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YEAST
VOL.
12: 505-514 (1996)
0o o o o
0' XIV % Yeast Sequencing Reports
0
0
0o o o o
The Sequence of a 24 152 bp Segment from the Left
Arm of Chromosome XIV from Succhuromyces
cerevisiae Between the BNIl and the POL2-Genes
MARK SEN-GUPTA, RUTH LYCKT, URSULA FLEIG, RAINER K. NIEDENTHAL AND
JOHANNES H. HEGEMANN*
Institute fur Mikrobiologie und Molekulurbiologie, Justus-Liebig- Universitat Giessen, Frankfurter Str. 107,
35392 Giessen, Germany
TInstitut f u r Zellbiologie, J. W. Goethe-Universitat Frunkfurt, Marie-Curie-Str. 9, 60439 FrunkfurtlM., Germany
Received 24 October 1995; accepted 11 November 1995
In the framework of the European Union programme for sequencing the genome of Saccharomyces cerevisiue we
have determined the nucleotide sequence of a region of 24 152 bp located on the left arm of chromosome XIV
between the BNIl and the POL2 genes. The sequence was obtained by directed sequence analysis using a mixture of
Ex0111 and primer walking strategies. Subsequent analysis revealed 13 open reading frames (ORFs) including four
small ORFs completely internal to, or partly overlapping with, other ORFs. Five of these ORFs have been described
previously (BNII, A P L I , L YPI, PIKI, POL2) and thus 74.8% of the 24 152 bp were already present in the databases
prior to this sequencing effort. Interestingly, all 13 identified ORFs are characterized by a low codon adaptation
index (0,04422). In addition, this region of chromosome XIV shows an unusually high gene density with about 88%
of coding DNA. This amounts to one gene per 2177 bp, which is significantly above the average gene length (about
1500 bp). For eight ORFs considerable homologies to 'Expressed Sequence Tags' derived from human cDNAs
located in the XREF database could be identified.'The complete nucleotide sequence of the 24 152 bp segment has
been deposited in the EMBL data library under the Accession Number X92494.
KEY WORDS
Sacchuromyces cerevisiue; chromosome XIV; ORF analysis; amino acid permeases; polymerase 11;
phosphatidylinositol-4-kinase
~
INTRODUCTION
Chromosome XIV from Saccharomyces cerevisiae
is one of the chromosomes currently being sequenced as part of the European Union BIOTECH I and I1 projects with the aim of obtaining
the entire sequence of the genome of this eukaryotic model organism. As part of this framework,
we report here the sequence and analysis of a
24 152 bp segment from the left arm of chromosome XIV. This segment starts about 132 kb
away from the left telomere with the previously
*Corresponding author.
CCC 0749-503X/96/050505-10
0 1996 by John Wiley & Sons Ltd
identified BNIl gene and ends about 470 kb away
from the centromere at the POL2 gene. The
telomere-proximal end of the DNA contig
described here is partly overlapping with part of
the insert from cosmid 14-3b sequenced by the
Glansdorff-Pierard laboratory.
MATERIALS AND METHODS
Plasmids and D N A manipulations
The DNA segment reported here is located on
two partially overlapping cosmids 14-3b pou and
14-4 which were obtained from Peter Philippsen
506
who is coordinator of the chromosome XIV sequencing group within the BIOTECH project.
Cosmid 14-4, which is about 44.75 kb in size and
carries a 36.9 kb insert, was obtained as follows:
yeast strain FY1679 was partially cleaved with
Sau3A and the fragments were cloned into the
BamHI-cleaved 8.2 kb cosmid vector pWE15
(Evans and Wahl, 1987) to obtain a yeast genomic
library (Thierry et al., 1995). Chromosome XIV
specific cosmids were identified and their position
along the chromosome was determined (Hamberg,
1993). Cosmid 14-3b pou is about 45.2 kb in size,
carries a 37-1 kb insert and was isolated from a
different yeast genomic DNA cosmid library. This
library was created by cloning a partial Sau3A
digest of genomic DNA from yeast strain FY1679
into the BamHI site of the pOU6lcos vector
(Knott et al., 1988; Thierry et al., 1995). We
verified the cosmid inserts by Southern blots on
EcoRI-cleaved cosmid DNA and cleaved yeast
genomic DNA of strain FY1679.
All DNA manipulations were done as described
(Sambrook et al., 1989) and enzymes were used
according to the supplier’s instructions. The
Ex0111 kit Erase-a-base was obtained from
Promega (Heidelberg, Germany). Oligonucleotides
were synthesized on an ABI 370B DNA synthesizer and used directly after ethanol precipitation
without further purification.
Strains and media
The Escherichia coli strain used for cloning
experiments was XLlblue (recAf, endAf, gyrA96,
thi-f, hsdRl7, supE44, relAl, lac [F’ proAB,
lac14ZdMf 5, Tnf 0 (tet‘)]). E. coli transformants
were selected on LB media supplemented with
0.1 gll ampicillin. The yeast strain was FY1679
( a h ura3lura3 trpllTRP1 leu21LEU2 his31HIS3
GAL2; from Bernard Dujon). Yeast media have
been described (Sherman et al., 1986).
Sequence analysis
Cosmids 14-4 and 14-3b were cleaved with
EcoRI, XhoI or SalI and the fragments subcloned
into pBluescript SKI1+ (Stratagene, Heidelberg,
Germany). All subclone inserts were sequenced at
their 5’ and 3’ termini using modified T3 (CAA
TTA ACC CTC ACT AAA G) and T7 (GTA
ATA CGA CTC ACT ATA G) primers. Oligonucleotides designed from these sequences were
used to sequence directly on the cosmids to determine physical linkage of individual subclones. For
M. SEN-GUPTA ET AL.
subclones with inserts longer than 2 kb a series of
nested deletions was constructed by limited digestion with Ex0111 according to the supplier’s
instructions and sequenced by using the modified
T3 and T7 primers. Sequence gaps were filled by
using appropriate oligonucleotides. Smaller subclones were sequenced by primer walking with
primers homologous to sequences 200 to 300 bases
apart.
Sequencing was performed on an ABI 373A
sequencer (Applied Biosystems Inc., Weiterstadt,
Germany) using the Taq DyeDeoxy Terminator
Cycle Sequencing Kit supplied by the manufacturer.
Sequence alignments and overlaps were done on
a Macintosh Quadra 650 computer using DNASTAR’S seqman program (Lasergene Ltd,
London, England). FASTA, MOTIFS and
PROSITE analyses were routinely performed by
MIPS (Martinsried, Germany). Further characterization was done by using DNASISlPROSIS
(Pharmacia, Freiburg, Germany) and the HUSAR
package (DKFZ, Heidelberg, Germany). Homology searches against the Expressed Sequence Tag
(EST) database were performed by the Genome
Cross-Referencing Group at the NIH, Bethesda,
U.S.A. This XREF database contains cDNAs
from humans, mouse and rat (Boguski et al.,
1994).
RESULTS AND DISCUSSION
Sequence determination
In total, 278 subclones and Ex0111 clones were
generated and sequenced to determine the
24 152 bp composition on both strands. The total
number of bases sequenced was 109 581. By using
a sequencing strategy of 45% primer walking and
55% nested deletions, the average reading number
per base was 4.5, with each base being sequenced
at least three times (upper and lower strand
together).
At the left end of the 24 152 bp contig, the
cosmid clone 14-3b has an overlap of 1701 bp with
cosmid clone 14-3b sequenced by the GlansdorffPierard laboratory (Figure 1). No sequence
discrepancies were found in this overlap.
Sequence analysis
A search for coding regions in the 24 152 bp
long contig revealed 13 open reading frames
(ORFs) longer than 300bp named NO646 to
507
24 152 bp SEGMENT FROM CHROMOSOME XIV
Telomere
Cosmid clone 14-4
Centrornere
---+
Cosmid clone 14-3b pou
N0665
NO670
NO790
NO795
10000
No825
NO800
15000
20000
24152
No647
0n
m ORF with intron
e w ~ ~ ~ s
also sequenced by the laboratory of Glansdorff-Pierard
I
I
Figure 1. Genomic organization of a 24 152 bp DNA fragment from the left arm of chromosome XIV. Localization of the inserts
from the relevant cosmid clones and localization and orientation of the open reading frames (ORFs) are indicated. Only ORFs
longer than 100 amino acids are shown. The preliminary ORF nomenclature was provided by MIPS and begins with N, indicating
chromosome XIV, followed by a consecutive number starting at the left end of this DNA fragment (the lowest number
telomere-proximal, the highest number centromere-proximal). Definitive numbering will be given after assembly of the entire
chromosome. Previously sequenced ORFs are indicated by shaded boxes and their names.
NU830 (Figure 1). This nomenclature is preliminary and will be revised once chromosome XIV is
assembled completely. ORF NO646 extends into
sequences flanking this contig to the left and is
thus incomplete. No tRNAs, T y elements, delta or
sigma sequences could be detected.
Determination of the codon adaptation index
(CAI; Sharp and Li, 1987) for each ORF revealed
that all 13 ORFs are probably expressed at a low
level, showing CAI values of 0.04 to 0.22 only
(Table 1). In particular the four ORFs NU647,
NU665, NU670 and NU830 have extremely low CAI
values between 0.04 and 0.10. These values are
below the minimal CAI value of 0.11 defined
previously for a ‘real’ gene (Dujon et al., 1994).
Taking the CAI value and the size as a measure
these four ORFs probably do not code for any
proteins. Furthermore, three of these four ORFs,
NU647, NU665 and NU830, were found in the
coding region of the previously identified genes
BNII (NU646), APLI (NU66U) and POL2 (NU825)
respectively.
Thus the very low CAI values and the fact that
thus far a gene within another gene has not been
described in S. cerevisiae strongly suggest that the
three internal ORFs are not real genes (Demolis et
al., 1993; Boles and Zimmermann, 1994). It should
be noted, however, that two of the questionable
ORFs (NU665 and NU67U) show a significant homology to human cDNAs, although the relevance
of this finding is at present unclear (Table 2).
Within the 24 152 bp long contig, five genes
have been identified previously, thus 74.8% of the
contig was already available in the databases.
Excluding the four questionable ORFs, the contig
carries nine ORFs. The mean length of these ORFs
is 2177 bp (726 amino acids). This number is above
the average ORF length of 1461 bp (487 amino
acids) described for the already published yeast
chromosomes (Oliver et al., 1992; Dujon et al.,
1994; Johnston et al., 1994; Feldmann et al., 1994).
The higher value for this particular region of
chromosome XIV may be due to the fact that
several larger genes (BNII, PIKl and POLZ) reside
within this region (Table 1). The nine ORFs cover
about 88% of the contig. Again this number is
significantly above the 72% described, for example,
for chromosome I1 (Feldmann et al., 1994) and is
probably due to the presence of the three larger
genes. Such a high coding capacity has also been
described for certain regions of chromosome VIII
(Johnston et al., 1994). The maximum length of an
+
-
NO809
-
+
-
-
+
+
+
+
255.7
13.7
0.15
0.10
0.20
0.16
40.7
35.5
350
314
14 839-15 891
16 172-17 116
2222
124
0.15
34.5
298
3556-14552
17488-24152
20535-20906
0.14
0.04
0.12
0.09
0.08
0.22
0.15
0.12
CAI2
174.5
12.5
64.0
13.8
15.3
68.1
119.9
15.5
MW
(kDa)
1553
103
573
123
131
61 1
1066
139
Size
(aa)
14659
68 1-992
52166937
5625-5996
6975-7370
78269661
0 1 5 4 1 3 354
3521-13940
Position
VEE350 (S.C.)
NADH
dehydrogenases
POL2 ( S . C . )
PIKl (S.c.)
NADH
dehydrogenases
N UFl (S.c.)
L YPl (S.c.)
ALP1 (S.C.)
BNIl (S.C.)
Homology to
11 210111210
93911825
11313029
12214276
3 12213134
488114881
1151709
298413006
706718870
Score
FASTA3
100
99.7 (3)
100
99.1 (5)
99.9 (2)
(aa)
100
99.6 (9)
100
99,3 (12)
99.9 (2)
(bP)
% Identity4
(changes)
Internal to NO825
Prokaryotic lipoprotein
attachment site
Putative intron at
pos. 14 425-14 531
Internal to NO660
Internal to NO646
Special
features
'
ORFs that are questionable because of their size and low CAI value are in bold.
+indicates that transcription of the ORF is centromere-directed; - indicates transcription towards the telomere.
'The CAI is the codon adaptation index calculated following Sharp and Li (1987).
3FASTA scores higher than 100 are listed. Only the highest score is shown. S.C.=Saccharornyces cerevisiae.
4The percentage of identity over the entire ORF is indicated. The numbers of amino acids or base pairs which are different to the previously published sequences are shown
in brackets.
NO825
NO830
NO815
NO820
-
NO646
NO647
NO660
NO665
NO670
NO790
NO795
NO800
-
Orientation'
ORF
name
Table 1.
F
.P
"1
b.
h
2
s
70
%
509
24 152 bp SEGMENT FROM CHROMOSOME XIV
Table 2. EST homologies.'
ORF
name
NO646
NO660
NO665
NO670
NO790
NO795
NO800
NO820
Highest
p-value
3.1e-2
7.9e-13
4.8e-5
3.8e-2
1.7e-13
7.8e-24
6.6e-2
6.2e- 11
cDNA
Origin
NCBI-ID
Human placenta RNA
Human RNA
Muscular atrophy patient; total brain RNA
Human fetal liver and spleen RNA
Human RNA
Total human brain RNA
Alu-primed human cDNA
Human fetal liver and spleen RNA
106 959
12 857
123 375
169 620
12 857
300 820
92 261
187 815
cDNA related protein
family
Amino acid permeases
Stress-related proteins
Amino acid permeases
Phosphatidylinositol 3-kinases
Cytochrome c oxidases
'Only homologies with a p-value better than 7.0e-2 are listed.
intergenic region, counted from the initiation or
the termination codon of the adjacent ORFs, was
found to be 889 bp (intergenic region between
A P L l and L Y P I ) and the minimum length was
167 bp (intergenic region between PIKl and
NOSOO). The average length of an intergenic region
was 383 bp, which is only 66% of the length found
for other intergenic regions (for example 574 bp on
chromosome 11, Feldmann et al., 1994). In summary, the low CAI values found for all ORFs can
be correlated with a high gene density in this
region of chromosome XIV. It remains to be tested
systematically, whether such a correlation can
be found also for other chromosomal locations.
Finally the G + C content of the 24 152 bp long
contig is 38.8%, a value slightly higher than that
found for the other published chromosomes
(Oliver et al., 1992; Dujon et al., 1994; Johnston
et al., 1994; Feldmann et al., 1994). For example,
for chromosome 11, the overall G + C content is
38.3%, while the G + C content of only the coding
regions is 39.6% (Feldmann et al., 1994). So the
higher value for our contig may be due to its high
gene density.
Analysis of ORF products
The location and orientation of the 13 ORFs on
the 24 152 bp contig, the CAI value as well as the
length and molecular weight of the deduced proteins are listed in Table 1. In addition, the results
from the FASTA analysis and the presence of
specific motifs in the putative proteins are summarized in Table 1. Finally, the amino acid sequence
of all ORFs was used to search the EST database
and EST homologues found for an ORF are listed
in Table 2 (XREF database, Boguski et al., 1994).
NO646 This ORF is identical to the first 4659 bp
of BNIl which was identified as a synthetic lethal
to CDC12 (Fares and Pringle, unpublished 1994;
GenBank Accession Number S48523). Mutant
forms of CDC12 show defects in cytokinesis and
formation of microfilament rings (Hartwell, 1971;
Byers and Goetsch, 1976). A comparison of NO646
and BNIl showed two base pair changes in
the ORF leading to two different amino acids.
Database searches revealed no further motifs to
be present in BNIl and only a weak homology
(p-value of 3.le-2) to an EST cDNA could be
detected.
NO660 This gene is identical to A P L l , which
encodes a putative permease for basic amino acids
(Sychrova and Chevallier, 1994; EMBL Accession
Number X74069). Comparison of the A P Ll sequence with NO660 revealed 12 base pair changes
and a deletion of three base pairs in our sequence.
The 12 base pair changes gave rise to four amino
acid variations between APLI and NO660 and
eight silent changes. One amino acid change at
position 517 is in a region not conserved among
known amino acid permeases L Y P l , A P L l , CANl
and GAPl (Figure 2). This may indicate that the
changes are due to strain differences rather than
sequencing errors. Two other changes are in regions (positions 260 and 548) where the permeases
are conserved and both exchanges fit the consensus
sequence better than the published A P Ll sequence
does. One exchange at position 126 within the
amino acid permeases signature changes an amino
acid which fits the consensus derived from the four
permeases L Y P I , A P L l , CANl and G A P l . However, this amino acid change is in a region not well
510
M. SEN-GUPTA ET AL.
ALPl
NO660
LYPl
NO790
M G R F S N I I T S N K W D E K Q N N I G E Q S M Q E L
M G R F S N I I T S N K W D E K Q N N I G E Q S M Q E L
CANl
GAPl
12857/2-243
128571242-304
ALPl
NO660
LYPl
NO790
CANl
GAPl
12857/2-243
ALPl
NO660
LYPl
NO790
CANl
GAP1
1285712-243
_ _ _ _ _ _ _ _ _ _ _ _ - _ _ - _ _ - _ _ _ _ _
T
ALP1
NO660
LYPl
NO790
F A Q R F L S P
T
T
T
1S S F T V
CANl
S
GAPl
TacdT
1285712-243
_ _ _ _ _
- - - - - - - -
- - - - - - - -
ALPl
NO660
LYPl
NO790
CANl
GAPl
1285712-243
ALPl
NO660
LYPl
NO790
P I G F R YW
P I G F R YW
P I G F R YW
W
R
R
R
R
N
N
N
N
P
P
P
P
G
G
G
G
A
A
A
A
W
W
W
W
G
G
G
G
P
P
P
P
G R P L G W V S S L
G R P L G W V S S L
CANl
GAP1
12857/2-243
12857/242-304
conserved in the amino acid permeases signature
(see Figure 2 legend). An additional lysine codon
was found in the published A P L l sequence. This
change was observed in the N-terminus of the
protein. In the APLl published sequence three
identical lysine codons were present at amino acid
position 91 to 93 while in our NO660 sequence only
two lysine codons could be found. As this sequence
variation is located at the N-terminus of the protein, it does not affect the homology regions to
other amino acid permeases. Thus the product of
F V G G - P Q
the NO660 gene sequenced here is 573 amino acids
long and one amino acid shorter than the
published Apllp sequence (574 aa).
The FASTA analysis revealed a high homology
of A P L l to ORF N0790, which is located upstream of Apll and which is identical to the L YPl
gene encoding a lysine-specific permease (see
below, Figure 2). The A P L l and L Y P l DNA
sequences show high homology to a gene on
chromosome V, CANl (Ahmad and Bussey, 1986),
and the general amino acid permease GAPl on
51 1
24 152 bp SEGMENT FROM CHROMOSOME XIV
ALPl
NO660
LYPl
I N A A F T Y Q G T E L V G I T A G E A A N P R K A L P R A I K K V V
I N A A F T Y Q G T E L V G I T A G E A A N P R K A L P R A I K K V V
P R K S V P R A I K K V V
P RK S V P m Q M F
IIG
ALPl
NO660
LYPl
NO790
IT A GE
AIK D B E ~ I K -
F Y I L S L F F I G L L V P Y N D
F Y I L S L F F I G L L V P Y N D
CANl
GAPl
1285712-243
128571242-304
L M I G L L V P Y N D
ALPl
NO660
LYPl
NO790
CAM
GAPl
1285712-243
128571242-304
ALPl
LYPl
NO790
GAPl
1285712-243
128571242-304
GAPl
1285712-243
128571242-304
ALPl
NO660
LYPl
NO790
CANl
GAP1
1285712-243
128571242-304
ALPl
NO660
LYPl
NO790
CANl
GAPl
1285712-243
128571242-304
ALPl
NO660
LYPl
NO790
CANl
GAPl
1285712-243
128571242-304
rn
M W E D
E P K N - - - - - - - - - - - _ _ _ _ _ _ _ _ _ _ _ -FWD FWNVVA
V W E D H E P K
L L K Q E I A E E K A I M A T K P R W Y R I W - N F W C
Figure 2. (Continued)
Figure 2. Alignment of the amino acid sequences of NO660 and NO7990 with the Apll,
Lypl, Can1 and Gap1 proteins and a sequence derived from the EST database with the
NCBI-ID 12857. Amino acids identical to the consensus sequence are boxed. Amino acids
that differ between N0660p and Apll or between N0790p and Lypl are shaded. The match
to the amino acid permeases signature is indicated. The signature is defined as: [STAGCI-
G-P-X(~,~)-[LIVMFYW](~)-X-[LIVMFYW]-X-[LIVMFSTA](~)-[SAG]-X(~)-[LIVMFYW]x-[LIVMS]-x(3)-[LMC]-[GA]-E-x(5)-[PS]
(taken from PROSITE). The numbers to the
right of the NCBI-ID of the cDNA indicate the translated bases. A possible frameshift in
the sequence is present at position 242.
chromosome XI (Jauniaux and Grenson, 1990;
Figure 2). In our search for human cDNA
homologues, significant homologies to peptide
sequences derived from several cDNAs were
found, all of which showed a high similarity to
amino acid permeases or amino acid transporters
512
NO795
67354/150-284
79696/1-240
216995/10-258
30082012-208
M. SEN-GUPTA ET AL.
LLVETITN
- --- --
A M S V H S I K K A L T K K M I E D A E L D D K G G I A S L N D H F L R A F G N P N G F K Y R R AODNa900
44
60
V V ~ V [ S 1 I ~ Q V -~ -X - X- - - - - - - - - - - -
_ _ _ _ _ _ _ _ - - - - _ _ _ _ _
PIK-Slgnaiure B
NO795
~ A ~ S L A A Y S V I C Y L L O V K D R H N G ~ I M I D N E G H V S H I D P G F M L S N S P G S V G F E A A P F K L T Y ~ 9 6 0
I
44
673541150-284
60
83
69
~~
Figure 3. Alignment of the amino acid sequence of NO795 with sequences derived from four different cDNAs (NCBI-IDS: 67354,
79696, 216995 and 300820) of the EST database. Amino acids identical to N0795p are boxed. The match to the two
and
phosphatidylinositol 3- and 4-kinases signatures, which are [LIVMFA]-K-~(~)-[DE](~)-[LIVM]-R-Q-[DE]-X(~)-[LIVMFY]-Q
S-~-A-~(~)-[LIVM]-~(~)-[FY]-[LIVM](~)-~-[LIVM]-X-D-R-H-X(~)-N
(taken from PROSITE), respectively, is indicated.
from different organisms (Table 2). The high
homology between the protein sequences derived
from A P L l , LYP1, CAN1 and GAP1 and the
cDNAs is demonstrated in Figure 2.
NO790 This ORF is identical to L Y P l coding
for a lysine-specific permease (Sychrova and
Chevallier, 1993; EMBL Accession Number
X67315). Compared to the LYPI sequence in the
database we observed nine base pair changes
which led to three different amino acids and six
silent changes. None of these changes lie within a
conserved region of the amino acid permeases
(Figure 2).
An homology search revealed strong similarities
to peptide sequences from the EST database
(Table 2). The peptide sequences themselves show
homology to amino acid permeases from different
organisms. In Figure 2, a multiple sequence alignment of Lypl, Apll, Canl, Gap1 and the protein
sequences derived from one cDNA with N0660p
and N0790p is presented. The aligned amino acids
identical to the consensus sequence are boxed. The
homology of the cDNA is not located exclusively
within transmembrane spanning or other known
functional domains.
NO795 This ORF is identical to PZKl, encoding
phosphatidylinositol-4-kinase, which catalyses the
first step in the biosynthesis of phosphatidylinositol4,5-bisphosphate (PtdIns[4,5]P2; Flanagan et al.,
1993; GenBank Accession Number L20220). No
changes in the DNA sequence were observed. In
the EST database, several human cDNAs were
found encoding peptides with highly significant
homologies to Pikl protein (Table 2). The amino
acid alignment revealed that the proteins derived
from two of the cDNAs show homology to one
domain from Pikl, which is found in protein kinases
(Figure 3). Two other cDNAs show very high
homology to a second motif located at the Cterminal end of N0795p. These phosphatidylinositol
3- and 4-kinase signatures seem to be distantly
related to the catalytic domain of protein kinases
(Kunz et al., 1993).
NO800 This ORF codes for a putative protein of
139 amino acids. ORF NO800 overlaps with ORF
NO809 by 382bp on the opposite strand. The
FASTA search revealed weak homologies to
several NADH dehydrogenases from different
organisms (Table 1). The motif search indicated a
sequence with similarity to prokaryotic lipoprotein
attachment sites. Search of the EST database
showed weak homology to a cDNA-derived
peptide, which shares some similarity with a group
of cytochrome c oxidases (Table 2). The CAI value
for NO800 was 0.12. ORF NO800 together with
NO809 was disrupted but no effect on viability or
growth was observed (Giildener and Hegemann,
unpublished).
NO809 This ORF encodes a putative protein of
34.5 kDa. The coding region appears to be interrupted by an intron (base pairs 21-127). The
putative gene product shows a moderate similarity
to NUFl from budding yeast, which functions as a
spacer protein in the spindle pole body (Mirzayan
et al., 1992; Kilmartin et al., 1993; Table 1).
Disruption of NO809 together with NO800 gave
24 152 bp SEGMENT FROM CHROMOSOME XIV'
no detectable altered phenotype (Giildener and
Hegemann, unpublished).
NO815 This hypothetical gene product bears significant similarity to another ORF from chromosome XIV, VEE350p (MIPS confidential database;
Table 1). No further significant homology was
detected by FASTA analysis or comparison to
the EST database. Disruption of NO815 resulted
in a slow growth phenotype, indicating that this
gene most likely is a real gene (Giildener and
Hegemann, unpublished).
NO820 The putative gene product shows moderate similarity to several NADH dehydrogenases
(Table 1). Search of the EST database revealed a
significant homology of 70% in a 48 amino acids
stretch with 41% identity to the translation product of a human cDNA sequence. This cDNA itself
shows n o homologies to other protein families
(Table 2). Disruption of this O R F was lethal,
indicating that NO820 is an essential gene
(Giildener and Hegemann, unpublished).
NO825 This O R F is identical to POL2 encoding
the catalytic subunit of DNA polymerase I1
(Morrison et al., 1990; GenBank Accession
Number M36724). N o sequence differences were
found within the 6666 b p long coding region. It
has been shown previously that Po12 has homologies to polymerases from higher eukaryotes.
ACKNOWLEDGEMENTS
We gratefully acknowledge the contribution of
Katrein Hamberg and Peter Philippsen, who provided the cosmid clones 14-4 and 14-3b. We thank
Susanne Heck for synthesizing oligonucleotides
and Lydia Karpfinger and Karl Kleine (MIPS,
Martinsried, Germany) for their help with the
sequence analysis. This work was supported by the
European Union Programmes BIOTECH I and 11.
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