analysis of a 26 756 bp segment from the left arm of yeast chromosome iv

YEAST VOL. 12: 1549-1554 (1996)

0 o o o o 0 0 0 IV '0 0 Yeast Sequencing Reports

Analysis of a 26 756 bp Segment from the Left Arm of Yeast Chromosome IV

The nucleotide sequence of a 26.7 kb DNA segment from the left arm of S(ic.c.h(~r.orii~.c.c.s cm-eiiricit~ chromosome IV is presented. An analysis of this segment revealed 11 open reading frames (ORFs) longer than 300 bp and one split gene. These ORFs include the genes encoding the large subunit of RNA polymerase 11, the biotin apo-protein ligase. a n ADP-ribosylation factor (ARF 2), the 'L35'-ribosomal protein, a rho GDP dissociation factor. and the sequence encoding the protein phosphatase 2A. Further sequence analysis revealed a short ORF encoding the ribosomal protein YL4IB. an introii in a 5' untranslated region and an extended homology with another cosmid (X83276) located on the same chromosome. The potential biological relevance of these findings is discussed. The sequence was submitted to the EMBL database under Accession Number X96876.

KEY WORDS - Seic~c~1icir~oiri~~c.c.s cewvisitit,; chromosome IV; BPLl: RPBl; ARF-7; L35; PPH-71: rho GDP dissociation factor; YL4IB; RGT2

INTRODUCTION

Almost the entire sequence of the yeast genome has now been sequenced. These efforts have mostly been undertaken by a consortium of European laboratories engaged i n the framework of the BIOTECH programme of the European Union. As part of this consortium of European laboratories, we sequenced a 26.7 kb region of the left a rm of chromosome IV. This 26 756 bp-long sequence was derived from the 32412 bp long insert of cosniid 21 12 and revealed 12 open reading frames (ORFs) and one split gene.

MATERIALS A N D METHODS

Pr.epcir.citiori of ,diotgiiii 1ihrii;ix. D N A prt>pur.titiori rind st~yiitwitrg

The cosmid 21 12. provided by Claude Jacq (Ecole Norniale Superieure, Paris, France) contained a fragment of 32 412 bp from the left arm of chromo-

*Corresponding authoi

some IV of Sucelzar.oiii~?ces cerevisirie and was used for the sequencing work presented in this paper. The cosmid DNA was purified by alkaline lysis followed by centrifugation in isopycnic caesium chloride/ ethidium bromide gradients for shotgun library preparation. The purified DNA was fragmented into segments of 800-1000bp length using a nebulizer (T. Pohl. Konstanz. Germany). These segments were ligated into the SiiiaI site of pTZl8r and used to transform the E.rcher.ichin coli strain KK2186. Approximately 2000 clones were pre- pared, out of which 1500 were selected for sequencing. Sequences were obtained by cycle sequencing according to the SequiTerm'" protocol. Approxi- mately 250 clones have been analysed manually using the GATC system and the other clones have been analysed on a Li-Cor sequencing system.

Sepence h t ( i assenihlJ~ and conipiiter. sepence uncllJ~.Si.s

The sequence data assembly was performed using the DNA,'3', SeqMan Software

CCC 0719-.503X/96/15 I549--06 # ( ' # 1996 by Joliti Wiley & Sons Ltd

1550

(DNASTAR, Inc.). Computer sequence analysis and comparisons of nucleotide and amino acid sequences were performed using the MacMolly ’ software package (SoftGene, Berlin), online FastA (Pearson and Lipman, 1988) and BLAST (Altschul et ( I / . , 1990) services at NCBI or EBI, and at MIPS (Martinsried Institute of Protein Sequences, Munich, Germany).

s. WOLFL ET AL.

RESULTS AND DISCUSSION Cosinid 2112 contained a fragment of 32.4 kb from the left arm of chromosome IV of S. cereiisicie. The nucleotide sequence of the insert was deter- mined and the sequence of the 26 756 bp segment presented was analysed as described in Materials and Methods. The average length per sequence was 500 bases and the average number of readings per base was 7.3. A large portion of the ins’ert of cosmid 2112 overlaps with the insert of cosmid 31B9 sequenced by Rieger and colleagues (unpublished).

The ORF-distribution map of the 26 756 kb fragment revealed 11 ORFs longer than 300 bp. One ORF generated by splicing and one ORF 78 nucleotides long. both encoding ribosomal proteins, were found by sequence comparison (Table 1) .

The ORFs D2140, D2160, D2165, D2170 (spliced). D2185, D2190 and D2195 were located within the plus strand and frames D2145, D2150, D2155, D2175, D2180 and the 78 bp ORF in the minus strand. The sequences of seven ORFs were identical to genes accessible in public databases (Table 1): D2140 from position 246 to 2315, encoding biotin apo-protein ligase (BPL1: Cronan and Wallace, 1995): D2 150 (position 7768-2570) codes for the largest subunit of RNA polymerase I1 (RP021; Allison et al., 1985); D2165 (position 13735-1 4277) encodes an ADP-ribosylation factor (Stearns et al., 1990); D2170 (position 14806- 15570; intron position 14809-1 5213) encoding the ’L35’ ribosomal protein (Song and Arndt, EMBL database: AC: L03328): D2175 (position 16494- l5889), the ‘rho GDP dissociation factor’ gene (Masuda er uf., 1994); D2180 (position 17977- 16871), encoding protein phosphatase 2A (PPHZI; Rome et al., 1991); the 78 nucleotide O R F (position 19017-18930) encoding YL41B. an extremely small basic ribosomal protein (Suzuki et al., 1990). Two additional ORFs (Table 1; D2160/D2195) were found to be homologous to known genes. Open reading frame D2160 (position 10558-

12846) is homologous to SNF3 (Celenza et al., 1988) and encodes Rgt2 (Ozcan ec al.. 1996). These genes play an important role in glucose sensing (Ozcan et ul., 1996). Open reading frame D3195 (position 24599-2591 8) shows a strong homology to the homocitrate synthase gene (similarities, see Table 2). In addition, four ORFs were found that could not be assigned to known genes (Table I ; D2145, D2155, D2185, D2190).

The analysis of the DNA segment described in this paper revealed several additional highly interesting features.

RNA yoljmerase II (RPBl ; see D2150) The sequence of the large subunit of the RNA

polymerase I1 gene (RPBI ) showed several mismatches and differences with previously published sequences (Allison et a/., 1985; Nonet el ill.. 1987; Figure 1). On the protein level these changes are confined to the C-terminal domain with its typical heptapetide sequence repeat. Nonet et al. (1987) showed that the number of repeats varies among different yeast strains. The sequence found on our cosmid, from yeast strain FY1679, was identical to that of strain S288C at the protein level. The yeast strain FY1679 is derived from strain S288C. Com- pared with the RPBl sequence from strain A364A (Allison er al., 1985) our sequence contained one additional heptapeptide repeat. At the DNA level, additional mismatches in the flanking sequences were found that do not alter the protein sequence. The domain of heptapeptide repeats (YSYTSPS) was found to be the target for phosphorylation (Peterson et ~ r l . , 1992), important for DNA binding (Blatter et al., 1994) and binding of co-factors (Suzuki et a/., 1990). The length of this peptide repeat varies significantly among species (Allison et al., 1985). In S. cererisiae, 26 or 27 heptapeptide repeats have been reported so far. Deletion of a large fragment leaving only ten heptapeptide repeats is lethal (Nonet et al., 1987).

Spliced genes Yeast has become the model system to study the

nuclear splicing mechanism. However, pre-mRNA splicing seems to be much less frequent in yeast than in other eukaryotes. Splicing is often found close to the 5’ end of the coding sequence in ribosomal genes (Leer et [/ I . , 1984). The gene encoding the ribosomal protein found in cosmid 2112 (Table I , D2170; ‘L35’) contained one intron separating the N-terminal AUG triplet from the

K 0 w P

Tabl

e 1.

B

rief d

escr

iptio

n of

the

OR

Fs o

f th

e an

alys

ed 2

6.7

kb D

NA

seg

men

t. T

he id

entif

icat

ion

and

size

(am

ino

acid

s) o

f th

e 12

OR

Fs a

nd o

ne sp

lit

gene

are

show

n. T

he p

ositi

ons

ofth

e ge

ne re

fer t

o th

e 26

.7 k

b D

NA

frag

men

t. Id

entit

y or

sim

ilarit

y to

kno

wn

gene

s ar

e in

dica

ted

toge

ther

with

acc

essi

on

num

bers

and

ref

eren

ces.

Iden

tific

atio

n A

cces

sion

an

d si

ze

Posi

tion

Gen

e Id

entic

al o

r si

mila

r to

nu

mbe

rs

Ref

eren

ces

D2 1

40 (S

ZC69

0)

D21

45 (

SZF3

02)

D21

50 (

SZE1

733)

D

2155

(SZE

247)

D

2 160

(SZA

763)

D

2165

(SZ

A18

1)

D21

7 (S

ZA12

0i)

D2 1

75 (S

ZF20

2)

D2 1

80 (S

ZE36

9)

78 n

t OR

F (S

ZE25

) D

2185

(SZ

A43

7)

D21

90 (

SZC

S15)

D

2195

(SZ

B44

0)

246-

23 1

5

7768

-257

0 93

25-8

585

1055

8-12

846

1480

6-14

808.

152

14-1

5570

14

809-

1521

3int

ron

1649

4-15

889

1797

7-16

871

1900

7-18

931

1963

3-20

943

2151

0-23

954

2459

9-25

918

3267

-236

2

1373

5-14

277

BP

LI

Bio

tin a

po-p

rote

in li

gase

RP

BI

RP

O2I

: R

NA

-pol

ymer

ase

I1 la

rges

t su

buni

t

RG

T2

AR

F2

‘L35

’ R

ibos

omal

pro

tein

L35

Hom

olog

y to

SN

F3:

glu

cose

tra

nspo

rt

AD

P-ri

bosy

latio

n-fa

ctor

2; G

TP

bin

ding

pro

tein

rho

GD

P d

isso

ciat

ion

fact

or

PP

H2l

Pr

otei

n ph

osph

atas

e 2A

YL

41h

Rib

osom

al p

rote

in Y

L41

U27

182

Cro

nan

er u

l. (1

995)

PO40

50

Alli

son

Ct a

/. (1

985)

Ozc

an e

t d. (1

996)

PI

9146

St

earn

s et

01.

(199

0)

LO23

28

D31

630

Mas

uda

ct 0

1. (1

994)

X

5885

6 R

onne

et

ul. (

1991

) X

1606

6 Su

zuki

et

(71.

(19

90)

Hom

olog

y to

hom

ocitr

ate

synt

hase

1552 s. WOLFL ET AL.

Table 2. Comparison of the six homologous ORFs of cosinid 2112 aiid cosmid X83276 (EMBL database: accession no. X83276; Verhasselt ct d.. 1995). Similarity of the aiiiiiio acid sequences was calculatcd using the MacMolly li software.

2112 X83276

Gene or Gene 01- Similarity: iden tifica tioii Position ideiitifica tion Position total (aligned part)

RGT? 10558-12816 S YF3 11115-13571 48.8'h (59 4Y) -1 RF2 13735-14277 '4 RFI 15658-16203 96.2'>,, (97.2';;)) 'L35' Join 14806-14808 'L35' Join 17001-17003 I00'!:1 D2 I70 and 15214-15570 (D1249) and 17495-17854 PPHJI 17977-16871 P P H 2 24335-3202 8 7');) ( 92.1 ' Y C 8 ) I'L4 1 B 19007-18931 YL411-I 29822-29746 1 OO!A,

D2195 24599-259 18 (D1298) 32711-34060 P6.4'h (97.2" 11)

zy;LsTg 1 S 2 8 8 C 1 A 3 6 4 4 1

FY1579 61 S28BC 51 9 3 6 4 B 61

71'1670 1 2 1

A364A 121

FYl579 191 S298C 181 A Z i 4 . 4 181

s2sec E L

Figure 1. Alignment of heptapeptide repeats from the large subunit of R N A polymerase I1 as found in strain FYI678 with tlie pre\iotisly published sequences of strains S288C (Nonet cf u/.. 1987) and A364A (Allison t'f u/., 1985).

rest of the coding sequence. Surprisingly, an additional intron was predicted using the EXPLORA program (Kalogeropoulos, 1995). However, splicing of this intron does not result in an additional ORF. This intron was located in the 5 ' - untranslated region of the ARF2 gene (Table 1 , D2165). Splicing of an irntranslated leader sequence has been reported previously for tlie COX4 gene (Schneider and Guarente. 1987). Two ex- pressed sequence tags (EST103251; EST102431) were found to correspond to the 5' end of the ARF2 gene. Both EST sequences were derived from spliced niRNA species containing splice sites a t position 13364 ( 5 ' ) and 13695 (3'). The EXPLORA program (Kalogeropoulos, 1995) predicted two potential 5'-splice sites and five potential _?'-splice sites. Only one pair of the calcu- lated splice sites was used by the two published EST-sequences described above.

Database searches revealed that the segment ot chroinosonie IV reported here exhibits extended sequence similarity with another region of the left arm of chromosome IV (cosniid X83376: EMBL database accession no. X83276; Vei-hasselt t't ul.. 1995). The region of sequence relatedness ektends over approxiiiiately 15 kb of our sequence and about 23 kb on the X83276 sequence. and encoin- passes six ORFs. The degree of similarity between cognate pairs of ORFs varied between 1OO':L ('L35' ribosonial proteins) and 49% (ORF D2 160ISNF3). Furthermore the position of the intron i n the two genes encoding ribosomal protein 'L35' is precisely conserved although the length of the two introns is different (this sequence. 405 bp; X83276. 492 bp). However. the two A R F genes differ since the

26 756 bp SEGMENT FROM LEFT ARM OF CHROMOSOME IV

I I J ~ I ~ O I4 I ? S I 8 250 22 375 26 500

1553

- r) -*-.*7 -> <--- . _t_ - DfI95. . -.-. . *

9

0

* I

* * ---

-*

~ R C R : A R E " U S " \, P P I ~ 2 L ~ PlAlR * * - -__ ~. 2112

. . . . ~. . . . . . . % . . . . . * . .~

I > \ I \ I \ I \

- ---- YLl lA "-..'7DL298) *s-< - -- ~ SNF3 ~ ' P K F I " L W .- . ~ 't'PH22 * - * ..*.#.# *< --- + X83276 I I I I 1

IOoOll 151nn) 20 inn) 2s I K X ) ininn) 35 001)

Figure 2. Alignment of homologous open reading frames of cosmid 2112 (top) and cosmid X83276 (bottom) (EMBL database: accession no. X83276: Verhasselt e r d.. 1995). Arrows indicate the position and orientation of open reading frames. Homologies are indicated by dashed lines ;ind the names of homologous genes (bold) are included. Numbers on the axes refer to base positions.

X83276 sequence ( A R F I ) contains no intron in the untranslated 5' region and lacks the conserved TACTAAC element (Keller and Noon, 1984). Alignment of the ARFI sequence with ESTs (EST 104287: EST 102395) confirmed that ARFI is unspliced.

Surprisingly the genes for ARF2 and 'L35' of cosinid 2112. as well as ARFI and 'L35' of cosinid X83267 were not separated by additional ORFs (Figure 2). This might be due to a conservation of signals relevant for processing of the ribosomal gene transcript. Although these genes were local- ized in close proximity on both cosniids, the splicing mechanism was conserved in one case only.

I t is remarkable that a comparison of this DNA region to other yeast sequences revealed no further homologies (Werner Mewes. MIPS. personal com- munication). It is well documented that clusters of homologous genes are found at various locations within the yeast genome (Johnston et nl., 1994; Lalo t'r (11.. 1993; Melnick and Sherman, 1993). In all these cases the homology was confined to ORFs, indicating that the associated regulatory motifs were variable. These differences in the regulatory region may be important to provide individual regulatory mechanisms for the various is o fo r m s .

Homologies I i.itli e.\prcssrtl .serlrience tags A comparison of the sequence of cosmid 2112

with ESTs revealed that several ESTs from yeast were related to ORFs. Therefore, i t was surprising that the human EST HSA45C041 was found to be highly homologous to a sequence mapping to a probably untranslated region. This EST matched almost perfectly with the sequence between the ORFs D2185 and D2190 (see Table I ) . Another human EST (one mismatch in 251 bp) corre- sponded with the 5' end of the rho-GDP- dissociation factor gene (see Table I , D2125). The

human gene encoding the rho-GDP-dissociation factor showed much less similarity. It is likely that these sequences found in databases as human ESTs are derived from contamination by yeast DNA.

ACKNOWLEDGEMENTS

We thank Werner Mewes and Paolo Zaccharias (Munich, Germany) for their help in parts of the sequence analysis and Albert Hiiinen and Heike Laanhardt (Jena, Germany) for helpful discus- sions. We are greatful to Andri. Rosenthal (Jena, Germany) for giving access to his computer and sequence software and Claude Jacq (Paris, France), for providing us with cosmid 2112. Finally, we would like to thank Thomas Pohl from Konstanz. Germany, for his excellent advice and help in preparing the shotgun libraries.

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1554 s. WOLFL ET AL.

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analysis of a 26 756 bp segment from the left arm of yeast chromosome iv

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