\./ 1993 Oxford University Press
Nucleic Acids Research, 1993, Vol. 21, No. 17 4039-4045
Non-universal decoding of the leucine
several Candida species
codon
CUG
in
Takeshi Ohama+, Tsutomu Suzuki1, Miki Mori, Syozo Osawa§, Takuya Ueda2,*,
Kimitsuna Watanabe2 and Takashi Nakase3
Department of Biology, School of Science, Nagoya University, Chikusa-ku, Nagoya 464-01,
1Department of Biological Sciences, Faculty of Bioscience and Biotechnology, Tokyo Institute of
Technology, Nagatsuta 4259, Midori-ku, Yokohama 227, 2Department of Industrial Chemistry, Faculty
of Engineering, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113 and 3Japan Collection of
Microorganisms, The Institute of Physical and Chemical Research, Wako, Saitama 351-01, Japan
DDBJ accession nosi
Received May 28, 1993; Revised and Accepted July 20, 1993
ABSTRACT
It has been reported that CUG, a universal leucine
codon, is read as serine in an asporogenic yeast,
Candida cylindracea. The distribution of this nonuniversal genetic code in various yeast species was
studied using an in vitro translation assay system with
a synthetic messenger RNA containing CUG codons inframe. It was found that CUG is used as a serine codon
in six out of the fourteen species examined, while it is
used for leucine in the remaining eight. The tRNA
species responsible for the translation of codon CUG
as serine was detected in all the six species in which
CUG is translated as serine. The grouping according
to the CUG codon assignments In these yeast species
shows a good correlation with physiological
classification by the chain lengths of the isoprenoid
moiety of ubiquinone and the cell-wall sugar contained
in the yeasts. The six Candida species examined in
which CUG is used as serine belong to one distinct
group in Hemiascomycetes.
INTRODUCTION
Since 1979, when a non-universal genetic code was first found
in human mitochondria (1), genetic code variations have been
reported not only for mitochondria but also for nuclear genes
(2). The non-universal codons in the nuclear genes had all been
related to termnination codons. CUG was believed to be a universal
leucine codon in Ascomycetes, such as filamentous fungi
(Euascomycetes) and yeasts (Hemiascomycetes), as in other
organisms. In 1989, however, Kawaguchi et al. (3) found that
CUG codes for serine instead of leucine in an asporogenic yeast,
Candida cylindracea (4). Amongst the nuclear genetic codes, this
is a unique instance in which the assignment of an amino acid
codon deviates from the universal genetic code. The present study
was undertaken to determine the distribution of the non-universal
serine codon CUG in various yeasts.
Translation of a synthetic mRNA having CUG codons in-frame
in cell-free extracts (the S30 fractions) from fourteen yeast species
indicates that in six species, including C.cylindracea, codon CUG
is translated as serine, while in the other eight species CUG is
read as leucine. Serine tRNA with the anticodon sequence CAG,
which is complementary to codon CUG, was found in all the
six species in which CUG is used as a serine codon. From
physiological classification by ubiquinone and cell-wall sugar in
these yeasts, it was deduced that these six Candida species belong
to a distinct group in Hemiascomycetes.
MATERIALS AND METHODS
Yeast species
Yeast strains used were obtained from the Japan Collection of
Microorganisms (JCM, RIKEN, Hirosawa, Wako, Saitama
351-01, Japan) and from the American Type Culture Collection
(ATCC) (Table 1). Candida albicans (strain C9) was from
Drs M.Homma and K.Tanaka of Nagoya University, School of
Medicine.
Cell culture
Each species was cultured aerobically in a rotatory air-shaker
(180-200 rpm) at 25°C, in a medium containing, per liter, 7 g
yeast extract (Difco), 3 g malt extract (Difco), 20 g peptone
(Difco) and 20 g dextrose. Cells were harvested between the early
log phase and mid-log phase.
To whom correspondence should be addressed
Present addresses: +Laboratory of Experimental Carcinogenesis, National Cancer Institute,
USA and §Biohistory Research Hall, 35 Murasaki-cho, Takatsu-shi, Osaka 569, Japan
1D14890, D12941, D14891, D14940
*
Building 37, National Institute of Health, Bethesda, MD 20892,
4040 Nucleic Acids Research, 1993, Vol. 21, No. 17
Preparation of cell-free extract (S30 fraction)
The preparation of the S30 fraction was essentially the same as
that for Saccharomyces cerevisiae (5-8), with the modifications
described below. Cells ( 2 g) were suspended in 5 ml of
extraction buffer (8.5% mannitol, 30 mM Hepes-KOH(pH7.4),
3 mM Mg(OAc)2, 100 mM KOAc, 2 mM dithiothreitol (DTT),
0.5 mM phenylmethanesulfonyl fluoride). The suspension was
transferred to a 125 ml screw-cap glass bottle with 8 g of acidwashed glass beads (0.5 mm-diameter), and shaken vigorously
up and down by hand in a vertical motion for 10- 15 minutes
with occasional cooling on ice for 20 seconds. The extract was
pooled, and the glass beads were washed with 3 ml of the
extraction buffer. The combined extract ( 8 ml) was centrifuiged
for 25 min. at 21,000 rpm (Beckman Type 40 rotor). Two-thirds
[r.
I
20
10
1
30
40
50
A TACTC &Q&TA&Z&
MQQGA=
&G&&Z
60
L
Met Met
s0
70
=i CTG
Lou Lou
CTG
CTG
CTG
CTG
TTC
90
TTC
TTC
TTC
100
TTC
TTC
TTC
TAK
Leu Leu Lou Lou Phe Phe Phe Phe Phe Phe Phe
***
AAGCTTGA
/Ser/Ser/Ser/Ser/Ser/Ser
Figure 1. Construction of the template DNA for a synthetic mRNA containing
CUG codons. Two single-stranded DNA fragments, strand-l (
;1 -64) and
;I11 -52), which have 13 complementary bases, were synthesized.
straid-2 (
The lacldng complementary strands were then synthesized with DNA polymerase
and the double- stranded DNA fragment was inserted between the Kpn I and
Hind HI sites of plasmid vector Bluescript KS (Strategene) downstream from the
T3 promoter. The resulting plasmid was linearized with Hind m and used as
a template for transcription.
of the supematant from the top was passed through a Sephadex
G-25 (medium) column (22 x 1.5 cm) in the absence of mannitol
to remove low-molecular weight components. After measurement
of A260, the main fraction ( 4 ml) was concentrated by
Centricon C-10 (10,000 MW cut-off, Amicon) so as to obtain
the S30 fraction of 5-10 Ag of protein per one
The S30
fraction so obtained was divided into 200 ,l aliquots, rapidly
frozen with liquid nitrogen, and stored at -70°C until use. The
S30 fraction was active for at least a month.
jd.
Preparation of T3 RNA polymerase-promoted transcript
DNA was synthesized, with the construction of the 5'-upstream
region of the initiation codon having a sequence similar to that
of native mRNAs of S. cerevisiae (9), so that the derived mRNA
could be translated efficiently in vitro. Two single-stranded DNA
fragments, strand-i (64 nucleotides) and strand-2 (60
nucleotides), having 13 complementary bases at the 3'-ends
(Fig. 1), were synthesized by a Gene-Assembler Plus DNA
Synthesizer (Pharmacia). Restriction enzyme sites for Kpn I and
for Hind IH were included at each 5'-end of the single-stranded
fragments for cloning.
The plasmid DNA containing the correct insert was linealized
by digestion with the restriction enzyme Hind HI. mRNA having
a capping structure at the 5'-end was synthesized under the
conditions recommended in the laboratory manual 'Molecular
Cloning'(10).
In vitro translation assay using synthetic mRNA
Cell-free translation was carried out in 50 yd of reaction mixture,
containing 25 mM Hepes-KOH(pH7.4), 150 mM KOAc, 2.5
mM Mg(OAc)2, 0.5 mM ATP, 0.1 mM GTP, 24g creatine
Table 1. Some properdes of Ascomycetes
Galactose In
G+C %4
Ubiquinone
type4
Swcharomyc. ctrevlsae (JCM 272S5)
40
0e
Candida utlIl
Plehia membrana.acMns
Candid. rugop.llculo.a (JCM 1593)
Candids divers (JCM 1848)
Candid. parapalloala (JCM 1785)
45
30
07
07
07
34;36
07
41
09
serine
Candida zeylanold.s (JCM 1627)
Candida albicons (C9)
Candid. cyllndrac.. (Arcc314830)
56
09
serinea
36
09
serine'
serinea b
50
09
serine
56
09
44
60
09
+
OS
+
Species
(strain
number')
Can0di rugos. (JCM 1619)
Candid. mellbloalca (JCM 1613)
Zygoacus hellankus (JCM 1698)
Candid. magnolle. (JCM 1446)
Candid. aryma (JCM 1691)
Yraowla Ilpolytka (JCM 2304)
Schlzossccharomyces pomb.
Genome
43
leucine
-
leucine
serlne
leucine'
leucine'
leucines
54
SO
42
Amino acid assignment
of codon CUG'
leucInea'b
63
09
010
AspergNu nidulana
Neuroapora cra
Cryptococcus humicolus (JCM 1457)
Trichosporon cutaneum (JCM 1533)
cell wall
+
leucine'
+
eucined
63
63
010
leucinee
'Used in this study.
2JCM: Japan Collection of Microorganisms, RIKEN, Wako, Saltama 351-01, Japan.
3ATCC: American Type Culture Collection.
4From Bamett et al. (17); Nakase, T. (unpublished) for asterisk.
5From Barnett et al. (17); Gorin and Spencer (18); Nakase, T. (unpublished) for asterisk.
6a: this study; b: from Kawaguchi et al. (3) and Yokogawa et al. (4); c: from Kalb et al. (30); D: from Germann
et al. (31).
Nucleic Acids Research, 1993, Vol. 21, No. 17 4041
Determination of tRNA sequences and charging assay
Low molecular weight RNAs were prepared by phenol extraction
from cells of Candida parapsilosis, C.zeylanoides, C.albicans,
C. rugosa and C.melbiosica (11) and the class II tRNA fraction
containing seine and leucine isoacceptor tRNAs was separated
from the class I tRNAs by electrophoresis with polyacrylamide
gel containing 7 M urea. The individual serine isoacceptors were
purified by RPC-5 column chromatography and gel
electrophoresis.
Nucleotide sequences of serine tRNAs were determined by the
methods of Donis-Keller (12) and Kuchino (13). The charging
activities were examined using seryl-tRNA and leucyl-tRNA
synthetases partially purified from the S100 fraction from
S. cerevisiae. Aminoacylation was at 37°C for 10 min. in 20 ,gl
of reaction mixture consisting of 100 mM Tris-HCI (pH7.5),
10 mM KCI, 5 mM MgCl2, 2 mM ATP, [3H]-labelled amino
acids as defined below, and appropriate amounts of tRNA and
the aminoacyl-tRNA synthetase fraction. The concentrations of
amino acid were 444 nM for [3H]serine (832.5 TBq/mmol) and
65.2 nM for [3H]leucine (5661 TBq/mmol), respectively.
phosphokinase (Sigma), 25 mM creatine phosphate (Sigma), 2
mM glucose-6-phosphate (Sigma), 2 mM DTT, 1.0 mM CaCl2,
and the S30 fraction (125yg protein).
The endogenous mRNA in the S30 fraction was removed in
two step; first by precipitation at 200C for 10 min.to perform
run-off translation of polysome, and then by digestion with
micrococcal nuclease (0.4 u/jul) (Pharmacia) for 20 min. at 20°C,
followed by the addition of EGTA (1.6 mM) to stop the nuclease
digestion.
Ten uM each of non-labelled methionine, non-labelled leucine
(or [3H]leucine, 1285.2 TBq/mmol) and non-labelled serine (or
[3H]serine, 1398.6 TBq/mmol), and 100 ytg of synthesized
mRNA were added and incubated at 20°C for 2 hr.
Twelve 1d aliquots were taken out, and after treatment with
1 N NaOH and 10% (w/v) trichloroacetic acid (TCA), the TCAinsoluble radioactivity was measured by a liquid scintillation
counter.
a
b
Saccharomyces cerevisiae
xlO 3cpm
Candida cylindracea
x10O3cpm
2c 6
0
Lo 4
'2
4
RESULTS
In vitro translation assay for the amino acid assignment of
codon CUG
/
2
Io2
.E
To determine whether codon CUG is translated as serine or
leucine in various yeast species, an in vitro translation system
was constructed by the S30 fractions from these species and a
synthetic mRNA having CUG codons in-frame which had been
transcribed from a synthetic DNA by T3 RNA polymerase
(Fig. 1). To test the reliability of the in vitro translation assay,
the S30 fraction from Saccharomyces cerevisiae (CUG =leucine)
and that from C.cylindracea (CUG = serine) were incubated with
the synthetic mRNA with [3H]leucine or [3H]serine. With the
Leu
2
1
TIME
3 hr
TIME
Figure 2. In vitro translation with the S30 fractions from S.cerevisiae and
CGcylindracea of mRNA synthesized by the transcription of template DNA shown
in Fig. 1. The transciption was carried out in the presence of [3H] serine (0)
or [3H] leucine (0).
Candida albicans
Candida parapsilosis
Candida zeylanoides
.3
Xla3cpm
xlO cpm
° 7.5
0
8c 05.01
°6
c
C0
.S
2.5
o'o3
E
1
2
TIME
3 hr
2
1
TIME
3 hr
TIME
Candida melibioscis
Candida rugosa
4
00
-
0
0
2
Leu.
_
E
1
2
TIME
I
3 hr
1
2
3 hr
TIME
Figure 3. In vitro translation of synthetic mRNA with the S30 fractions from five Candida species.
For
legends,
see
Fig.
2.
4042 Nucleic Acids Research, 1993, Vol. 21, No. 17
Candida diversa
Yarrowla lypolitica
Candida magnoliae
Candida rugopelliculosa
x04cpm
oc4
u
c
2
E
Candida azyma
TIME
Zygoascus hellenicus
Trichosporon cutaneum
xl0
6l
x14epm
xlcpm
4-6
1
2
6
~~~~~~~~~~~~~~~4
4
3 hr
1
TIME
2
TIME
1
3 hr
c
C
C
G
O-C
0-C
A-U
U a
A-U
G-C
A-U
U*G
A-U
A-U
C-O
C-G
C-0
O-C
mWAa
uA-U COUCC
A
OC
AGG
U
G-C
A
0I0I1I1 I1
DGAA
A
A
C
C
O-C
D
species. For legends, see Fig. 2.
0
C
act
3 hr
A
A
A
2
TIME
the S30 fractions from seven Candida
Figure 4. In vitro translation of synthetic mRNA with
D
ncpm
L
12 6
4
TIME
TIME
TIME
DG
O
A
uA- G Goc
A G
CU
U
A-AU
A-U
O-C
D GAG
C
a a
O-C
A-p
u
Gm
actCGG G
G
oG O C
A
DD
DDAA
41c
no
GUcc
*AUUC
A-U
G
0-C
c
00a
C
A-IF
UU
IV
U
A U
IF
U
m'G
m'0
C
TTC
iC
G-C
A-U'
AU
U
IIYII
OCAOG
0-C
U
C
o
G- itc
0C
C
Cc
GCCA-'
A-U
G0A0
CC
a-c
at
ml3a
G
O-C
CAO
A O
Candida parapsilosis
Candida
C
A
Candida albicans
O-
zeylanoldes
A
A
A
C
C
C_
c
C
C
G
U
°-C
O-C
0-C
G-C
A-U
c-O
A-U
U G
U -G
A-U
A-U
C-0
C-0
U-A
A-U
G-C
AD
o
C
O-C
u-UUGUCCCM'A
A0
UG
Co
0-c
o-
UC
A
U-A
0-C
GM
AAt
mCG
Cr
0-C
0-C
A-IF
C
A-IF
U
UOUCC
GC A' G° G°
U
G
m'A
TC
O3I-A GUucC
D A
0-C
U-A °C
C
00a
A
*et C G
oG
D
A-U
U
00G
C
UU
0-C
A
U
C
IF
m'O
C A O
U
G
A
CA0a
Candida cylindracea
Candida rugosa
Candida meliblosica
Figure 5. Nucleotide sequences of serine tRNA with anticodon CAG, fiom six Candida species: C.parapsilosis, C.zeylanoides, C.albicans, C.cylindracea (4), C rugosa
and C.melibiosica.
S30 fraction from S. cerevisiae a time-dependent incorporation
of [3H]leucine occurred, while almost no incorporation of
[3H]serine was observed (Fig. 2a). With the S30 fraction from
C.cylindracea, the situation was reversed, i.e., [3H]serine was
incorporated, and [3H]leucine was not (Fig. 2b). These profiles
were observed with magnesium concentrations between 2.5 mM
and 4.0 mM, the maximum incorporation being with 2.5 mM.
When no mRNA was added to the assay system, no appreciable
incorporation of [3H]leucine or [3H]serine was detected, so that
contribution of endogenous mRNA in the S30 fraction
could be ruled out. Thus, with this present in vitro system it is
possible to determine whether codon CUG is for leucine or serine
in various yeast species.
A predominant incorporation of [3H] serine was observed
with the S30 fractions from Candida parapsilosis, Candida
zeylanoides, Candida albicans, Candida rugosa and Candida
a possible
Nucleic Acids Research, 1993, Vol. 21, No. 17 4043
(T)
G-C
A-T
C-a
A-T
C-G
C-G
G-C
tf>
7
TG
Aa
Tt QCTC
TA T
GGG
T-C
T
AC
CT
cG-C
A-T Ac
G-C
a
G
C
aG
A-T
A-T
T-A
C
C
T T
C. mellbloslcs
C. cyllndraces
C. rugoss
G
TA-GT
C-G
G-C
T
T A
a
CCG
CGTCC
GGC
AGA
G
T
C
C
TC
T
A-T
C.
C
GCAGGT
T
G
aG
Aa
zeylanoldes
C. albicans
C. perapelloIs
Figure 6. Nucleotide sequences of tRNAserCAG genes from six Candida species: C.parapsilosis, C.zeylanoides, C.albicans(14, 15), C.cylindracea (4), C.rugosa
and C.melibiosica. The genes were amplified by PCR using primers indicated by solid lines and sequenced. Splicing sites are shown by arrows.
melibiosica, in addition to C. cylindracea (Fig. 3), whereas a
predominant [3H] leucine incorporation was evident for
Zygoascus hellenics, Candida magnoliae, Candida azyma,
Yarrowia lipolytica, Candida diversa, Candida rugopelliculosa
and Trichosporon cutaneum (Basidiomycetes) in addition to
S. cerevisiae(Fig. 4).
Serine tRNA with anticodon sequence CAG complementary
to codon CUG
Serine isoacceptor tRNAs were isolated from C.parapsilosis,
C.zeylanoides, C.albicans, C.rugosa and C.melbiosica and
sequenced. In each Candida species, tRNA having the anticodon
sequence of CAG, that had been identified from C.cylindracea
(4), was found among the serine isoacceptor tRNAs. Because
the tRNA is as abundant in amount as other serine tRNA
isoacceptors in C cylindracea and C.zeylanoides, it is likely that
the tRNA with anticodon CAG is one of the major serine tRNA
species in these Candida species, which have GC rich genomes.
The gene sequences for these tRNAs have been determined for
C.cylindracea (4); C.albicans (14,15) and all other Candida
species described above (Fig. 6), all of which confirmed the
tRNA sequences presented here. It is noted that all the
tRNAserCAG species have a similar structural feature; position
33, the 5'-adjacent position to the anticodon, is occupied by a
guanosine residue instead of a uridine or cytidine residue as in
most other tRNAs (16). This would imply that the genes for these
tRNAs were derived from a single ancestor.
The amino acid-charging activities of these tRNAs were
examined by seryl-tRNA and leucyl-tRNA synthetases partially
purified from S. cerevtsiae. All the tRNAs having anticodon CAG
accepted serine to a considerable extent (more than 1,000
pmoles/A260), without appreciable acceptance of leucine (less
than 5 pmoles/A260). tRNAserCAG from C.cylindracea was a
good substrate for seryl-tRNA synthetase, in spite of having U
at the discriminator position where G is located in the other five
tRNAs and eukaryotic serine tRNAs reported so far. As described
above, it is clear that these tRNAs from Candida species have
enough tRNA identity elements as serine tRNAs, though we have
not determined enzymatic parameters of these tRNAs for seryltRNA synthetases. Thus it can be concluded that the tRNA with
anticodon CAG from these Candida species is a serine tRNA
corresponding to codon CUG.
Distribution of non-universal CUG codons in Hemiascomycetes
By comparing the amino acid assignments with the chain length
of the isoprenoid moiety of ubiquitine (17) and the composition
of cell-wall sugar (18), the yeast species could be classified into
three groups. The first group is one in which CUG codes for
leucine, the ubiquinone type is either Q-9 or Q-10, and the cell
wall contains galactose (+Gal) (18). -Group I; Zygoascus
hellenicus, C.magnolidae, C.azyma, Yarrowia lipolytica and
Schizosaccharomyces pombe*. The second group contains six
Candida species, all of which use CUG as a serine codon; the
ubiquinone type is Q-9 as in Group I, but the cell wall lacks
Gal(-Gal) -Group II; C.parapsilosis, C.zeylanoides,
C.albicans, C.cylindracea, C.rugosa and C.melibiosica. The
third group comprises yeasts in which CUG is used as a leucine
codon, the ubiquinone type is Q-6 or Q-7, and the cell wall lacks
Gal (-Gal) -GroupIll; yeasts such as C.utilis*, S.cerevisiae
and Pichia membranaefaciens*, (The amino acid assignments
for the species marked with an asterisk are not known). On the
basis of the isoprenoid chain length of ubiquinone, Group II (Q-9)
is closer to Group I (Q-9/Q-10) than Group HI (Q-6/Q-7),
whereas Group II (-Gal) is similar to Group IH (-Gal) and
different from Group I (+Gal) with respect to the composition
of cell-wall sugars (Table 1). Group II may intervene between
Group I and Group 11.
4044 Nucleic Acids Research, 1993, Vol. 21, No. 17
DISCUSSION
We have established an in vitro trnslation assay system for amino
acid assignments of codons in Hemiascomycetes. The genetic
codes in certain organisms are usually detennined by comparing
the amino acid sequence of a protein and the nucleotide sequence
of its gene DNA. However, the purification of a protein and
determination of its amino acid sequence are often laborious tasks.
There is also the possibility of RNA editing (19). In such cases,
in vitro translation assay is useful.
From the in vitro translation experiments, we conclude that
the following six Candida species use codon CUG for serine:
C.parapsilosis, C.zeylanoides, C.albicans, C.cylindracea,
C. rugosa and C.melibiosica. This conclusion is consistent with
the facts that [1] C.cylindracea has tRNAseCAG that translates
codon CUG as serine, while tRNAL'- for the translation of
CUG as leucine has not been detected (4), and [2] a similar serine
tRNA with the anticodon sequence CAG exists in all the C
species in which codon CUG is translated as seine. In
the following eight yeasts, codon CUG codes for leucine as in
the universal genetic code: S. cerevisiae, C. diversa,
C.rugopelliculosa, Zygoascus hellenicus, C.magnoliae, C.azyma,
Y lipolytica and Torichosporon cutaneum (Basidiomycetes).
Recently Santos et al. (15) reported the nucleotide sequences
and characterization of tRNAserCAG and its gene from
C.albicans. The sequences are identical with those reported by
us (14, 20). Santos et al. (15) reported unusual initiation of
translation occasionally caused by the tRNAserCAG in
C.albicans cell-free extras using a-, fl-globin and Bromo mosaic
virus coat protein mRNAs, and yet translation of codon CUG
by tRNA1erCAG has not been reported. The nucleotide
sequence of tRNAserCAG in the six Candida species (Group I)
is very similar; the sequence homologies of the tRNA from
C.parapsilosis, C.albicans and C.zeylanoides are more than 90%,
whereas those of C.melibiosica, C.cylindracea and C. rugosa are
about 70-80% (see Fig. 5). These similarities correlate to the
phylogenetic relationship predicted from their 5S rRNA sequences
(our unpublished observation). All tRNAs except that from
C. cylindracea have G at position 73 (discrinminator nucleotide),
which is one of the characteristics of serine tRNAs, whereas they
show a feature of leucine tRNA that m'G is located at the 3'
adjacent position of the anticodon. However, it is notable that
G33 is present in all the species. This unique feature and the
phylogenetic relationship of theses tRNAs,deduced from their
sequence similarities would imply that tRNAserCAG is derived
from a single ancestor.
We have proposed that tRNAserCAG is derived from an
intron-containing serine tRNA based on the nucleotide sequence
of tRNAseCAG of C.cylindracea. Here the sequences of
RNAserCAG genes of the Candida species in which CUG is
translated as serine.are determined. Among these six tRNA
genes, the tRNA genes of two species, C. cylindracea and
C.melibiosica, are interupted by introns, while the other four
tRNA genes lack intron. According to the similarities of 55 rRNA
sequences it is suggested that C. cylindracea and C.melibiosica,
which have the intron-containing tRNAS-rCAG genes, are
phylogenetically closer to Group I than the others (unpublished
data). Thus, assuming that these tRNA genes are of the same
origin, it is likely that intron have disappeared during the
evolution of the tRNAserCAG
According to the codon capture theory (2, 21), production of
an unassigned codon is the first step for neutral genetic code
change. Unassigned codons may be produced by directional
mutation pressure (AT- or GC- pressure), which primarily
determines the synonymous codon choice, especially in extremely
high or low G+C bacteria (2). There is also a strong correlation
between the relative amount of isoacceptor tRNA for an amino
acid and the usage of the corresponding synonymous codons
(22,23). In its extreme form, a codon becomes unassigned by
removal of the codon and the corresponding tRNA, as
exemplified by CGG in Mycoplasma capricolum (G+C: 25%)
(24) or by AGA and AUA in Micrococcus luteus (G+C: 74%)
(22).
One remarkable feature of yeasts resides in the wide range of
their genomic G+C content (e.g., 27.1 % for Hanseniaspora
valbyensis and 63.4% for Rhodotorulafujisanensis). The GC
contents of the genomes of these yeasts (Table 1) are highly
diversified, even in closely related species. For example,
C.albicans is very closely related to C.zeylanoides based on the
sequence homology of 5S rRNA (94%)(unpublished), and yet
the G+C contents are 36% and 56%, respectively. This suggests
that the direction of the mutation pressure has rapidly changed
within a very short time, even in closely related species in yeasts.
The range of genomic G+C-content in Group II used in this study
is 34% to 63%, while that in Group I is 43% to 60%. It is thus
possible that CUG became unassigned during the emergence of
Group II yeasts under strong AT-pressure by converting to
another A+T-rich synonymous codon, accompanied by a loss
of the corresponding tRNA. A relaxation of AT-pressure, i.e.
an increase in GC-pressure, could have brought the reappearance
of CUG as serine upon emergence of a tRNA translating the
codon as serine.
Naturally, such an unassigned codon capture by the serine
tRNA is acceptable at any dispensable site in a gene. For example,
a dispensable UUG leucine can mutate to CUG serine, etc.
However, in one of the lipase genes of C. cylindracea, CUG is
used as a serine codon in the catalytic center (25,26), showing
that a serine codon, even at an important site, can change to CUG
by a nearly neutral process. The change of the universal serine
codon UCN (N: U, C, A, or G) or AGY (Y: U or C) to CUG
cannot occur by a single mutation - it must pass through an
intermediate codon such as UUR (Leu)(R: G or A) or CCN (Pro).
The gene having such an intermediate codon becomes functionally
inferior or inactive if it occurs at an important site. However,
most of the eukaryotic genes exist multiply in the genome and
therefore deleterious mutation in one of the genes would not affect
the viability of the cell. The 'inferior' gene would become a
pseudogene at one time, but at another time it would be revived
by a second mutation at the site originally occupied by a serine
codon such as UCG [e.g., UCG (Ser) - UUG (Leu) -CUG
(Ser)]. The latter process would not be impossible if the creation
of tRNA has preceded the change of UUG (Leu) to CUG (Ser).
Indeed, there would be no other way of near-neutral generation
of CUG serine codons at important sites.
Naturally, the higher the GC-pressure, the higher the usage
of codon CUG, regardless of its assignment, serine or leucine
(3, 27). For example, CUG is a rare leucine codon in S.cerevisae
(G+C: 40%) (28) and is also a rare codon (for serine) in
C.albicans (G+C: 34%)(29), while it is a predominant serine
codon in C. cylindracea (G+C: 63%) in which several copies
of the genes for tRNAserCAG have been detected (unpublished).
Propagation of codon CUG for serine in Group H yeasts is
also possible through duplication of the genes containing CUG
after its reassigmnent.
Nucleic Acids Research, 1993, Vol. 21, No. 17 4045
Peggie, M. W. and
4298.
19,
Res.
Acids
Nucleic
(1991)
K.
R.
Swoboda,
arose through individual mutations of various other codons,
30. Kalb, V. F., Woods, C. W., Turi, T. G., Dey, C. R., Sutter, T. R. and
neutral process
umvesalsere
inclu
codons, bya
nearly neuralproessLoper,
by anarl
guniversal serinecdons
J. C. (1987) ANA 6, 529-537.
including
31. Gemnann, U. A., Muller, G., Hunziker, P. E. and Lerch, K. (1988) J. Biol.
as described above. Indispensable serine sites in a protein gene,
Chem., 263, 885 -896.
only a single copy of which exists in an organism, would not
have been directly involved in this type of reassignment, because
a mutation, if it occurred at these sites, would be removed by
negative selection. Therefore, such code changes could occur only
when multiples of the same gene exist.
We infer that all of the serine CUG codons in Group II yeasts
ACKNOWLEDGEMENTS
We thank Dr T.H.Jukes (University of California, Berkeley) for
discussion and to Drs M.Homma and K.Tanaka (School of
Medicine, Nagoya University) for the strain of C.albicans strain
C9. This work was supported by a Grant-in-Aid for Scientific
Research on Priority Areas from Ministry of Education, Science
and Culture of Japan.
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