termini, and noneof
the genes are interruptedby intervening sequences. The close
proximity and common polarity of these genes is consistent
with the possibility that some or all of the genes might be
Taq I
79 100
47
cl
6k8E51 I 1 1 2 5 7
515
I 355 I
545
12001
initially transcribed as a polycistronic transcription unit. This
and the
seems mostlikely for the tRNAH'"-tRNAM"'-tRNATrl'
Hlnl I
du
I
850
I
tRNA';'U-tRNA"'Y genes, which are separated by intergenic
10
spacers of only 4-14 bp. We arecurrentlyattemptingto
Dde I
el
435 I 4 8 5
1>03
920
I I 2578201
identify the primary transcriptsof these genes.
This is the second tRNA gene cluster identified in Euglena
FIG. 1. Restriction endonuclease map. Map of the Barn-Sal 9
fragment of E. gracilis chloroplast DNA. This fragment containsall chloroplastDNA. The coding location is 1.6 kb from the
of EcoV andsections of EcoH and EcoG. Distances betweencleavage
previously characterized four gene tRNA cluster
of chloroplast
sites aregiven in base pairs.
fragment EcoG. With thesesix genes in BS9, the total number
of different known Euglena chloroplast tRNA genes is now
(Fig. 1) has previously been reported (6). This is a portion of 12, the largest number for any chloroplast DNA. Since the
the data from1.6 kb of Euglena chloroplast DNA containing tRNA"" and tRNAA'"genes are each present three times/
a cluster of four tRNA genes (6).
genome (l),each gene located in each of threetandemly
Location of Transfer RNA Genes withinBS9-In order to repeated rRNA transcription units (l), the total number of
locate the tRNA coding locus (or loci) within BS9, Southern known Euglena chloroplast tRNA genes is now 16.Allof
(15) hybridizations were used. TotalEuglenachloroplast
these genes, exceptthe tRNAL""of EcoG (6), are of a common
tRNAs were purified. They were radioactively labeled at the polarity.
3'-CCA termini with [a-'"P]ATP as previously described (16).
tRNA"' Gene-The tRNAM" gene most likely encodes an
The tRNA probe was hybridized to membrane fiiter blots of
elongator tRNA rather than an initiator tRNA
for several
BS9 restriction endonuclease fragments (1,6, 16). The region
reasons. First,the predicted tRNA"' secondarystructure
of tRNA hybridization was localized to a 600-bp region be(Fig. 4) contains 7 bp in the aminoacid acceptor expected for
tween the two AvaII sites of BS9 (data not shown). This isa an elongator, rather than the 6 bp expected for an initiator
larger coding locus than waspreviouslydescribedfor
this tRNA (23). Second, the base sequence homology of the Euregion for the bacillaris strain of E. gracilis ( 3 ) . In another glena tRNAMe', although similar for both the tRNAM" and
experiment, the synthetic oligonucleotide 5"CTACCAACTtRNA"' initiator of spinach chloroplasts(59 and 62%,respecGAGCT was found to hybridize to both EcoH and the frag- tively), is significantly higher for Escherichia coli tRNA""
ment H i n d I I B (6) which contains all of BS9.3 This synthetic (70%) than for tRNAM"' initiator (56%). Finally, a putative
probe is complementary to the D-stem and loop of Euglena
Euglena chloroplast tRNA"' initiator gene has recently been
chloroplast tRNA''h' (17), tRNAA1"(4, 5), and tRNA""' (6). ~equenced.~ This tRNA
would have a 6-bp aminoacid accepFollowing nucleotide sequence analysis(described below),the tor stem,84%sequence homology with the spinach chloroplast
identical 14-base sequence was identified within the D-Stem tRNAM"' initiator,and
68% homology withtheE.
coli
and loop of a tRNA"'" gene. TheDdeIsequence
of the tRNA"' initiator.
synthetic 14-mer (5"CTGAG) is the DdeI cleavage site betRNASecondaryStructures-Thesecondarystructures
tween the 485- and 201-bp DdeI fragments (Fig. le).
predicted for the primary transcripts of the six tRNA genes
DNA Sequence Analysis of the tRNA Coding Locus in
are shown in Figs. 4 and 5. All six genes can be represented in
BS9-The region within BS9 that hybridized with chloroplast the tRNA cloverleaf structure proposed by Holley et al. (20).
[,"P]tRNAs was sequenced by the method of Maxam and
However, two of the tRNAs would have single mismatched
Gilbert (12). The portion of BS9 analyzed and the sequence
base pairs. The tRNA"'" would be predicted to have a U:W
strategy used is shown in Fig. 2. DNA sequence was deter- U40mismatch internally in the anticodon stem. The tRNAM"
minedfor fragments 5'-end-labeled at the following sites: would be predicted to have an
A x - G ~mismatch
~
at theclosure
EcoRI, AuaII, AuaI, HhaI, TaqI, AluI, HinfI, and DdeI. Forof the anticodonloop (Fig.4). One of the base assignments for
100% of the data obtained, the sequenceof both strands was this A81-G39mismatch, A:i15in the tRNA"' gene, is confirmed
obtained and/or the same fragment
was sequenced more than as part of the HinfI cleavage site GRIIA~IT:~~T:IBC:~~,
which was
once. The continuous sequence
of 889 bp fromBS9, containing located by restriction endonuclease analysis (Figs. 1 and 4).
637 bp of the 1.2-kb EcoV and 252 bp of the 1.1-kb EcoH, is An autoradiogram of a 20% DNA sequencing gel for a DNA
given in Fig. 3.
fragment from the
tRNA"' gene radioactively labeled at this
Transfer RNA Gene Organization-There are six tRNA HinfI cleavage site is shown in Fig. 6. The DNA sequence is
geneswithin the tRNA coding locus of BS9 identified by
5'-TRRC34A3r,TR6A3;ARHGR9C40C41C42
. . . and contains both the
tRNA hybridization. These six genes, uhich all have the same anticodon 5'-CR4A3RT3ti
and base Gas. The GXI assignment is
polarity, are encoded within 557 bp of Euglena chloroplast
apparent.
DNA. The DNA sequence of this tRNA gene cluster, as well
There are other examples of either tRNAs or tRNA genes
as of the proximal and distal flanking DNA, is given in Fig. 3. with mismatches at these sites in the anticodon stem. The
The tRNA genes were identified by comparison of the posi- phage T4 tRNAArgcontains a U:JII-$~,I
mismatch(21);the
tions of invariant and semi-invariant bases and secondary
Saccharomyces cerevisiaetRNAG1" gene, C:IO-T40
a
mismatch
structure tc- the knowngeneral structure of other tRNAs
(22), and the phage T5 tRNAAs", a C:ill-T4~,
mismatch (23).
(described below) (18, 19). Theidentity of the geneswas
There is no precedent for an AX-GRSpair, but the yeast (24,
predictedfrom theanticodonsequenceandthesequence
25) and mammalian (26, 27) elongator tRNA"' and the Neuhomologies to known tRNAs (describedbelow). This gene
organization is as follows: tRNA;'!GA-64
bp spacer-tRNA#&; -14
' G. Karabin and H. B. Hallick, unpublished observations.
The nucleotide positionsare numbered according to the positions
assigned yeast tRNA'"" (23).
' J. Nickoloff and K.B. Hallick, unpublished observations.
E c o V - Eco ti t R N A Gene C l u s t e r
FIG. 2. Sequencingstrategy
for
of BS9.
the tRNA gene cluster region
Fragmentsweresequencedfromthe
sitesindicated in thedirection o f the
urrou'c. tRNA genes are shownhekJl1, in
alignment with the map. They would be
transcribed from right t o left.
20
40
40
40
40
80
100
GGAATTTTC~TTTCTTTGA~GTTTTATTTGTAAGACTTGGAAGTTTTCC~ATTCTACGAACTATTCGCAATCGTGGTCC~CTATATCTTGACATAGTTT~
CCTTAAAAGAAAAGAAACTACAAAATAAACATTCTGAACCTTCAAAAGGATAAGATGCTTGATAAGCGTTAGCACCAGGAGATATAGAACTGTATCAAAA
140
120
tRNATyr+'
8'
200
ATCTATATT~TTTTTGATC~TAAATAACT~TTTCTATGT~AATTGTTTT~GTTGTTTAA~ATCTAATTG~GAGTTGTTGCCCGAGTGGT~AATGGGCGC~
TAGATATAAAAAAAACTAGAATTTATTGAAAAAGATACAATTAACAAAAACAACAAATTGTAGATTAACGCTCAACAACGGGCTCACCAATTACCCCC~C
40
40
12797
E. gracilis Chloroplastt R N A Gene Cluster
220
GATTGTAAATCCGCAGTTCATCTTTCGCTCGTTCGAATCCAGCACGACTC~AAATATTT~TATATTTAA~CA~ACAGTT~TCGA~TTATAATAAAT
CTAACATTTAGGCGTCAAGTAGAAAGCGACCAAGCTTAGGTCGTGCTGA~TTTTATAAAAATATAAATTAGTCTGTCAAAAGCTCAATATTATTTAAAAA
380
3 6 0+
'
3
340
ATTATATTT~TTATAGGTGGGTGTAGCCAAGTGGTAAGGCAAAGGACTGTGACTCCTTCATTCGCGGGTTCGATCCCCGTCATTCACCT~CTATTAATT~
TAATATAAAAAATATCCACCCACATCGGTTCACCATTCCGTTTCCTGACACT~AG~AAGTAA~CGCCCAAGCTAGGGGCA~TAA~T~~AA~ATAATTAAA
420
500 480tRNATrP+.
t RNAMel.
TTAGGCTCAGTAGCTCACAGGATAGAGCAGGGGATTCATAAGCCCTTGGTCACAGGTTCAAATCTTGTCTGAGCCAAACTGCGCTTTTAGTTCAATTGG~
AATCCGAGTCATCGAGTCTCCTATCTCGTCCCCTAAGTATTCGGGAACCAGT~TCCAAGTTTAGAACAGACTCGGTTTGACGC~AAAATCAAGTTAACCA
520
5 8 0 t R N A G 1"+.
600
AGAACGTAGGTCTCCAAAACCTGATGTAGTAGGTTCGAATCCTACAGAG~GCGTTTGTT~TTTTTCTTTATCTTAAATTTGCCCCCATCGTCTAGAGGCC
TCTTGCATCCAGAGGTTTTGGACTACATCATCCAAGCTTAGGATGTCTC~CGCAAACAAAAAAAAGAAATAGAATTTAAACG~~GGTA6CAGATCTCC~G
620
'tRNAG
. 700
680
6
'+
TAGGACATCTCCGTTTCACGGAGGCAACGGGGATTCGAATTCCCCTGGG~GTAATTTATGCGGGTATAGCTCAGTTGGTAGA~CGTGG~~CTTCCA~GTC
ATCCTGTAGAGGGAAAGTGCCTCCGTTGCCCCTAAGCTTAAGGGGACCCCCATTAAATACGCCCATATCGAGTCAACCATCTCGCACCAGGAAGGTTCAG
720
CAATGTTGCCTGTTCGAATCACGTTACCCECTTTTAACT~TTTGATCTT~ATA~AAAGT~AATTTTTAA~TTTTAT~ATATTTTTAATA
GTTACAACGCACAAGCTTAGTGCAATGGGCGAAAATTGAAAAACTAGAAATATCTTTCAATTAAAAATTAAAAATACTATAAAAATTATAT~ATACCCTA
820
860
880
GTGCGATTCGTAAAATTTGGATCTTATTT~TAAAAAAAT~TTTTGCCATGTAAATTAAA~TTTTACTCT~AATTTTCCGATATTTTAGT
CACGCTAAGCATTTTAAACCTAGAATAAAAATTTTTTTAAAAAACGGTACATTTAATTTAAAAAT~AGAATTAAAAG~CTATAAAATCA
FIG. 3 . Sequence of the tRNA gene cluster and flanking UNA of restriction fragment BS9 from Euglena chloroplast DNA. 'I'he
top strandof the DNA is written left to right in the 5' to 3' direction. The tRNA-like sequencesare in italics. Italics on both strands rellresent
restriction sites used for 5'-end labeling in Maxarn-Gilbert (12) sequence analysis.
rospora crassa mitochondrial tRNA""" (28) contain the pair
RI;, YN,,R,,, R.'4, Y,,, Rztj,(G or C).i,l, Y,v, K.j7,(A or C ) , I ~( G
,
~.ll-+~l~+.
The phage T s tRNA""" hasa C.II-+:~!, pair ( 2 3 ) . In or C)I(I, Y,", R;?, R,;, Y,,,,,Y,,,, and R;:l. Some of the six Euglena
addition, the S. cerezisiae (29) and Aspergillus nidulans ( 3 0 ) chloroplast tRNA genes have different bases at invariant and
mitochondrial
genes
contain
the
pair
Tjl-T,l!,, and
a
semiconserved positions. The tRNA""' gene codes for C I ~ ~ - G ~ ~ ,
rat mitochondrial tRNA'"" gene (31) has the bases A:lI-Cl!,.
in the D-stem rather than Glll-Y2:. This reciprocal base pair
Invariant and Semiconseroed Bases-Based on an analysis change also occurs in E. coli tRNA"'" ( 3 2 ) .The tRNA';'" gene
of procaryotic, eucaryotic, and phage tRNAsinvolved in pro- codes for A~.~-Ulil
base pair rather than the invariant G5:I-C,jI.
tein synthesis, Singhal and Fallis (19) have described both
These base assignments for tRNA"'" are confirmed by restricinvariant and semiconserved basesin tHNAs that occur with tion endonuclease analysis. The A:,I and tJ,;l are within HinfI
greater than 90% probability. The 16 invariant bases are Ux, and EcolEI cleavage sites, respectively. This is a very rare
base change among known tKNAs and tRNA genes. Only the
Glo, AI,, GI,, GI!,, ALI,U{.I,G ~ IT:,,,
, $x,,
Cx;, Am, CSI,' 2 7 8 , Cyr,,
and A;+,.The 20 semiconserved bases are (G or C)?, Rc,,
Yll, tRNA:ip and tHNAM"' genes of A. nidulans mitochondria are
E.Chloroplast
gracilis
12798
tRNA Gene Cluster
A
G *C
G *C
C .G
A
G-C
A.U
G-C
U.A
u .A
U.G
G- C
C *G
A*U
GATCL
-c
"
G*U
tRNATYr
U.A
"ismatch
-A
-A
c
G*C
G*C
A*U
c
"c
U
A
GUG
tRNAn"
FIG.4. Nucleotide sequence of tRNATyr,
tRNA"'",
-T
and
tRNA"'.
The primary structures are deduced from the DNA sequence and shown in the normal tRNA cloverleaf secondary structures.
.@
u
G
C *G
G*C
C -0
U* A
.G
G.C
C *G
G*C
G*C
G*C
u
G*C
G*C
U*A
C
A
AA'C
FIG.6. Autoradiogram of a 20% sequencing gel of a portion
of the tRNAM"'.The DNA was 5"end labeled at the Hinocleavage
site in the anticodon stem. The data arefor the tRNA-like strand of
the gene. The G-base-labeled Mismatch is G:w,which is opposite A:I~
of the HinfI cleavage site in the normal tRNA cloverleaf secondary
structure.
G.C
U G
G.6
C'0
C-G
C*G
C'G
C
A
U
A
UC C
TABLE
I
Sequence homologies h e r cent) between tRNAs specific for the
same amino acid
Most sequence data are from Ref. 23. Only tRNAs with the same
anticodon are compared.
S. cereui- A. nidu- Mammal
E. grac"'
chloroplast
chondria
Plant ~ " 0 roplast
E. coli
siae
His 47
Met
74"
5gh
Tw
88h
Glu
FIG.5. Nucleotide sequence of tRNATm, tRNA'"", and
tRNA"IY. The primary structures are deduced from the DNA sequence and shown in the normal tRNA cloverleaf secondary structures.
Glv UCC
" Chloroplast from maize.
70
70
66
78
62
Inns
mito- cytoplasm
chondria
42
58
64
TYr
tRNAG'"
mito-
55
47
53
42
49
58
56
62
51
Chloroplast from spinach.
parisons between Euglena and plant chloroplast tRNAs (Tasimilar (30). In addition, the tRNAH"and tRNAM"'genes code ble I and Ref. 6), the average primary sequence homology is
76%,with a range of 49-93%. Second, the Euglena chloroplast
for GI6and Ale, respectively, rather thanY16;and the tRNA"'"
tRNAs
are next most similar to procaryotic tRNAs. The six
gene codes for &*,rather than theY48which is normally part
of the correlated semiconserved pair R15-Y4". Finally, the tRNAs of BS9 have an average sequence homology of 68%
tRNAHi",tRNAM"', and tRNA';'" genes code for U or C ~ R , with the same species from E. coli. The average for 12 Euglena chloroplast tRNAs (Table I and Ref. 6) is 70%, with a
rather than Ria.
range of 62-78%. Third, the homology of chloroplast tRNAs
Sequence Homology with tRNAs from Other OrganismsThe sequence homology of the six tRNAs from the BS9 to those of the mitochondria and cytoplasm of eucaryotes is
fragment of E. gracilis chloroplast DNA to thecorresponding muchlower thanthe homology to procaryotic andother
tRNA species from higher plant chloroplasts, E. coli, mito- chloroplast tRNAs. Additional analysis of the tRNA cluster
chondria, and mammalian cytoplasm is shown in Table I. The DNA sequence data is presented in the Miniprint Supplement
same comparison was previously made for seven additional to this paper.
Euglena chloroplast tRNAs (6).Some generalizations may be
would like to thank DE. Patrick W. Gray
made about the relatedness of Euglena chloroplast tRNAs to andAcknowledgments-We
Emil M. Orozco, Jr. for the initial cloning and characterization,
those encoded in other genomes. First, in most cases, Euglena respectively, of pPG76. We also thank Tim Heuser for expert advice
chloroplast tRNAs are most closely related to thesame tRNA in computer programming and Alice Sirimarco for preparation of the
species from plant chloroplasts. For the eight possible com- manuscript.
12799
E. gracilis Chloroplast tRNA Gene Cluster
REFERENCES
1. Orozco, E. M., Jr., and Hallick, R. B. (1982) J . Biol. Chem. 257,
3258-3264
2. Hallick, R. B., Rushlow, K. E., Orozco, E. M., Jr., Stiegler, G. L.,
and Gray, P. W. (1979) ICN-UCLA Symp.Mol. Cell. Biol. 15,
127-141
E. T., Helling, R. B.,
3. El-Gewely,M. R.,Lomax,M.L.,Lau,
Farmerie, W., and Barnett, W. E. (1981) Mol. Gen. Genet. 181,
296-305
4. Orozco, E. M., Jr., Rushlow, K.E., Dodd, J. R., and Hallick, R. B.
(1980) J . Biol. Chem. 255, 10997-11003
E. (1980) Nature (Lond.) 286,
5. Graf, L., Kossel, H., and Stutz,
908-910
6. Orozco, E. M., Jr., and Hallick, R. B. (1982) J . B i d . Chem. 257,
3265-3275
7. Koch, W., Edwards, K., and K6sse1, H. (1981) Cell 25, 203-213
8. Schwarz, Z., Kossel, H., Schwarz,E., and Bogorad,L. (1981)Proc.
Natl. Acad. Sei.U. S. A . 78, 4748-4752
Jolly, S. O., Steinmetz, A. A., and Bogarad, L. (1981)
9. Schwarz, Z.,
Proc. Natl. Acad. Sci. U. S. A . 78,3423-3427
10. Todoh, N., Shinozaki, K., and Sugiura, M. (1981) Nucleic Acids
Res. 9, 5399-5406
M. (1981)
11. Kato, A., Shimada, H., Kusada,M.,andSugiura,
Nucleic Acids Res.9, 5601-5607
12. Maxam, A. M.,andGilbert, W. (1980) Methods Enzymol. 65,
499-560
13. Gray, P. W., and Hallick, R. B.(1977) Biochemistry 16, 1665-1671
14. Hallick, R. R. (1982) in The Biology of Euglena (D. Buetow, ed.)
Vol. IV, Academic Press, New York
15. Southern, E. M. (1975) J . Mol. Biol. 98, 503-517
16. Orozco, E. M., Jr., Gray, P. W., and Hallick, R. B. (1980) J. Biol.
Chem. 255, 10991-10996
17. Chang, S. H., Brun, C. K., Silberklang, M., RajBhandary, U. L.,
Hecker, L. I., and Barnett, W. E. (1976) Cell 9, 717-723
L. (1976) Annu. Reu. Biochem.
18. Rich, A,, and RajBhandary. U.
45,805-860
19. Singhal, R. P., and Fallis, P. A. M. (1979) Prog. Nucleic Acids
SWPLElIENTARY MATERIAL TO
Euglena yracllin Chloroplast Transfer RNA Transcription Unite.
line phosphatase, 2.5 units Of T-4 polynucleotide klnase, 5 0 W Trip (pH 1 . 6 1 ,
10 mM EqCl , 5 W DIT, 0.1 W spernldine, and 1 mM EDTA. The reaction was
allowed to proceed at 37-C f o r 1 5 min. Another 2.5 units of enryrne yere
added and the reactlon was continued at 37-c for 15 mi".
The mixture was
then heated t o 68.C t o inactivate the kinase. Alternately, 75 picomoles of
Nvcleotide Sequence Analysis Ofa tRNAmr-tRNhmi*-tFXAettRNATIP-tRNAGIU-tRNAG1y Gene Cluster
M. J. Hollingnworth and Richard 8 . Hallick
EXPERIMENTAL P R O C E D U W S
Materlaln: II-'~PIATP ~ 3 0 0 0 C i / m o l e ) was Obtained from New England
NYCleaZ and hmersham. T-4 polynucleotide kinase was p u r c h a ~ e d f r?TEN
m
and
New England Biolabs. Restriction endonucleases were also purchased from New
England Biolabs. Lar melting point agarose, a g ~ o x e ,and E.
alkaline
a
Phosphatase were purchased from Sigma. M r y l a m i d e and bis-aorylanide were
obtained from Fisher, Kodak andPolysciences. Dimethyl *+fate was a l w from
Xodak. Hydrazine and piperdlne were from Baker. P01ypropylcr.c collloy~s with
fiberglass dxskswere purchased from Isolab, Inc.
Plasmld DNA Construction, Isolation and'Lapping: The plasmid pPG76 was
Constructed by dlgeoting Evqlena chloroplast DNA and pBR322 ( 3 3 ) with Bpm 8-I
and
I. The products were ligated wlth T-4 DNA ligase and transt0-d
Into
E_.
HBlOl (34).
AmpicllllnTeSIStant,tetracyclinesensitive
clones
were selected by replica plating. Sizes of the insertse r e detemined by
restriction endonuclease digestion and gelelectrophoresis.
plasmid DNA yds
isolated by the cleared lydate method ( 3 5 1 and purified by ethidium b r o d d e cesium chlorlde density centrlfugation ( 3 6 1 . Restriction endonuclease digestiona and electrophoretic techniques were perforned ais previously described
123,241. Salt concentrations used for the endonucleases were those rec-nded by New England Biolabs.
Purification of MIA Fragments:
Res. Mol. Biol. 23, 227-290
J . T.,Marquisee,
20. Holley, R.W., Apgar, J., Everett, G. A., Madison,
M., Merrill, S. H., Penswick, J . F., and Zamir,A. (1965) Science
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21. Mazzara, G. P., Seidman, J. G.,McClain, W. H.,Yesian,H.,
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22. Nobrega, F. G., and Tzagoloff,A. (1980) FEBS Lett. 113, 52-54
23. Sprinzl, M., and Gauss, D. H. (1982) Nucleic Acids Res. 10, rlr55.
24. Gruhl, H., and Feldmann, H.(1976) Eur. J . Biochem. 68,209-217
(1976) J. Biochem.(Tokyo) 80,
25. Koiwai, 0.. andMiyazaki,M.
951-959
26. Piper, P. W. (1975) 6 u r . J . Biochem. 51, 283-293
27. Petrissant, G., and Boisnard,M. (1974) Biochimie 56, 787-789
Yin, S., and
28. Heckman, J . E., Sarnoff, J., Alzner-de Weerd, B.,
RajBhandary, U. L. (1980) Proc. Natl. Acad. Sci. U. S. A . 77,
3159-3163
29. Berlani, R. E.,Pentella, C., Macino, G., and Tzagoloff, A. (1980)
J. Bacteriol. 141, 1086-1097
30. Kochel, H., Lazarus,C. M., Basak,N., and Kuntzel, H.(1981) Cell
23,625-633
31. Saccone, G., Cantatore, P., Gadateta, G., Gallerani, R., Lanave,
C., Pepe, G. and Kroon, A. M. (1981) Nucleic Acids Res. 9,
4139-4148
32. Goodman, H. M., Abelson, J., Landy, A., Brenner, S., and Smith,
J. D. (1968) Nature (Lond.)217, 1019-1024
R. L., Greene, P. d., Betlach, M. C.,
33. Boliver,F.,Rodriguez,
Heyneker, H. L., and Boyer, H. W. (1977) Gene (Amst.)2,95113
34. Rodriguez, R. L., Bolivar, R., Goodman, H. M., Boyer, H.
W., and
Betlach, M. C. (1976) ICN-UCLA Symp. Mol. Cell. Biol. 471477
35. Guerry, P., LeBlanc, D. J., and Falkow, S. (1973) J . Bacteriol.
116, 1064-1066
36. Cohen, S. N., and Miller, C. A. (1970) J . Mol. Biol. 50, 671-687
37. Wienand, U., Schwarz, Z., and Feix, G. (1979) FEBS Lett. 98,
319-323
[T-'~PIATP and 20 units Of klnase in the same reaction mixture e r e incubated
at 37.C for 90 mi".
was incubated with 0.01
Phosphatase ReaCtlM: DNA (20-200 pm 5"ends)
m l t s phosphatase in 10 mM Tris (pH 81 for 45 min at 68'C.
The phosphatase
was inactivated by phenol extraction.
cipitatlon.
The DNA was collected by ethanol pre-
Sequence M a l y s i s : Sequencing wae carried out v ~ a
the method o f w x a m
and Gilbert (12).
SUPPLEMENTARY RESULTS AND DISCUSSION
Conparison of Euglena gracilis strain
2 and strain baclllar~s tRNA
coding
et al. (3) recently reported a X I/&
I map Of the Ec0 x
fragment Of chloroplastDNA o f the bacillaris Strain of Euglena gracilis.
X Of the bacillaris strain is the same sire as E V of the 2 strain.
It
also has the 8chloroplast DNA maplocation and Internal
I 1 cleavage
slte as B
Z
v.
x maps within a
n 1 - x I fraqment e q u ~ v a l e n r to B S ~
( 3 ) and hybridizes wlth chloroplast tRNAs. It is likely that
v of the
2 straln and
X of the baclllarls strain are the same DNA.
It has been
proposed that a l l of the signiflcant differencesbetween the chloroplast
DNAs Of Euglena yracllis L and bacillaris occur in the rRNA Codlngreglon
(14). SurDrisingly, the rePtriCtlOn nuclease nap and the tRNA codlng region
loci: El-Gewely
e
+
e
e
e
Three methods were used to purify individ-
by El-Gewely et al. 1 3 ) for
X do not
agree wlth
OUT
data on
e
X
Initially, fragments were purified from low melting p i n t
agarose gels. The gel slice containing the fragment was melted in buffer.
The TeSYltlng Solut10nwas extracted once with phenol and twlce with ether.
The DNA Yae collected from the aqueous phaseby ethanol precipitation. DNA
fragments were also purified by an adaptation Of the'Laxam-Gilbert crush and
v.
soak Procedure 112). Instead Of fllterinq the acrylamide solution throuqh
Was Pelleted by centrifugatron. and the llquld was
graV1t-Y frltered through POlypropylene ColVlonP (ISolabl plugged wrrh glass
fiber disks. DNA fragments were also eluted from gels electrophoretically
ization was described for
H Of baclllarla chloroplastDNA ( 3 1 , w h x h IS
probably Identical to
H Of Z strain chloroplast DNA. In our study, we
have mapped the X I alten without amhlgulty,and confirmed the location Of
tRNA genes and most cleavage srtes by DNA sequence analysis. Elther the
Z strarn and bac~lldrls chlorOplaSt DNAs arew r e different than PrevlDuSlY
believed. or the analy818 Of tRNA coding loci for the region
O f bacillaris
DNA corresponding t o BS9 needs to be re-evaluated.
ual DNA fragments.
glass W O l , the acryl-lde
1311.
m:The
mOlel
2-8
reactlon mlxture COnSISted Of 20 yM [ y - ' 2 p ] ~ ~ p
(3000
mole DNA that had been p r e v l ~ u s l ytreated wlth bacterial =lka-
we obtalned a different arrangement Of
number of cleavage sites and fragment
sizes
I fragments. although the
were the same. Another differ-
X tRNA coding locus was mapped to a single 120
nucleotide
I fraqment, which is only large enough for one complete tRNA
gene, as opposed to the 551 base palr reglo" of Ec0 V. Also. no tRNA hybrid-
ence 1s that the
e
e