close

Вход

Забыли?

вход по аккаунту

?

779

код для вставкиСкачать
YEAST
VOL.
12: 493499 (1996)
-oooo
0
0’ xrv % Yeast Sequencing Reports
0
0
0o o o o
The Sequence of 12.8 kb from the Left Arm of
Chromosome XIV Reveals a Sigma Element, a
pro-tRNA and Six Complete Open Reading Frames,
One of Which Encodes a Protein Similar to the Human
Leukotriene A, Hydrolase
*
F. NASR, A,-M. BECAM AND
c. J. HERBERT*
Centre de GPnttique Moliculaire, Laborntoire propre du CNRS associC a I’Universitt Pierre et Marie Curie,
F-91198 Gij-sur- Yvette, France
Received 8 September 1995; accepted 14 October 1995
We have determined the nucleotide sequence of a 12.8 kb fragment from the left arm of chromosome XIV carried
by the cosmid 14-16d. An analysis of the sequence reveals the presence of a sigma element, a pro-tRNA gene and
eight open reading frames, six of which are complete. All of the eight open reading frames correspond to new genes.
Of the eight new genes, two show strong similarities to a pair of new genes from chromosome IX, suggesting an
ancestral duplication, and one gene encodes a protein similar to mammalian leukotriene A, hydrolase.
--,The sequence
has been deposited in the EMBL data bank under the Accession Number X94547.
KEY WORDS - Saccharomyces
cerevisiue; genome sequencing; chromosome XIV; leukotriene A, hydrolase
INTRODUCTION
As part of the EEC BIOTECH I project to sequence chromosomes X and XIV of Saccharomyces cerevisiae, we have determined the sequence of
12 8 kb from the left arm of chromosome XIV
carried by the cosmid 14-16d, using a combination
of random and oligonucleotide-directed procedures. The sequence reveals eight open reading
frames (ORFs), six of which are complete and all of
which correspond to new genes.
MATERIALS AND METHODS
Escherichia coli
The E. coli strain used was XL1 blue {recAl,
endAl, gyrA96, thi-1, supE44, hsdRl7, relA1, lac,
F [proA+B+,lac@, lac Z6M15, TnlO (tetr)]}.
*Corresponding author.
CCC 0749-503X/96/050493-07
0 1996 by John Wiley & Sons Ltd
D N A manipulations
Routine DNA manipulations: plasmid preparation, subcloning, nick translation, agarose gel
electrophoresis and transformation were carried
out as described in Maniatis et al. (1982). Restriction endonucleases, T4 DNA ligase and alkaline
phosphatase were used in accordance with the
manufacturers’ instructions. Hybridization screening of bacterial colonies to identify recombinant
plasmids was performed as described in Maniatis
et al. (1982).
Sequencing strategy
The starting material for the sequencing was the
cosmid 14-16d which was provided by Professor P.
Philippsen, the DNA coordinator for chromosome
XIV. This cosmid bridges the gap between cosmids
14-16 and 14-17. Only the central section of 14-16d
494
F. NASR ET AL.
N2510
N2505
N2515
Figure 1. ORF map of the 12.8 kb fragment from the cosmid 14-16d. The ORFs
marked above the line are translated from the 5'-3' sequence (EMBL: X94547),
ORFs marked below the line are translated from the complementary strand. The
sigma element (N2660, coordinates 10460-10799) and the pro-tRNA gene (N2665,
co-ordinates 10816-10918) are shown as half-height boxes.
needed to be sequenced; this was contained in a
12 kb XhoI fragment. In order to sequence this
section the whole cosmid was sonicated, end repaired with T4 DNA polymerase and size fractionated on an agarose gel. Fragments between 0.82 kb were purified and cloned in HincII-cut
pUCl8. Recombinant plasmids were detected
by hybridization against a probe made from the
purified 12 kb XhoI fragment. The majority of the
sequence was determined using the Pharmacia
automatic sequencer (ALF) in conjunction with
the Pharmacia fluorescent sequencing kit. Custom
oligonucleotide primers were used in conjunction
with fluorescent dATP and the ALF sequencer
or with manual sequencing, ( u [ S ~ ~ ] ~ Afrom
TP
Amersham and T7 DNA polymerase from
Pharmacia). The sequence data from individual
clones were assembled using the Staden-plus@
software package (Amersham).
Computer analysis
Routine sequence analysis was performed using
the UWGCG programs (Devereux et al., 1984),
DNA Strider (Marck, 1988) and BLAST on the
NIH server (Altschul et al., 1990). For the calculation of the BESTFIT derived Z-score, the
PAM250 matrix of Dayhoff was used with a gap
weight of 3, a gap length weight of 0.3 and 100
randomizations.
RESULTS AND DISCUSSION
We have determined the sequence of 12.8 kb which
is part of the insert carried by the cosmid 14-16d.
This cosmid was isolated from a library constructed by cloning a Sau3A partial digestion
of genomic DNA from the strain FY1679, a
derivative of wS288c (Dujon et al., 1994), in the
BamHI site of the vector pWEl5. An analysis of
the sequence reveals a sigma element (N2660), a
pro-tRNA gene (N2665) and eight ORFs, none of
which correspond to previously described genes
(Figure 1). Among the eight ORFs, six are
complete. A summary of the data concerning the
ORFs is given in Table 1.
As part of our routine analysis, the deduced
amino acid sequences were compared with the data
banks using the FASTA and BLAST programs. Of
the eight ORFs, four show significant similarities
to previously known sequences: N2.510, N251.5,
N2535 and the incomplete ORF N2505.
N2505 and N25 15
The deduced sequence of the n2505p fragment is
similar to yillO9cp (P40482.SWISSPROT, Z-score
95.6) and yhr098cp (P388 lO.SWISSPROT, Z-score
27.7), proteins of unknown function identified
during the systematic sequencing of chromosomes
IX (unpublished) and VIII (Johnston et al., 1994)
respectively. N2505p is also similar to a human
protein of unknown function (D38555.GP, Z-score
42.0). An alignment of the three yeast sequences
is shown in Figure 2. The conservation of this
sequence in Man implies that the protein has an
important basic function in the cell. This hypothesis is supported by the observation that the
dbest database contains human, nematode and
plant cDNA tags which significantly match the
n2505p fragment [Homo sapiens (T79533.GB),
Caenorhabditis elegans (D27679.GB), R. callus
(D21986.GB) and A . thaliana (T13977.GB)I.
The deduced n2515p sequence shows significant
similarities to three other yeast proteins: yillO5cp
from chromosome IX (P40485.SWISSPROT,
Z-score 85.7), a hypothetical protein from chromosome XVI (U32445.GP, Z-score 37.8) and asklOp
495
12.8 kb FROM LEFT ARM OF CHROMOSOME XIV
Table 1. Co-ordinates, molecular weight, codon bias (Bennetzen and Hall, 1982) and
codon adaptation index (Sharp and Li, 1987) of the ORFs encoded by the 12.8 kb fragment
from the cosmid 14-16d. The co-ordinates refer to the first nucleotide of the initiator codon
and to the third nucleotide of the last sense codon.
Co-ordinates
Molecular weights
Gene
First
Last
N2505
N2510
N2515
N2530
N2535
N2650
N2655
N2670
1633
1895
5603
6026
6685
9123
9631
11822
nd
3538
3636
6541
8697
9596
93 14
nd
~~
Incomplete
63.1
74.7
19.3
77.3
17.4
11.3
Incomplete
Codon
0.039
0.030
- 0.012
0.284
0.341
- 0.055
0.144
0.121
0.077
0.238
0.222
0.096
~
CAI, codon adaptation index; nd, not determined.
n2505p
yillO9cp
yhr098cp
1
1
1
n2505p
yillO9cp
yhr098cp
22
61
54
n2505p
yillO9cp
yhr098cp
61
120
103
n2505p
yillO9cp
yhr098cp
109
176
162
n2505p
yillO9cp
yhr098cp
166
233
222
n2505p
YillO9CP
yhr098cp
224
291
281
n2505p
YillO9CP
yhr098cp
281
348
341
n2505p
yillO9cp
yhr098cp
341
386
372
n2505p
yillO9cp
yhr098cp
394
438
431
n2505p
yillO9cp
yhr098cp
454
498
484
n2505p
yill09cp
yhr098cp
514
558
542
Figure 2. An alignment of the n2505p fragment and the N-terminal fragments of yillO9cp
(P40482SWISSPROT) and yhr098cp (P38810SWISSPROT). The alignment was made
using the MULTAL program of Corpet (1988) with gap creation and elongation penalties of
10. Similar and identical residues are on a grey background and residues conserved in all
sequences are on a black background.
from chromosome VII (U27209.GP, Z-score 34.6).
AsklOp is reported to enhance Skn7p-dependent
transcription from a LexA-HIS3 reporter construction (unpublished); no information is
available concerning the function of the other
proteins. An examination of the comparisons of
these four proteins shows that the chromosome IX
and XIV sequences are closely related and that the
chromosome VII and XVI sequences are also
closely related, but that the similarity between
496
F. NASR ET AL.
yill0 5cp
n2515p
yillO5cp
157
181
n2515p
yillO5cp
277
301
n2515p
yillO5cp
337
361
n2515p
yillO5cp
457
n2515p
yillO5cp
627
660
480
Figure 3. An alignment of n2515p and yil105cp (P40485SWISSPROT), a hypothetical
protein from S. cerevisiue chromosome IX. The alignment was made and is presented as
described in Figure 2.
these two groups is significantly less. An alignment
of n2515p and yil105cp sequences is shown in
Figure 3.
Interestingly, N250.5 and N2.515 are transcribed
from the same strand and show strong similarities
to a pair of genes, YIL109c and YIL105c, which
are close and also transcribed from the same
strand on chromosome IX. These similarities are
suggestive of an ancestral duplication between
chromosomes XIV and IX. However, no solid
conclusions can be drawn until more extensive
sequence comparisons are made.
N2510
The deduced n2510p sequence shows significant
similarity with a hypothetical protein of unknown
function from C. elegans identified during the
systematic sequencing of part of chromosome 111
(Wilson et al., 1994). A comparison of the two
sequences gives a Z-score of 28.1. An alignment of
n2510p and the C. elegans protein is shown in
Figure 4.
N2535
The N2.535 gene is predicted to encode a protein
of 77.3 kDa, and has a codon bias of 0.284 (calculated according to Bennetzen and Hall, 1982),
indicating that it is relatively highly expressed.
When the deduced n2535p was compared to the
data banks, strong similarities with leukotriene A,
hydrolases (LTA, hydrolase) from Guinea pig (Zscore 58.7), Mus musculus (Z-score 57.7), Rattus
norvegicus (Z-score 68.0) and Homo sapiens (Zscore 60.6) were detected. The alignment of n2535p
and human LTA, hydrolase (Figure 5) shows that
they are very similar. LTA, hydrolase is a cytosolic
enzyme involved in the biosynthesis of leukotrienes
(Wetterholm et al., 1992; Medina et al., 1991).
Leukotrienes are bioactive compounds which
act as mediators of hypersensitivity and inflammation (Samuelson, 1983; Samuelson et al., 1987).
Recently, sequence comparisons revealed a zincbinding motif in the amino acid sequence of
LTA, hydrolase, a motif which is found in
aminopeptidases and neutral proteases (Malfroy
et al., 1989; Vallee and Auld, 1990). Accordingly,
LTA, hydrolase was found to contain one atom
of zinc per molecule and to exhibit a peptidase
activity towards synthetic substrates and was thus
characterized as a metalloenzyme (Minami et al.,
1990; Haeggstom et al., 1990; Wetterholm et al.,
1991). The zinc atom appears to have a catalytic
function, as its removal leads to the loss of both
enzymic activities. The three amino acids involved
in zinc binding or coordination were determined
by site-directed mutagenesis and correspond, in
the mouse LTA, hydrolase, to H-295, H-299 and
497
12.8 kb FROM LEFT ARM OF CHROMOSOME XIV
n2510p
C.elegans
231 k g
204 s n
n2510p
C. elegans
;;;~ ~ ~ ~ B s g asc
l 5 9 " h " 8 " 1 : ~ ~ ~ 8 " n - - - - - - - - - - - - - - - - - - - - - - - - - snd g
kdy
n2511Jp
C.elegans
441 _ _ _ _ _ _ . - - -n -g - _ - _ - - - - - - - - -el
439 r n d t r k y a r k s l t r f g e a a f e v s r f k 1 kngiffnrk kkltgtkkskkccehfqfi
n251Qp
C.elegans
454
499 inhifsrflyflygfllyntm
n2510p
C.elegans
539 c k
679 8 s
I
ghcvgylsitkeeyvetilkiveeglkk
0 1 5------pqE---------------
.....................
_._______._______--------------
lddlvkksenlsdqskmfytsarkmnkccnyv
Figure 4. An alignment of n2510p and the C. elegans protein (U00031.GP). The alignment
was made and is presented as described in Figure 2.
etphkmlplsiegrrpsrspeydqst
__________________________
n2535p
H.sapiens
177 -gk
127 keh
n2535p
232
n2535p
H.sapiens
470 f
425 w
n2535p
H.sapiens
529 t
482 -
n2535p
588 d
n2535p
H.sapiens
646
585
--
eykhladwlgtv
Figure 5. An alignment of n2535p with the human leukotriene A, hydrolase (A33886.PIR).
The alignment was made and is presented as described in Figure 2.
E-318 (Medina et al., 1991). The analysis of
n2535p revealed the presence of the zinc-binding
motif and the alignment of n2535p with human
LTA, hydrolase shows that the three residues
are in a highly conserved region and could be
unambiguously identified in the n2535p sequence
as H-340, H-344 and E-362 (Figure 5).
Another feature of the zinc-binding motif is the
presence of a conserved glutamic acid residue
shared by some zinc-proteases and aminopeptidases including LTA, hydrolase (Pangburn
and Walsh, 1975). X-ray crystallographic data
suggested that a conserved glutamic acid residue
is involved in the proteolytic mechanism of the
498
n2535p
G.pig
X. eapiens
M.musculus
R.norvegicus
L.helveticus
H.sapiens
L. lactis
S.cerevisiae
F. NASR ET AL.
337
293
293
293
292
286
390
285
297
Figure 6. An alignment of the zinc-binding motifs from
different proteins. The first five sequences are derived from
n2535p and mammalian LTA, hydrolases. The remaining
sequences are derived from aminopeptidases. From top to
bottom: n2535p, Guinea pig (D16669.DBJ), H. supiens
(A33886.PIR), M. musculus (P24527,SWISSPROT), R.
norvegicus (P30349,SWISSPROT), L. helveticus (U08224.GP),
H. sapiens (A47531,PIR), L. luctis (P37896,SWISSPROT), S.
cerevisiue (P37898,SWISSPROT). The alignment was made and
is presented as described in Figure 2.
enzyme. This was confirmed by in vitro mutagenesis of glutamic acid-296 in the mouse
LTA, hydrolase (Kester and Matthews, 1977;
Wetterholm et al., 1992). This residue, which corresponds to E-296 in human LTA, hydrolase, is
also conserved in n2535p where it corresponds to
E-341. Figure 6 shows an alignment of the zincbinding motifs from four LTA, hydrolases and
four aminopeptidases.
These results strongly suggest that n2535p is a
zinc-metalloenzyme with an aminopeptidase activity. However, in sequence comparisons, n2535p
shows a much stronger resemblance to LTA,
hydrolases than other zinc aminopeptidases. This
may suggest that n2535p also has a hydrolase
activity. It is difficult to imagine that the physiological r81e of leukotrienes is conserved in
yeast, but n2535p may be involved in the metabolism of structurally, if not functionally related
compounds.
Other genes
N2650 and N26.55 are overlapping ORFs on
opposing strands; both ORFs are predicted to
encode small proteins and do not show any similarity to proteins in the data banks. According
to Bennetzen and Hall (1982), N26.50 has a high
codon bias of 0.341 whilst N2655 has no codon
bias. This may suggest that N2650 is a more
probable gene than N2655. This is supported by
the finding, in the dbest data bank, of a human
cDNA tag showing similarity to N2650
(T64688.GB, Z-score 7.75). When the deduced
protein sequences of the remaining ORFs were
compared with the dbest data bank, yeast cDNA
tags corresponding to N2510 and N2.535 were
found, indicating that these genes are expressed.
ACKNOWLEDGEMENTS
We would like to thank Prof. P. Philippsen for the
distribution of the clones, Drs L. Karpfinger and
H. W. Mewes for the coordination of the DNA
submissions, Dr J.-L. Risler for maintaining the
computer facilities at the Centre de Gknetique
Moleculaire and Mme D. Menay for the synthesis
of the oligonucleotides. We are grateful to Prof. P.
P. Slonimski for his encouragement and support
throughout this work and for critically reading
the manuscript. This work was supported by
the Commission of the European Communities
under the BIOTECH program of the Division of
Biotechnology and the Groupement de Recherches
et d’Etudes sur les Genomes. F.N. was financed
in part by a predoctoral fellowship from the
Foundation HARIRI Lebanon.
REFERENCES
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. and
Lipman, D. J. (1990). Basic local alignment tool.
J. Mol. Biol. 215, 403410.
Bennetzen, J. F. and Hall, B. D. (1982). Codon selection
in yeast. J. Biol. Chem. 257, 302G-3031.
Corpet, F. (1988). Multiple sequence alignment with
hierarchical clustering. Nucl. Acids Res. 16, 1088110890.
Devereux, J., Haeberli, PO. and Smithies, 0. (1984). A
comprehensive set of sequence analysis programs for
the VAX. Nucl. Acids Res. 12, 387-395.
Dujon, B., et al. (1994). Complete DNA sequence of
yeast chromosome XI. Nature 369, 371-378.
Haeggstrom, J. Z., Wetterholm, A,, Shapiro, R., Vallee,
B. L. and Samuelsson, B. (1990). Leukotriene A,
hydrolase: a zinc metalloenzyme. Biochem. Biophys.
Res. Commun. 172, 965-970.
Johnston, M., et al. (1994). Complete DNA sequence of
Saccharomyces cerevisiae chromosome VIII. Science
265, 2077-2082.
Kester, W. R. and Matthews, B. W. (1977). Crystallographic study of the binding of dipeptide inhibitors
to thermolysin: implications for the mechanism of
catalysis. Biochemistry 16, 2506-2516.
Malfroy, B., Kado-Fong, H., Gros, C., Giros, B.,
Schwartz, J.-C. and Hellmis, R. (1989). Molecular
clonding and amino acid sequence of rat kidney
aminopeptidase M: a member of a super family of
zinc-metallohydrolases. Biochem. Biophys. Res. Commun. 161, 236-241.
Maniatis, T., Fritsch, E. F. and Sambrook, J. (1982).
Molecular Cloning: A Laboratory Manual. Cold
Spring Harbor Laboratory, Cold Spring Harbor, New
York.
Marck, C. (1988). “DNA Strider”; a “C” program for
the fast analysis of DNA sequences on the Apple
12.8 kb FROM LEFT ARM OF CHROMOSOME XIV
Macintosh family of computers. Nucl. Acids Res. 16,
1829-1 836.
Medina, J. F., Wetterholm, A., Radmark, O., et al.
(1991). Leukotriene A, hydrolase: determination of
the three zinc-binding ligands by site-directed mutagenesis and zinc analysis. Proc. Natl. Acad. Sci. USA
88, 7620-7624.
Minami, M., Ohishi, N., Mutoh, H., et al. (1990).
Leukotriene A, hydrolase is a zinc-containing aminopeptidase. Biochem. Biophys. Res. Commun. 173, 620626.
Pangburn, M. K. and Walsh, K. A. (1975). Thermolysin
and neutral protease: mechanistic considerations.
Biochemistry 14, 40504054.
Sammuelsson, B. (1983). Leukotrienes: mediators of
immediate hypersensitivity reactions and inflammation. Science 220, 568-575.
Samuelsson, B., Dahlen, S.-E., Lindgren, J. A,, Rouzer,
C. A. and Serhan, C. N. (1987). Leukotrienes and
499
lipoxins: structures, biosynthesis and biological
effects. Science 237, 1171-1 176.
Sharp, P. M. and Li, W. H. (1987). The codon adaptation index: a measure of directional synonymous
codon usage bias, and its potential applications. Nucl.
Acids Res. 15, 1281-1295.
Vallee, B. L. and Auld, D. S. (1990). Zinc coordination,
function and structure of zinc enzymes and other
proteins. Biochemistry 29, 5647-5659.
Wetterholm, A., Medina, J. F., Radmark, O., et al.
(1991). Recombinant mouse leukotriene A, hydrolase:
a zinc metallo-enzyme with dual enzymatic activities.
Biochim. Biophys. Acta 1080, 96-102.
Wetterholm, A., Medina, J. F., Radmark, O., et al.
(1992). Leukotriene A, hydrolase: abrogation of the
peptidase activity by mutation of glutamic acid-296.
Proc. Natl. Acad. Sci. USA 89, 9141-9145.
Wilson, R., et al. (1994). 2.2 Mb of contiguous
nucleotide sequence from chromosome 111 of C.
elegans. Nature 368, 32-38.
Документ
Категория
Без категории
Просмотров
2
Размер файла
621 Кб
Теги
779
1/--страниц
Пожаловаться на содержимое документа