YEAST VOL. 12: 145-148 (1996)

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0" 0 VII % 0 Yeast Sequencing Reports

Sequence Analysis of a 10 kb DNA Fragment from Yeast Chromosome VII Reveals a Novel Member of the DnaJ Family ESTHER RODRIGUEZ-BELMONTE, ANA M. RODRIGUEZ-TORRES, BELEN TIZON, JOSE L. CADAHIA, ISABi5.L GONZALEZ-SISO, ELVIRA RAMIL, MANUEL BECBRRA, MONICA GONZALEZ-DOMINGUEZ AND ESPERANZA' CERDAN*

Departamento de Biologia Cchlur 1' Molecuhr, Universidad de A Coruca, 15071, Spain

Received 24 June 1995. accepted 5 August 1995

We report the sequence analysis of a 10 kb DNA fragment of Saccharomyces cereiisicrc chromosome VII. This sequence contains four complete open reading frames (ORFs) of greater than 100 amino acids. There are also two incomplete ORFs flanking the extremes: one of these, G2868, is the 5' part of the SCS3 gcne (Hosaka et nl., 1994). ORFs G2853 and G2856 correspond to the genes CEGl, coding for the alfa subunit of the mRNA guanylyl transferase and a 3' gene of unknown function previously sequenced (Shibagaki et ul., 1992). G2864 is identical to SOHl also reported (Fan and Klein. 1994).'The translated sequence of G2861 is similar to the human dnaJ homolog. The nucleotide sequence reported here has been entered in the EMBL Data Library undcr the Accession Number X87252.

KEY WORDS - genome sequencing, ~ r ~ c r h a r o n ~ ~ c e s terevisiae, chromosome VII; CEGl, S O H I , DnaJ, SCS3

INTRODUCTION

The sequence analysis reported here forms part of the European BIOTECH I 1 project to sequence the genome from Sacchrrrot~i~ic,c..c cerevisiae. The se- quence was determined using nested-deletions and double-strand sequencing strategies. This fragment contains the previously sequenced CEGl and SOHl genes and a fragment of the SCS3 gene (Shibagaki et a/., 1992; Fan and Klein, 1994, Hosaka et al., 1994), an open reading frame (OKF) of unknown function and a new member of the yeast DnaJ family.

MATERIALS A N D METHODS

Bacterial straiiu Eschericliiu coli HBlOl and JM109 were used for

subcloning and sequencing work. Bacterial cells

*Corresponding author

were transformed using the procedure of Hanahan and Meselson (1983). Cells werc grown and main- tained on selective media by conventional methods (Sambrook et a/., 1989).

Sequencing strategy

The cosmid clone, pEGH348, was kindly provided by H. Tettelin (DNA coordinator). The sequenced area presented here is shown in Figure 1A.

The contiguous restriction fragments Cld- BarnHI (3149 bp), BanzHI-EcoRI (1432 bp), EcoRI-EcoRI (1663 bp) and EcoRI-EcoRI (5600 bp) Bere subcloned in pSK, deleted by the DNaseI method (Sambrook et a l , 1989) and sequenced on both strands by the method of Sanger et al. (1977). The junctions between contiguous fragments and some gaps not covered by the deletions were

CCC 0749-503X/96/020145-04 1996 by John Wiley & Sons 1 td

146 E. RODRIGUEZ-BELMONTE ET AL.

A left te1omere

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B 7000 4000 6000 8000 1 n n n n

3 1

2

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Figure 1. (A) EcoRI restriction sites in the Saccharomyces cerevisiae DNA insert of cosmid clouc pEGH348. The bar indicates the sequenced region presented here. (B) DNA Strider plot revealing relative positions of ORFs within the contiguous sequence. AUG codons are represented by half-height vertical bars and stop codons by full-height vertical bars in all six reading frames.

sequenced using synthetic primers and overlapping subclones as templates.

Srquen ce an a Iy s is The complete nucleotide sequence was analysed

using DNA-Strider (Marck, 1988), DNASIS- HITACHI and FASTA (Pearson and Lipnian, 1988) software. Additional analyses were performed at MIPS (Martinsrieder Institiit fur Protein Sequenzen).

RESULTS AND DISCUSSION

Sequence analysis The complete nucleotide sequence of the frag-

ment has been entered in the EMBL Data Library under Accession Number X87252. The DNA Strider plot in Figure 1B illustrates the relative positions of the ORFs. ORF G2850 and G2870 are not complete and overlapping with adjacent frag- ments will be necessary in order to complete their analysis. G2853 and G2856 correspond exactly to the previously published sequences of CEGl and a putative gene located 3’ to it (Shibagaki et al., 1992). The sequence of G2861 has significant homology to the DNJ1 human gene (Oh et al., 1993). The sequence of G2864 corresponds to the SOH1 gene (Fan and Klein, 1994). There are two expressed sequence tags which match to the

region 31 52-3244 and to the region 5962-6303 respectively.

Analysis of ORF products G28.53 The product of (32853 corresponds to the inRNA guanylyl transferase (EC 2.7.7.50). The yeast mRNA capping enzyme is composed of two separate chains of 52 (a) and 80 kDa (b), respon- sible for the activities of mRNA guanylyl trans- ferase and RNA 5’-triphosphatase, respectively. The a subunit has 459 amino acid residues and is encoded by the CEGl gene (Shibagaki et al., 1992).

G28.56 In the region corresponding to the G2856 ORF, the sequence reveals one discrepancy (at position 7396) with that previously reported (Shibagaki et al., 1992). This change, the addition of an A, causes an enlargement of the coding region. Translation of G2856 gives the amino acid sequence of a protein of unknown function con- taining two recognizable motifs (Figure 2). Motif I is an ATPIGTP-binding site motif A (P-loop), a glycine-rich region, which is conserved in an appreciable proportion of proteins that bind ATP or GTP, and probably forms a flexible loop, between a beta-strand and an alpha-helix, and interacts with one of the phosphate groups of the nucleotide (Hodgman, 1988; t inder et al., 1989). Motif I1 has the consensus PXEXLKXLV that is

NEW MEMBER OF DnaJ FAMILY 147 G2856 488 AMINO ACIDS 02861 347 AKIN0 ACIDS

H Y K N I Y I C I L Y I Y I N F Y T S R E N l N K R T S T S H V V l H l

R I H L R M S T S H T I G Q R L F T T A R s L Q A A K p A p K G K T Q G H N I Y T H T H I Y I S H I Y I Y I C I Y I C K P X Y F Y I S F L C F P F S K K S S S V S S Y S S A K R V T P G S L Y K N W T N T T H T A Q L Q Q T A V P L A L P I F N F D D I S K T L N K V V S Y S N K Q Y K S L H H C 1 L S R K T L E D E S K 1 K K Q V H R R T T 8 L A T T M p G H E L E D L G S F K K S Q F N E L F Q K P V C L V R E D A T N S F L K K L V S H P V I N P R L N L Y D V L E L P T P L D V H T I Y D D L P Q I K H K Y R T

v K K F 1 l T Q v G Q E P K T V L L S Q A H A Y A v D ~ K Q 1 l l N 1 ~ L A L K Y H P D K H P D N P S I I H K F H L L S T A T N I L T N I D V R

Y P E L F L N G R N D F S Y D D D L K L F I Q p M y L K K L I K K I L K P H Y D R W L I E F L R K T N D I E R N K L I Q K L E E S E S S T I P T A N D P A L L K S I E L S K D Y K F S N A N P K N A S V K P F V T L N K T K N T V L D L L S V M T H P H N R G K L K K A I I D E L S V Q S K V P T T P H P D L L Q I Q R H G E L L H K L K H F N L p Y G D w K H L N T Q I H F T V D N F S K V L T T A Y S A Y R N T E N K Q I Y S L D L Q H G K D Q E N A S Q H P Y Y D C S T L R I V L D N F L Q S N N K S N C L S H L

*** Motif I *.*

L n H D 1 l S G E T K F A N G E S S T I L A I S G Y D R T N K T L P Y R H N Q V F I T L S A N E l Y D I Y F S E R N N Y S K D D S l f I Y T V F L G K I P V D P Y V T R Y H Y E P K F V E L L Q K G N V T E F E V P K L N K Q E V N E L I D Y Y K Q S N V L L D K D I T C K K W E N L ~ D E K Y D T P I T A Q H V P R N W S S G N L I P T V K D I S P L I P L H Y ~ S D F L S G N G N P R E L L K S L V L S H R

**. Motif II .**

Figure 2. Amino acid sequence corresponding to the transla- tion of G2856. ATPIGTP-binding site motif A or P-loop (motif I) and the sequence matching the consensus pattern of substrate carrier proteins involved in energy transfer (motif 11) are in bold letters.

found in different types of substrate carrier pro- teins involved in energy transfer and located in the inner mitochondria1 membrane (Sulliwan et ul., 1991; Colleau et al., 1992).

G2861 The 1041 bp sequence of the G2861 nucleotide sense strand and predicted protein sequence reveal homology with the DnaJ human homolog (Oh et al., 1993) and other members of the dnaJ family (Figure 3). DnaJ is implicated in the heat-shock response (Rowley et al., 1994) and cooperates with DnaK in its chaperone function (Cyr et al., 1994). In the yeast S. cerevisiue, several genes encoding DnaJ homologs have been charac- terized so far: SEC63 (Sadler et al., 1989), SCJl (Blumberg and Silver, 1991), YDJl (Caplan and Douglas, 1991), SISI (Luke et ul., 1991), ZUOl or

F N L E T E L N D D I A H L V S N E P I L L D

Figure 3. Predicted protein sequence of G2861.

MAS5 (Zhang et al., 1992; Atencio and Yaffe, 1992) MDJl (Rowley et al., 1994) and YIB4W (Vos et al., 1995). These genes appear to fulfil different roles in protein transport and heat-shock response. The DnaJ common domain consists of only 70 amino acids, suggesting that it is a single common domain within otherwise dissimilar pro- teins. In the conserved DnaJ domain, G2861 has 37% identical residues with human DnaJ and among the yeast sequences the highest coincidence, 37%, is found with YDJl and the lowest, 27%, with SEC63 and ZUOl (Figure 4).

G2864 The ORF G2864 corresponds to the sequence of SOHl , a gene cloned by complemen- tation of a mutation that suppresses the temperature-sensitive growth of the hprl mutant of S. cerevisiue (Fan and Klein, 1994). The deduced amino acid sequence shows slight homology to DNA- and RNA-directed RNA polymerases.

DNAJ-H

KDJl

YDJl

SISl

SCJl

YI84W

SEC63

zuo1 02861

DNAJ-H

HDJl

Y D J l

SISl

SCJl

Y184W

SEC63

ZUOl

02861

Figure 4. and human DnaJ1.

Alignment of the common domain of the DnaJ family in yeast

148 E. RODRIGUEZ-BELMONTE ET AL.

ACKNOWLEDGEMENTS This work was supported by the Commission of the European Communities under the Biotech I1 program and by CICYT (BI0941333-CE). We are grateful to H. Tettelin for the clone and preliminary information. We thank MIPS, es- pecially K. Kleine, for help with sequence analysis; the Servei of Bioinformatics of the Valencia University for access to the GCG package; and A. Santos from the Servicios Informaticos de Apoyo a la Investigacion, Universidad de A Coruiia. E.R.-B. and M.B. were supported by fellowships from Xunta de Galicia, E.R. by a fellowship from Diputacion de A Coruiia and M.G.-D. has an FPI fellowship from MEC (Spain).

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