yeast carbamyl phosphate synthetase

Yeast Carbamyl Phosphate Synthetase STRUCTURE OF THE YEAST GENE AND HOMOLOGY TO ESCHERICHIA COLI CARBAMYL PHOSPHATE SYNTHETASE*

(Received for publication, July 5, 1983)

C. J. Lusty$, Esther E. Widgren, Karen E. Broglie, and Hiroshi Nyunoyas From the Molecular Genetics Laboratory, The Public Health Research Institute of The City of New York, Inc., Neu: York. New York 10016

A cloned fragment of yeast chromosomal DNA carrying the gene CPAB coding for the large subunit of arginine-specific carbamyl phosphate synthetase has been sequenced. The cloned DNA has a 3,354-nucleotide long continuous reading frame coding for a polypeptide of 1,117 amino acids. The calculated molecular weight of the encoded polypeptide is 123,787, in good agreement with the reported molecular weight of the yeast carbamyl phosphate synthetase large subunit. The amino acid sequence of yeast carbamyl phosphate synthetase is homologous to the recently determined sequence of Escherichia coli carbamyl phosphate synthetase (Nyunoya, H., and Lusty, c. J. (1983) Proc. Natl. Acad. Sei. U. S. A. 80, 4629-4633) over almost the entire length of the protein. Like the E. coli large subunit, the yeast enzyme exhibits an extensive internal homology between its NH2- and carboxyl-terminal halves. The internal homology in both the yeast and E. coli proteins indicates that the gene coding for the large subunit of carbamyl phosphate synthetase was derived from a tandem duplication which occurred prior to the divergence of eukaryotes and prokaryotes.

Carbamyl phosphate is the first precursor of both the arginine and pyrimidine biosynthetic pathways. In Esche- richia coli and other bacteria (1, 2), carbamyl phosphate is synthesized from glutamine, bicarbonate, and two molecules of ATP by a single enzyme, carbamyl phosphate synthetase. Enzyme activity is regulated allosterically by intermediates of both pathways (1-3). The prokaryotic enzyme is composed of two different subunits. The smaller subunit (-42 kDa) catalyzes the hydrolysis of glutamine (4), and the larger subunit ( - 130 kDa) catalyzes the formation of carbamyl phosphate from NH:{, bicarbonate, and ATP (4). In fungi, including Saccharomyces cerevisiae (5) and Neurospora crassa (6), as well as in higher eukaryotes (7), carbamyl phosphate for arginine and pyrimidine biosynthesis is synthesized by two separate enzymes, each enzyme being pathway-specific and separately regulated (1, 7, 8).

In S. cerevisiae, the pyrimidine-specific enzyme is part of a polyfunctional protein, localized in the nucleus (9). The ar-

* These studies were supported by Grant GM 25846 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed. 5 On leave from the Department of Biology, Okayama University,

Okayama, .Japan.

ginine-specific enzyme is generally a mitochondrial constitu- ent, although in S. cereuisiae, the enzyme is located in the cytoplasm (10). Arginine-specific carbamyl phosphate synthetase catalyzes the same overall reaction and has the same subunit structure as the prokaryotic enzyme (11). The small subunit (36 kDa) hydrolyzes glutamine, and the large subunit (130 kDa) can utilize NH:+ as amino donor for carbamyl phosphate synthesis from bicarbonate and ATP (11). The two subunits are encoded by two unlinked genes, CPAl and CPAB, respectively (5).

The yeast gene, CPA2, coding for the large subunit of arginine-specific carbamyl phosphate synthetase has been cloned in yeast and E. coli (12). The cloned gene has been shown to complement yeast strains carrying mutations in the structural gene of the large subunit (12). The genes carAB coding for the small and large subunits of carbamyl phosphate synthetase of E. coli have also been cloned (13, 14), and the nucleotide sequence of carB coding for the large subunit has recently been determined (15). The E. coli large subunit is 1072 amino acid residues in length, and has a calculated molecular weight of 117,710. An internal homology between the NH,-terminal and carboxyl-terminal halves of the polypeptide indicates that carB was formed from a gene half the size of carB (15). The availability of the derived amino acid sequence of the E. coli large subunit allows us to establish the relation of the yeast and prokaryotic carbamyl phosphate synthetases through comparison of their amino acid sequences.

In this report, we present the complete nucleotide sequence of the CPAB gene, coding for the large subunit of arginine- specific carbamyl phosphate synthetase of yeast. The DNA sequence codes for a polypeptide of 1117 amino acids with a calculated molecular weight of 123,787. Comparison of the derived amino acid sequence of the yeast arginine-specific large subunit with the amino acid sequence of the large subunit of the E. coli carbamyl phosphate synthetase shows extensive homology between the two proteins. Internal homology in the primary structure of the yeast enzyme is similar to that previously observed in the E. coli enzyme. The amino acid sequence homology indicates that the two enzymes are evolutionarily related, and further suggests that the genes coding for the two proteins evolved from a common ancestral gene.

MATERIALS AND METHODS

JL2 ( a leu2-3 leu2-112 ura2-2 cpa2-3) and LL2 (a leu2-3 ku2-112) Yeast and Bacterial Strains and DNA Preparation-Yeast strains

and E. coli K12 strain RR1 (pro- leu- thi- lacy- hsdR- endA- rpsL20 ara-14 galKP xyl-5 mtl-1 supE44) carrying the recombinant plasmids pJL2/T3 and pJL2/T5 with the yeast gene CPAP have been previously described (12). The recombinant plasmids consist of the chi-

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Yeast Carbamyl Phosphate Synthetase 14467

meric vector YEpl3 (16) and 5.9-kb' fragments of yeast chromosomal DNA (17) containing the CPA2 gene inserted into the BamHI site of the pBR322 component of the vector (12). RR1 clones carrying the recombinant plasmids pJL2/T3 and pJL2/T5 were grown in LB broth supplemented with 0.1% glucose and 20 pg/ml of ampicillin. Plasmid DNA was isolated by the cleared lysate method (18) and purified by chromatography on Sepharose-GB.

DNA Sequence Determination-The plasmids pJL2/T3 and pJL2/ T5 have inserts with a common sequence of 4.6 kb (cf. Fig. 1). DNA fragments from both plasmids were used to sequence the entire 4.6- kb overlapping region. pJL2/T3 was digested with BamHI which cleaves the plasmid into three fragments of 10.7 (vector), 3.9, and 2.1 kb. The DNA fragments were separated on preparative agarose gels and purified. pJL2/T3 was also cleaved with Hind111 to yield seven fragments of 10.7 (vector), 2, 1.4, 1.2, 0.7, 0.6, and 0.4 kb that were purified on preparative agarose gels. pJL2/T5 was digested with BamHI plus Sal1 to yield fragments of 13.7 and 2.9 kb; the 2.9-kb fragment was isolated as described above.

The purified DNA fragments were cleaved with the appropriate restriction endonucleases and the secondary cleavage products were 5'-end labeled with [r-R2P]ATP (5000 Ci/mmol, Amersham) and polynucleotide kinase (19). The labeled single strands were separated by electrophoresis on polyacrylamide gels, and the DNA sequence was determined by the method of Maxam and Gilbert (19).

S, Nuclease Mapping of Yeast Transcripts-A wild type strain of S. cereuisiae, LL2, was grown to stationary phase in 1% yeast extract, 2% peptone, and 2% glucose. The cells were digested with Zymolyase 5000 (Miles Laboratories, Elkhart, IN) a t 3 mg of Zymolyase/g wet weight of cells. The resultant spheroplasts were washed two times with 1.2 M sorbitol and suspended in a 1:l mixture of phenol/ chloroform/isoamyl alcohol (25:24:1) and 50 mM Tris/HCl, pH 7.6, 0.15 M sodium acetate, 3% sodium dodecyl sulfate. The mixture was homogenized for 15 s in a Waring blendor, and centrifuged for 10 min a t 6000 X g. The aqueous phase was re-extracted three times with phenol/chloroform/isoamyl alcohol, adjusted to 0.3 M sodium acetate, and precipitated with 3 volumes of ethanol. The nucleic acids were dissolved in 10 mM Tris/HCI pH 7.6, 1 mM EDTA, 0.1% sodium dodecyl sulfate, and total RNA was precipitated with 2 M LiCl (20). The poly(A)-containing fraction was isolated by chromatography on oligo(dT)-cellulose (21).

The size of the RNA transcripts was estimated by Northern blot hybridization of total yeast RNA after separation on agarose gels containing methylmercuric hydroxide (22). After electrophoresis, RNA was transferred to nitrocellulose and hybridized as described by Thomas (23) with a radioactive probe prepared by nick translation (24) of a 1.2-kb EcoRI-Hind111 fragment contained within the coding sequence of the CPAS gene.

SI nuclease mapping of the 5'-end of the yeast transcripts was performed as described by Berk and Sharp (25). 5'-end labeled single- stranded fragments of DNA extending beyond the anticipated transcriptional start site of the CPA2 gene (for details, see the legend to Fig. 7) were hybridized with 200 pg of total yeast RNA. RNA-DNA hybridization was carried out in a total volume of 50 pl of 40% formamide containing 5 X SSC and 1 mM EDTA for 3 h a t 45 "C. The annealed samples were diluted 1:10 into SI nuclease buffer (25) and treated with different amounts of S, nuclease (60-500 units/ml). After incubation for 30 min at 37 "C, DNA was recovered by ethanol precipitation. The RNA-DNA hybrids were denatured in formami& and the DNA was separated on a sequencing gel adjacent to the chemically derivatized DNA probe.

RESULTS

Nucleotide Sequence of the CPA2 Gene-The nucleotide sequence of the CPA2 gene was derived from the recombinant plasmids pJL2/T3 and pJL2/T5. These plasmids consist of the chimeric vector YEpl3 (16) with 5.9-kb fragments of yeast chromosomal DNA carrying the CPA2 gene (12). As shown in Fig. I , the DNA inserts of the two plasmids have an overlapping region of 4.6 kb with common Hind111 sites. Since both pJL2/T3 and pJL2/T5 have been shown to complement yeast strains with mutations in the CPAS structural gene (12), it is assumed that the CPA2 gene lies within the overlapping

' The abbreviations used are: kb, kilobase pairs or kilobases in the

~ ~" ~"

case of RNA; bp, base pairs.

pJL2/T5/

E a m H l , + "+

E c o R I +4 "

+ Mno I

t

A

Rsa I t -+

A c c I 4 4

I 1000 2 0 0 0 3000 -

N U C L E O T I D E S

FIG. 1. Restriction map of the DNA insert carrying the CPAB gene in the recombinant plasmids, pJL2/T3 and pJL2/ T5. Yeast chromosomal DNA, approximately 5.9-kb long, is indicated by the thin line; the YEp13 vector is shown as the open bar. The seqtlencing strategy is illustrated in the schematic drawing below. The arrows above the restriction sites indicate the direction and the lengths of the sequences obtained. The limits of the CPA2 gene within the sequenced region are indicated by the open bar above the restriction map. The large arrow indicates the direction of transcription.

region. The entire 4.6-kb overlap region was sequenced. The DNA fragments used for sequence analysis were obtained by preparative digestions as described under "Materials and Methods." The purified fragments were used for secondary cleavages using the restriction sites shown in Fig. 1. More than 90% of the DNA sequence was confirmed from the complementary strands. All of the labeled sites were crossed with a second set of overlapping fragments. The nucleotide sequence of the 4.6-kb overlapping region of pJL2/T3 and pJL2/T5 is shown in Fig. 2. The nucleotide sequence has a continuous reading frame of 3354 nucleotides. None of the other five possible reading frames codes for a polypeptide of any significant length (>lo0 amino acids). The 3354-nucleotide long coding sequence begins with an ATG initiation codon at nucleotide +1, and ends with a TGA termination signal at nucleotide +3355, followed by a second TAA termination codon at nucleotide +3383.

Characterization of the CPAB Structural Gene-The 3354- nucleotide long reading frame (excluding the ATG initiation codon) encodes a protein with a molecular weight of 123,787, a size consistent with the value of 130,000 previously reported for the large subunit of yeast carbamyl phosphate synthetase based on gel filtration (11). In view of its lability and low abundance, the yeast enzyme has not been purified (ll), and consequently, no protein structure data have been reported. In order to confirm the correct assignment of the gene sequence, we have relied on the homology of the protein se-


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14468 Yeast Carbamyl Phosphate Synthetase

CCTGCGGCATTCTATAGAT;~~~GCGAATGACTCTTATTGATGAGATGGCAATAACTTTTGAATATCAGAGATAGGAACCTCCATGTCGTAACGATTGTGTCACCTT - 2 5 0

GAGTAAGCATCGA~~~AATCCAATCTTTTTTTTTCCGTCATAAGCATTTCTGCCATGCTATT;~S~TA~ATATAATA~TAATA~GTCTTCTATAGTATGCCTTATC

T C T T T T ~ ~ ~ A A G C G C T A T T T A A G T T T A A G C A T C G A A A A A ~ T A A ~ A T ~ T A T A G T T A A A ~ T T A G T T ~ T A T A A A G G A A G A G ~ A A ~ ~ ~ ~ ~ ~ A ~ A ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ A . L J . J. -5 0 3

- 1

- - - - - - - f l

Thr ser i l e t y r thr ser thr q l u p r o thr a s n ser a l a p h e thr thr g l u a s p t y r l y s p r o g l n l e u v a l g l u g l y ATG ACA TCG ATT TAT ACA TCA ACA GAG CCT ACG AAT TCT GCT TTT ACT ACC GAG GAC TAC AAA CCT CAA TTA GTT GAA GGA

GTA AAT TCT GTA CTT GTC 4TT GGA TCA GGA GGG CTC TCT ATT GGT CAA GCT GGT GAA TTC GAT TAC AGT GGT TCT CAA GCT v a l a s n ser v a l l e u v a l i l e g l y ser g l y g l y l e u ser i l e g l y g l n a l a g l y g l u p h e a s p t y r ser g l y ser g l n a l a

i l e l y s a l a l e u l y s g l u a s p a s n l y s p h e thr i l e l e u v a l a s n p r o a s n i l e a l a thr a s n g l n thr ser h i s ser l e u ATC AAA GCT CTG AAG GAA GAT AAC AAG TTT ACT ATA TTG GTT AAC CCA AAT ATC GCT ACT AAC CAG ACT TCT CAT TCC CTG

a l a a s p l y s i l e t y r t y r l e u p r o v a l thr p r o g l u t y r i l e thr t y r i l e i l e g l u l e u g l u a r g p r o a s p a l a i l e l e u GCG GAC AAG ATT TAT TAC TTG CCC GTT ACA CCA GAA TAC ATC ACA TAT ATC ATT GA! CTT GAA AGG CCG GAT GCT ATA CTT

l e u thr p h e g l y g l y g l n thr g l y l e u a s n c y s g l y v a l a l a l e u a s p g l u ser g l y v a l l e u a l a l y s t y r a s n v a l l y s TTA ACC TTC GGT GGT CAA ACA GGT CTA AAT TGT GGG GTG GCT CTG GAT GAA TCT GGT GTT TTG GCT AAA TAC AAC GTC AAA

v a l l e u g l y thr p r o i l e l y s thr l e u i l e thr ser g l u a s p a r g a s p l e u p h e a l a ser a l a l e u l y s a s p i l e a s n i l e GTT TTA GGT ACT CCT ATC AAA ACT TTG ATC ACT TCT GAA GAT AGG GAT CTT TTC GCA TCT GCG TTA AAG GAT ATC AAC ATT

p r o i l e a l a g l u ser p h e a l a c y s g l u thr val a s p g l u a l a l e u g l u a l a a l a g l u a r g val l y s t y r p r o v a l i l e v a l CCC ATC GCA GAA TCA TTT GCT TGT GAA ACC GTG GAT GAA GCT TTG GAG GCT GCT GAA AGG GTC AAA TAC CCA GTT ATT GTC

a r g ser a l a t y r a l a l e u g l y g l y l e u g l y ser g l y p h e a l a a s n a s n a l a ser g l u m e t l y s g l u l e u a l a a l a g l n ser AGA TCT GCA TAC GCT TTG GGT GGG TTA GGC TCA GGT TTC GCT AAC AAT GCA AGT GAA ATG AAG GAA CTT GCC GCA CAG TCC

l e u ser l e u a l a p r o g l n i l e l e u v a l g l u l y s ser l e u l y s g l y t r p l y s g l u v a l g l u t y r g l u v a l v a l a r g a s p a r g TTG TCG TTG GCC CCA CAA ATT CTT GTT GAA AAA TCT TTG AAA GGT TGG AAA GAA GTT GAA TAT GAA GTG GTC AGA GAT AGG

v a l g l y a s n c y s i l e thr val c y s asn m e t g l u a s n p h e a s p p r o l e u g l y v a l h i s thr g l y a s p ser m e t v a l p h e a l a GTT GGT AAC TGT ATT ACA GTA TGT AAT ATG GAA AAT TTC GAC CCA CTT GGT GTT CAT ACT GGT GAT TCT ATG GTT TTT GCT

CCT TCG CAG ACC CTA TCA GAT GAA GAG TTT CAT ATG TTA AGA TCC GCC GCA ATT AAA ATC ATT AGA CAC CTT GGT GTT ATT p r o ser g l n thr l e u ser a s p g l u g l u p h e h is m e t l e u a r g ser a l a a l a i l e l y s i l e i l e a r q h is l e u g l y v a l i l e

g l y g l u c y s a s n v a l g l n t y r a l a l e u g l n p r o a s p g l y l e u a s p t y r a r g v a l i l e g l u v a l a s n a l a a r g l e u ser a r g GGT GAA TGT AAT GTC CAA TAC GCT TTG CAA CCT GAT GGG CTA GAC TAT AGA GTT ATT GAA GTG AAC GCA CGT TTA TCT CGT

ser ser a l a l e u a l a ser l y s a l a thr g l y t y r p r o l e u a l a t y r thr a l a a l a l y s i l e g l y l e u g l y t y r thr l e u pro TCC TCT GCA TTG GCG TCT AAG GCC ACT $GT TAT CCC TTA GCA TAC ACT GCC GCC AAA ATT GGG CTA GGC TAT ACT TTG CCA

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

6 0 0

7 0 0

8 0 0

9 0 0

1 0 0 0

1 1 0 0

g l u l e u p r o a s n p r o i l e thr l y s thr thr v a l a l a a s n p h e g l u p r o ser l e u a s p t y r i l e v a l a l a l y s i l e p r o l y s GAA TTG CCA AAC CCA ATC ACA AAA ACT ACA GTG GCT AAC TTT GAG CCA TCT TTG GAT TAT ATT GTG GCA AAA ATA CCT AAG

1 2 0 0

t r p a s p l e u ser l y s p h e g l n t y r v a l a s p a r g ser i l e g l y ser ser m e t l y s ser v a l g l y g l u v a l m e t a l a i l e g l y TGG GAT CTT TCT AAG TTC CAA TAC GTG GAC AGA TCC ATT GGT TCC TCT ATG AAA TCA GTT GGA GA! GTT ATG GCT ATT GGT

a r g a s n t y r g l u g l u a l a p h e g l n f y s a l a l e u a r g g l n v a l a s p p r o ser l e u l e u g l y p h e g l n g l y ser thr g l u p h e AGA AAC TAT GAA GAA GCC TTT CAA AAA GCA TTA AGA CAG GTG GAT CCA TCA TTA TTG GGA TTC CAA GGT TCT ACT GAA TTC

1 3 0 0

GGC GAT CAA CTT GAT GAA GCC TTG AGA ACT CCA ACT GAT AGA AGA GTC CTT GCC ATT GGT CAG GCC TTA ATC CAT GAA AAC g l y a s p g l n l e u a s p g l u a l a l e u a r g thr p r o thr a s p a r g a r g v a l l e u a l a i l e g l y g l n a l a l e u i l e h i s g l u a s n

t y r thr v a l g l u a r g v a l a s n g l u l e u ser l y s i l e a s p l y s t r p p h e l e u t y r l y s c y s m e t a s n i l e v a l a s n i l e t y r TAT ACT GTT GAG AGA GTT AAT GAA TTG AGT AAA ATT GAT AAA TGG TTT CTT TAC AAG TGC ATG AAC ATT GTT AAT ATC TAT

1 5 0 0

l y s g l u l e u g l u ser v a l l y s ser l e u ser a s p l e u ser l y s a s p l e u l e u g l n a r y a l a l y s l y s l e u g l y p h e ser a s p AAA GAG CTT GAA TCA GTT AAA TCT TTA AGT GAC TTG AGT AA! GAT CTC TTG CAG AGA GCC AAG AAA TTA GGG TTT TCA GAT

1 6 0 0

l y s g l n i l e a l a v a l thr i l e a s n l y s h i s a l a ser thr a s n i l e a s n g l u l e u q l u i l e a r g ser l e u a r g l y s thr l e u AAG CAG ATT GCG GTT ACT ATA AAT AAA CAC GCC TCC ACA AAC ATT AAC GAA CTG GAA ATC !GA AGT TTA AGA AAA ACG TTA

1 7 0 0

g l y i l e i l e p r o p h e v a l l y s a r g i l e a s p thr l e u a l a a l a g l u p h e p r o a l a g l n thr a s n t y r l e u t y r thr thr t y r GGT ATA ATC CCT TTT GTC AAG AGA ATC GAT ACT TTG GCC GCA GAA TTT CCA GCA CAA ACC AAT TAT TTG TAT ACC ACT TAC

a s n a l a thr f y s a s n a s p v a l g l u p h e a s n g l u asn q l y m e t l e u v a l l e u q l y ser q l y val t y r a r q i l e g l y ser ser AAT GCT ACA AAG AAC GAT GTG GAG TTC AAC GAA AAT GGT ATG CTG GTT TTA GGC TCT GGT GTC TAT CGT ATT GGT TCA TCT


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1 8 0 0

v a l g l u p h e a s p t r p c y s a l a v a l a s n thr a l a l y s thr l e u a r g a s p qln g l y l y s l y s thr i l e met i l e a s n t y r a s n GTA GAA TTT GAT TGG TGT GCC GTG AAC ACC GCG AAG ACA TTA AGA GAT CAA GGC AAG AAA ACT ATC ATG ATA AAT TAT AAC

pro g l u thr v a l ser thr a s p p h e a s p g l u v a l a s p a r q l e u t y r p h e g l u g l u l e u ser t y r g l u a r g v a l m e t a s p i l e CCA GAA ACA GTT TCC ACA GAT TTC GAT GAA GTT GAT AGA TTA TAC TTT GAA GAA TTA TCG TAT GAA AGA GTG ATG GAC ATT

t y r g l u l e u g l u g l n ser g l u g l y c y s i l e i l e ser v a l g l y g l y g l n l e u p r o qln a s n i l e a l a l e u l y s l e u t y r a s p TAT GAG TTG GAG CAA TCT GAG GGT TGC ATT ATT TCT GTC GGT GGT CAA TTA CCT CAA AAC ATT GCC TTG AAA CTT TAC GAT

a s n g l y c y s a s n i l e m e t g l y thr a s n p r o a s n a s p i l e a s p a r q a l a g l u a s n a r q h i s l y s p h e ser ser i l e l e u a s p AAC GGC TGT AAT ATA ATG GGT ACC AAT CCA AAC GAT ATT GAT AGA GCT GAG AAC AGA CAC AAA TTC TCA TCT AT? TTG GAT

TCT ATT GAT GTT GAC CAA CCT GAA TGG AGT GAA TTA ACA TCA GTA GAA GAA GCA AAA TTA TTT GCT TCT AAA GTT AAC TAC ser i l e a s p v a l a s p g l n p r o q l u t r p ser g l u l e u thr ser v a l g l u g l u a l a l y s l e u p h e a l a ser l y s v a l a s n t y r

pro v a l l e u i l e a r g p r o ser t y r v a l l e u ser g l y a l a a l a m e t ser v a l v a l a s n a s n g l u g l u g l u l e u l y s a l a l y s CCT GTG TTG ATT CGT C C C TCA TAT GTT CTT TCC GGT GCG GCA ATG AGT GTT GTT AAT AAT GAG GAG GAA CTG AAG GCT AAA

l e u thr l e u a l a ser a s p v a l ser p r o a s p h is p r o v a l v a l m e t ser l y s p h e i l e g l u g l y a l a g l n g l u i l e a s p v a l TTA ACT TTG GCA TCT GAC GTT TCT CCA GAC C t T CCA GTC GTC ATG TCT AAA TTT ATT GAA GGT GCT CAA GAA ATT GAT GTG

a s p a l a v a l a l a t y r a s n q l y a s n v a l l e u v a l his a l a i l e ser q l u h i s v a l q f u a s n a l a g l y v a l h i s ser g l y a s p GAC GCC GTT GCT TAT AAT GGT AAT GTC TTG GTA CAT GCC ATT TCC GAG CAT GTT GAA AAT GCG GGT GTG CAC TCC GGT GAT

a l a ser l e u v a l l e u p r o p r o g l n h is l e u ser a s p a s p v a l l y s i l e a l a l e u l y s a s p i l e a l a a s p l y s V a l a l a l y s GCT TCT TTA GTC TTA CCG CCA CAA CAT CTT TCT GAC GAT GTG AAG ATT GCC CTA AAA GAC ATT GCT GAT AAG GTC GCA AAA

a l a t r p l y s i l e thr g l y p r o p h e a s n m e t qln i l e i l e l y s a s p g l y g l u h i s t h r l e u l y s v a l i l e g l u c y s a s n i l e GCT TGG AAG ATC ACT GGC CCC TTC AAT ATG CAA ATC ATC AAG GAT GGG GAG CAT ACA TTG AAA GTG ATT GAA TGT AAC ATT

AGA GCT TCT AGA TCA TTT CCA TTC GTT TCA AAA GTT TTA GGC GTT AAT TTT ATT GAA ATT GCT GTC AAG GCA TTT TTG GGC a r g a l a ser a r q ser p h e p r o p h e v a l ser l y s v a l l e u q l y v a l a s n p h e i l e q l u i l e a l a v a l l y s a l a p h e l e u g l y

g l y a s p i l e v a l p r o l y s p r o v a l a s p l e u m e t l e u a s n l y s l y s t y r a s p t y r v a l a l a thr l y s v a l pro g l n p h e ser GGT GAC ATT GTA CCA AAA CCT GTT GAT TTG ATG CTC AAC AAA AAG TAC GAC TAT GTT GCT ACT AAA GTT CCT CAA TTT TCC

p h e t h r a r g l e u a l a q l y a l a a s p pro p h e l e u g l y v a l g l u m e t a l a ser thr g l y g l u v a l a l a ser p h e q l y a r g a s p TTT ACA AGG TTG GCT GGT GCA GAT CCT TTC TTA GGG GTT GAA ATG $A TCA ACT GGT GAA GTT GCT TCA TTT GGT AGA GAT

l e u i l e g l u ser t y r t r p thr a l a i l e g l n ser thr m e t a s n p h e h i s v a l p r o l e u p r o p r o ser g l y i l e l e u p h e g l y TTA ATT GAA AGC TAT TGG ACT GCT ATT CAA AGT ACC ATG AAC TTC CAT GTA CCA CTA CCT CCA AGT GGT ATA TTA TTT GGA

1 9 0 0

2 0 0 0

2 1 0 0

2 2 0 0

2 3 0 0

2 4 0 0

2 5 0 0

2 6 0 0

2 7 0 0

2 8 0 0

2 9 0 0

GGT GAT ACA TCT CGA GAA TAC TTG GGC CAA GTG GCT TCC ATA GTG GCC ACT ATT GGT TAC AGA ATA TAC ACA ACT AAT GAG g l y a s p thr ser a r g g l u t y r l e u g l y g l n v a l a l a ser i l e v a l a l a thr i l e g l y t y r a r q i l e t y r t h r thr a s n g l u

3 0 0 0 thr thr l y s thr t y r l e u qln g l u h is i l e l y s g l u l y s a s n a l a l y s v a l ser l e u i l e l y s p h e pro l y s a s n a s p l y s ACC ACT AAA ACG TAT CTA CAG GAA CAC ATC AAA GAA AAG AAC GCA AAG GTT TCT TTG ATT AAA TTT CCA AAG AAT GAT AAG

a r q l y s l e u a r g g l u l e u p h e qln q l u t y r a s p i l e l y s a l a v a l p h e a s n l e u a l a ser l y s a r g a l a g l u ser thr a s p AGA AAA TTG CGT GAA CTA TTT CAA GAA TAT GAC ATA AAA GCT GTT TTC AAT TTA GCC TCC AAG AGA GCT GAG AGC ACT GAC

a s p v a l a s p t y r i l e met a r q a r g a s n a l a i l e a s p p h e a l a i l e p r o l e u p h e a s n g l u p r o g l n thr a l a l e u l e u p h e GAT GTT GAC TAT ATT ATG AGA AGG AAT GCT ATT GAT TTT GCT ATC CCA TTG TTC AAT GAA CCT CAA ACG GCT TTG TTA TTT

a l a l y s c y s l e u l y s a l a l y s i l e a l a g l u l y s i l e l y s i l e l e u q l u ser his a s p v a l i l e v a l p r o p r o g l u v a l a r g GCA AAG TGT TTG AAG GCA AAA ATT GCA GAA AAG ATC AAA ATT TTG GAA TCT CAT GAC GTT ATA GTT CCA CCA GAA GTC CGT

ser t r p a s p q l u p h e i l e g l y p h e l y s a l a t y r TCC TGG GAT GAA TTT ATT GGT TTC AAA GCA TAT TGA TTG TGC AAA AGA AAA ACC TGC CTC TAA TTGTATTACTGTATATTATATA

3 1 0 0

3 2 0 0

3 3 0 0

3 4 0 0

3 5 0 0

ATGGATTTTTTTAGAGATATTTTTAATTTCAAAGAGACTTCTTAGAGTATATTCTACCCCAGTATTATTATCTTGAACAAAAGTAGATCGCAAAACTTATCACTAAA

ATTCAAATGTATTTTCGAAGTGAGTAACGCATGGGCCGTGGAAAATAATTCATCAATCAATACCTCAGGCAA~T~TA~TTACTATGCCTTTATATCTTCC~TAT~

- AATAGAGCGTTGATTAATTGAGATGTGCAACAA~GTGACCAGTACACGTAGATTATAGCATTTTTCACCGATC P A

FIG. 2. Nucleotide sequence of the CPA2 gene. The sequence is that of the nontranscribed strand. CPA2 begins at nucleotide +1 and ends at nucleotide +3354. The amino acid sequence is shown above the D N A sequence, and presumes the initiator methionine to be cleaved post-translationally. A TATA box (boned), pyrimidine-rich block (underlined), and the sequence PuTACATA (dashed underline) are indicated in the figure. 1 denotes the transcriptional start sites mapped with SI nuclease. Downstream of the termination codon, two AATAA sequences are underlined. PA represents the putative poly(A) addition sites deduced from the consensus sequence of Bennetzen and Hall (32).


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14470 Yeast Carbamyl Phosphate Synthetase 1

Yeast TSIYTSTEPTNSAFTTEDYKPQLVEGVNSVLVIGSGGLSIGQAGEFDYS~SQAIKALKEDNKFTILVNPNIATNQTSHSLADKIYYLPVTPEYITYIIE~

E. coli

5 0 1 0 0

I I I I I l l 1 I I I I I I I1 I l l I I l l 1 I I1 I I I 1 I I I l l _ _ _ PKRTDIKSILILGAGPIVIGQACEFDYSGAQACKALREEGYRVILVNSN~ATIMTDPEMADATYIEPIH~WRKIIEK

1

1 5 0 5 0

? o n

ERPDAILLTFGGQTGLNCGVALDESGVLAK~GTPIKTLITSED~LFASAL~INIPIAESFACETVDEALEAAER~YPVIVRSAYALGGLGS~ l l l I l I l l l I I / l l I I l l I I I I I I I I I I 1 I I I I I I I I I I l l 1

" _

ERPDAVLPTMGGQTALNCALELERQGVLEEFGVTMIGATADAIDKAEDRRRFDVAMKKIGLETARSGIAHTMEEALAVAADVGFPCIIRPSFTMGGSGGG , . , ,

100 I 5 0 2 5 0

FANNASEMKELAAQSLSLAPQI LVEKSLKGWKEVEYEWRDRVGNCITV~N~NFDPLGVHTGDSM~APSQTLSDEEFHMLRSAAIKIIRHLGVI G I I I I I I I I I I 1 I l l / I I I I I I I l l I I I I I I I I I I I I I 1 I l l I I I I I I I I IAYNREEFEEICARGLDLSPTKELLIDESLIGWKEYEMEWRDKNDNCIIVCSIENFDAMGIHTGDSITVAPAQTLTDKEYQIMRNASMAVLREIGVETG , , .

3 0 0 2 0 0

3 50 2 5 0

EC~QYALQPDGLDYRVIEVA~SRSSALASKATGYPLAYTAAKIGLGYTL~ELPNPITKT TVANFEPSLDYIVAKIPKWDLSKFQYVDRSIGSSMKS I l l I I I l l I I I I I I I I I I I I I I I I I l l I I I I I I I I 1 I I I l l 1 I I I I l l I I I l l

GSNVQFAVNPKNGRLIVIEMNPRVSRSSALASKATGFPIAKVAAKLAVGYTLDELMNDITGGRTPASFEPSIDnrVTKIPRFNFEKFAGANDRLTTQMKS 3 0 0 3 5 0

4 0 0 4 5 0

VGE~AIGRNYEEAFQKALRQVDPSLLGFQGSTEFGDQLDEALRTPTDRRVL A~GQALIHENYT I l I I I I 1 I I I I i l I I I1 I I l l I1 I I I I I I I / I 1 1 I

VERVNELSKIDKWFLYKCMNIVNIYKEL

VGEVMAIGRTQQESLQKALR$LEVGATGFDPKVSLDDP EALTKI RRELKDAGADRIWYIADAFRGLSVD$VFNLTNIDRhTLVQIEELVRLEEKV

5:O 4 0 0

5 5 0 4 5 0

ESVKSLSDLSKDLLQRAKKLGFSDKQIAVTINKHASTNINELEIRSLRKTLGIIPFVKRID~LAAEFPAQTNYLYTTYNATKNDVEFNENGML VLGSGV I I I I I I I I I I I I I I I I I I 1 I I ( I l l I I I I I I l l I

AEVGITGLNADFLRQLKRK GFADAPA KLAGVR EAEIRKLRDQYDLHPVYKRVDTCAEFATDTAYMYSTYEEEFEANPSTDREKIMVLGGGP 5 0 0

6 0 0 6 5 0

5 5 0

YRIGSSVEFDWCI;VNTAKTLRDQGKKTIMINYNPETVSTDFDEVD~YFEELSYERVMDIYE~EQSEGCIISVGGQLPQNIALKLYDNGCNIMGTNPNDI I l l I l l i I I I I I l l I I I I I I I I I I I I I I I I I I I I I I I l l I I I I I I I I

NRIGQGIEFDYCCVHASLALREDGYETIMVN~NPETVSTDYDTSDRLYFEPVTLEDVLEIVRIEKPKGVIVQYGGQTPLKLA~LEAAGVPVIGTSPDAI 6 0 0 6 5 0

7 p O

DRAENRHKFSSILDSIDVDQPEWSELTSVEEAKLFASKVNYPVLIRPSYVLIRPSYVLSGA~S~NEEELKAKLTLASDVSPDHP~SKFIEGAQEIDVDAVAY 7 5 0

I l l 1 I I I 1 I I I I I I I I I I I I I / I I I I I I I I I 1 I I I I l l 1 DRAEDRERFQHAVERLKLKQPANATVTAIEMAVEKAKEIGYPLWRPSWLGGRAMEIVYDEADLRRYFQTAVSVSNDAPV4LDHFLDHFLDDAVEVDVDAICD

7 0 0 7 5 0

8 0 0 NGNVLVHAISEHVENAGVHSGDASL~PPOHLSDD~IALKDIADKVA~~ITGPFNMQIIKDGEHTLKVIECNIRASRSFPFVSKVLGVNFIEIA~

s > o

I I I I I I I I I I I I I I / I I I I I I 1 I I I I I I I I I I I I I I GEMVLIGGIMEHIEQAGVHSGDSACSLPAYT~SQEIQDVMROQVQKLAFELQVRGLVQFAVKNNEVYL IEVNPRAARTVPFVSKATGVPLAKVAARV

B O O 8 5 0

FLGGDIVPKPVDLMLNKKYDWATKVPQFSFTRLAGADPFLGVE~STGEVASFGRDLIESYWTAIQ STMNFHVPLPPSGILFGGDTSREYLGQVAS 9 0 0 9 5 0

I I I I I I I I I I 1 1 I i I I I I 1 I I I l l I I I I I I MAGKSLAEQGVTKEVIPP Y Y S V K E W L P F N K F ~ G V D P L L G P E M R S T G E V M G V G R T F A E GDKERWDLA AK

9 0 0 9 5 0

IVATIGYRIYTTNETTKTYLQEHIKEKNAKVSLIKFPKNDKRKLRELFQEYDIKAVFNLASKRAESTDDVDYIMRRNAIDFAIPLFNEPQTALLFAKCLK 1 0.0 0 10.5 0

I I I I I I I I I I 1 1 I 1 / 1 1 I I LLKQ GFELDATHG TAIVLGEAGI NPRLVNKVHEGRPHIQDRIKNGEYTYII NTTSGRPAIE DSRVI RRSALQYKVHYDTTLNGGFATtMALN

l o o 0 1 0 5 0

AKIAEK IKILESHDVIVPPEVRSWDEFIGFKAY-COOH 1 l.0 0

I I l l 1 1 I ADATEKVISVQEMHAQIK-COOH

FIG. 3. Homology of the derived amino acid sequences of the large subunit of carbamyl phosphate synthetase of yeast and E. coli. The two sequences have been aligned for maximal homology. Identical amino acids are indicated by the vertical lines. Gaps in the sequences represent insertions/deletions.

quences encoded in the yeast gene and the carB gene of E. coli (15). The deduced amino acid sequences of the yeast and bacterial proteins are presented in Fig. 3. A comparison of the two sequences indicates an overall homology of 39.7% based on identical amino acid residues, and 64.3% if conservative replacements are included in the calculation. Of particular significance is the extensive homology at the NH2-terminal end showing a clustering of conserved amino acids up to almost the NH2-terminus of the E. coli protein. The reading frame in the yeast DNA, however, extends for an additional 63 nucleotides before reaching the proposed methionine initiator codon. An in-frame TAA termination codon occurs 51

nucleotides upstream of this initiator. The DNA sequence in this region has been confirmed several times from complementary strands, leading us to believe that the ATG codon at nucleotide +1 is the start of the gene. As discussed in a subsequent section, the assignment of the ATG at nucleotide +1 is consistent with the results of S1 mapping of the yeast transcripts. Although the homology in the carboxyl-terminal regions of the two proteins is less extensive, it is nonetheless significant, based on both identities and alignment of the yeast and E. coli sequences by the ALIGN (26) computer program.

Internal Homology of the Large Subunit of Yeast Carbamyl


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Phosphate Synthetase-In previous studies (15), we noted that the large subunit of E. coli carbamyl phosphate synthetase had an internal homology encompassing almost the entire two halves of the protein. These results strongly indicated the carB gene to have arisen from a duplication event (15). A computer analysis of the protein sequence encoded in the yeast CPA2 gene revealed a similar internal duplication. The dot matrix (27-29) comparing the amino acid sequences of the two proteins is presented in Fig. 4. Three lines of homology

200 400 600 800 1000

E COLI CPS

FIG. 4. Homology between the amino acid sequences of the large subunits of yeast and E. coli shown by dot matrix analysis. The center line represents the homologies between the yeast and E. coli proteins. The shorter lines above and below the main line represent reciprocal internal homologies between the yeast and E. coli sequences.

are evident in this matrix. The central line represents the homology of the primary sequences of the E. coli and yeast proteins. The two shorter lines lying above and below the central line denote the interspecies homologies between the NH,- and carboxyl-terminal halves of the E. coli and yeast sequences. The extent of the internal homology in the yeast sequence is shown in Fig. 5, where the two halves of the protein have been aligned to maximize identities and conservative replacements. The two halves (residues 1-549 and residues 550-1117) have 28.5% identical residues and an additional 27.2% conservative replacements. The alignment shown in Fig. 5 includes only ten small deletions/insertions in the entire sequence. The internal homology was confirmed by computer analysis. The probability that the two sequences are unrelated is <lo-””. These data indicate that the yeast gene, like the carB gene of E. coli, has an internal duplication.

S , Mapping of the CPA2 Transcripts-The size of the yeast carbamyl phosphate synthetase transcript was estimated by electrophoresis on agarose gels containing methylmercuric hydroxide. Total yeast RNA and the poly(A)-containing fraction from wild type S. cereuisiae LL2 were denatured in 10 mM methylmercuric hydroxide and separated on 1% agarose, and the RNA was blotted onto nitrocellulose as described under “Materials and Methods.” Hybridization of the nitrocellulose blot with a nick translated probe from an internal region of the gene revealed a single major radioactive band of 3.7-3.8 kb (Fig. 6). These data indicate that the major transcript is only 300-400 nucleotides longer than the predicted coding sequence of the gene.

To more accurately estimate the 5’ start of the transcript, total wild type RNA was hybridized to a 5’-end labeled single- stranded DNA fragment containing the sequence from nucleotides +9 to -73 (cf. Fig. 2). Following SI nuclease diges- tion, the products were denatured and separated on a sequenc-

1 5 0

TSIYTSTEPTNSAFTTEDYKPQLVEGV NSVLVIGSGGLSIGOAGEFDYS~;SQAIKALKEDNKFTILVNPNIATNQTSHSLADKIYYLPVTPEYITYIIE

I I I I I I I I I I I l l I I I l l I I I I I I I I I I I I I I TLAAEFPAOTNYLYTTYNAT~D~FNENGML~GSG~RIGSS~FDW~AVNTAKTLRD~G~TIMINYNPETVSTDFDEVDRLYFEELSYERVMDIYE 550 6 0 0

i o 0 1 2 0

LERPDAILLTFGGQTGLNCGVALDESGVLAKYNVKVLGTPIKTLITSEDRDLFASALKDINIPIAESFACETVDEALEAAERVKYPVIVRSAYALGGLGGLGS I / I l l I I I I I I I I l l I I I l l I I I I I I I l l $EQSEGCIISVGGQLPQNIALKLYDNGCNIM GTNPNDIDRAENRHKFSSI~DSIDVDQPEWSELTSVEEAKLFASKVNYPVLIRPSYVLSGAAM

6 5 0 7 0 0 2.0 0 2:o

GFANNASEMKELAAQSLSLAPQILVEKSLKGWKEVEYEWRDRV GNCITVCNMENFDPLGVHTGDSMVFAPSQTLSDEEFHMLRSAAIKIIRHLGVI

SVVNNEFELKAKLTLASDVSPDHPVVMS KFI EGAQEIDVDAVAYNGNVLVHAISEHIrENAGVHSGDASLVLPPQHLSDDVKIALKDIADKVAKAWKIT I I I I I I I I I I I I I I I I l l I 1 I I I l l I I I

7 5 0

3.0 0 8 0 0

GECNVQYALQPDGLDYRVIEVNARLSRSSALASKATGYPLAYTAAKIGLGYTLPELPNPITKTTVANFEPSLDYIVAKIPKWDLSKFQYVDRSIGSSMKS

GPFNMQIIPGEHTLK VIECNIRASRSFPFVSKVLGVNFIEIAVKAFLGGDIV PKPVDYL NKKY DWATKVPQFSFTRLAGADPFLGVEMAS

3.5 0

I I I I l l I I I l l I I I I I I I I I I I I I I I I I I 8 5 0

4.0 0 9 0 0

VGEVMAIGRNYEEAFQKALRQVDPSL LGFQGSTEFGDQLDEALRTPTDRRVLAIGQALIHENYT VERVNELSK IDKWFLYKCM NIVNIYKELES 4 > 0

I l l I I I I I I I / I I I I I I I I I I I I I I I TGEVASFGRDLIESYI?’TAIQSTMNFHVPLPPSGILFGGDTSFEYLGQVASI VATIGYRIYTTNETTKTYLQEHIKEKNAKVSLIKFPKNDKRKLRELFQ

9 5 0 1 0 0 0

5.0 0 VI(SLSDLSKDLLQRAKKLGFSDKQIAVTINKHASTNINELEIRSLRKTLGIIPFVKR1D

I I I I I I I I EYDIKAVFNLASKRAE~TDDVDYIMRRNAIDFAIPLFNEPQTALLFAKCLKAKIAEKIKILESHDVfVPPEVRSWDEFIGFKAY-COOH

1 0 5 0 1 1 0 0

FIG. 5. Internal homology in the amino acid sequence of the yeast large subunit. The derived amino acid sequence of the NH2-terminal half (residues 1 to 549) and the carboxyl-terminal half (residues 550 to 1117) of the protein have been aligned for maximal homology. Identical amino acids are indicated by the vertical lines. Gaps in the sequence indicate postulated insertions/deletions.


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I 2 3

- - 9.4 k b

6.5 - 4.4

- 2.0

1 . 1

- - - -

FIG. 6. Sizing of the CPAP transcripts from wild type S. cereuisiaestrain LL2. Total RNA and poly(A)-enriched RNA were denatured in methylmercuric hydroxide and fractionated on a 1% agarose slab gel under denaturing conditions. After transfer to a nitrocellulose filter, the RNA was hybridized with a radiolabeled nick-translated DNA probe (1.2-kb EcoRI-Hind111 fragment) which was internal to the coding region of the CPA2 gene. A mixture of a Hind111 digest of X DNA and a HaeIII digest of 6x174 replicative form DNA was used to calibrate the gel. Lune I , 5 pg of total RNA from LL2; lane 2, 1.8 pg of poly(A)-enriched RNA from LL2; lane 3, 0.9 p g of poly(A)-enriched RNA from LL2.

TABLE I Frequency of codons in the CPA2 gene

The initiation codon is not included in the table. UUU Phe 27 UUC Phe 18 UUA Leu 31 UUG Leu 38

CUU Leu 16 CUC Leu 3 CUA Leu 8 CUG Leu 6

AUU Ile 50 AUC Ile 22 AUA Ile 12 AUG Met 18

GUU Val 44 GUC Val 17 GUA Val 8 GUG Val 18

UCU Ser 35 UCC Ser 15 UCA Ser 17 UCG Ser 4

CCU Pro 15 CCC Pro 5 CCA Pro 25 CCG Pro 2

ACU Thr 29 ACC Thr 10 ACA Thr 19 ACG Thr 4

GCU Ala 39 GCC Ala 20 GCA Ala 25 GCG Ala 7

UAU Tyr 25 UAC Tyr 19 UAA Term“ 0 UAG Term 0

CAU His 11 CAC His 5 CAA Gln 25 CAG Gln 8

AAU Asn 29 AAC Asn 28 AAA Lys 45 AAG Lys 30

GAU Asp 46 GAC Asp 19 GAA Glu 64 GAG Glu 18

UGU Cys 9 UGC Cys 2 UGA Term 1 UGG Trp 8

CGU Arg 6 CGC Arg 0 CGA Arg 1 CGG Arg 0

AGU Ser 10 AGC Ser 2 AGA Arg 28 AGG Arg 6

GGU Gly 42 GGC Gly 10 GGA Gly 6 GGG Glv 8

Term, termination.

ing gel alongside the chemically derivatized probe. As a con- trol, the probe was also digested with SI nuclease in the absence of added RNA. The results of these analyses (Fig. 7) indicate four different transcripts. The two more prominent have 5’ starts at nucleotides -37 and -65. When a similar experiment was done with a probe extending from nucleotides -74 to -202, no protection was offered by the RNA, suggesting that the longest transcript has a 5‘-end a t nucleotide -65.

The presence of an A a t -65 and the A at -37 in the two transcripts is consistent with two separate transcriptional starts at pyrimidine residues. The minor transcripts start a t -59 and -70. The 5’ leader sequences of the carbamyl phosphate synthetase transcripts share certain common features with other recently described yeast genes (30-35). The first 25 nucleotides upstream of the initiation codon are A-rich

(35). The sequence PuTACATA a t -16 is similar to the PuCACACA sequence found at the same location in other yeast genes (33, 34). The sequence upstream of CPAQ also includes the invariant A a t -3 (34). A TATA or Goldberg- Hogness box (TATATAAT) is located a t -139. Between the TATATAAT sequence and the sequences coding for the mapped 5’-ends is a pyrimidine-rich block (34, 35) between -114 and -100. The sequence CAAG, indicative of a high efficiency promoter (34), is not present in the CPAQ leader sequence.

Although we have not determined the 3’-end of the carbamyl phosphate synthetase transcripts, there are two likely polyadenylation sites in the sequence. Based on the consensus sequence for adenylation (T. . .TAAATAAG. A . .T. . .A. . .AT)

- FIG. 7. Mapping of the 5‘-ends of CPAP transcripts. SI nu-

clease mapping of the 5’-ends of the CPA2 transcripts was performed as described under “Materials and Methods.” The DNA probe used for the protection experiments was a Tag1 fragment with the sequence from nucleotides +9 to -73 (see Fig. 2). Lane I , DNA probe alone; lane 2, DNA probe plus 200 pg of total yeast RNA from LL2; lane 3, DNA probe treated with 500 units/ml of SI nuclease; lane 4, 200 pg of total yeast RNA from LL2 hybridized with DNA probe and treated with 500 units/ml of SI nuclease. The chemically derivatized probe shown in the last four lnnes was used as the sizing ladder. The arrows point to the positions of the radioactive bands present only in the RNA-DNA hybrids treated with SI nuclease. The numbers of the corresponding nucleotides in the DNA sequence are shown for each band. One nucleotide has been subtracted from the sequence positions to correct for the displacement of the 3’ terminus in the sequencing ladder.


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Yeast Carbamyl Phosphate Synthetase

W H M O W

14473

4

B-, L I :: s ? M d

4 I

0

a 3


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proposed by Bennetzen and Hall (32), the 3' terminus of the transcripts could be either a t nucleotide +3591 or at +3654. Assuming the message to have 230-290 extra nucleotides at the 3'-end, a 5' leader of 37-65 nucleotides, and 50 residues of poly(A) (36), the overall length of the transcript falls in the range of 3.7-3.8 kb, which is in good agreement with the size calculated from its migration in agarose (3.7-3.8 kb).

Codon Utilization in the CPA2 Gene-Codon usage within the CPAB gene does not show the strong bias observed in other yeast genes that are highly expressed (37, 38). AGA is preferred for arginine, GGU for glycine, and the UUA and UUG codons for leucine, however, 59 of the possible 61 codons are utilized in the CPAB gene (Table I). Based on the method of Ikemura (38), the frequency of optimal codon usage in CPA2 is 0.61, a value similar to those calculated for TRPS (33) and CYCI (38), both of which are modestly expressed genes in yeast. These data imply that the CPAB gene is also moderately expressed.

Possible Adenine Nucleotide Binding Sites in the Large Subunit of Yeast and E. coli Carbamyl Phosphate Synthetme- It has been shown that the purified large subunit of E. coli (39) and yeast (11) carbamyl phosphate synthetase catalyzes the formation of carbamyl phosphate from NH3, bicarbonate, and ATP. These studies imply that the function of the small subunit is to provide NHCr from glutamine, the physiological donor of the amino group. Since the stoichiometry of carbamyl phosphate synthesis requires 2 mol eq of ATP/mol of carbamyl phosphate formed (40,41), a number of studies have been directed toward determining the number of ATP binding sites on the enzyme. Both pulse-labeling (42) and affinity labeling (43) experiments with the E. coli carbamyl phosphate synthetase indicate the existence of two separate ATP binding sites.

1

To identify possible ATP binding sites in the large subunit of yeast and prokaryotic carbamyl phosphate synthetases, we have searched for primary sequence homologies with other adenine-nucleotide utilizing enzymes. In particular, we were interested to see whether there are homologies with sequences known from crystallographic data to be involved in ATP binding. Such sites have been identified in adenylate kinase (44, 45), phosphoglycerate kinase (46), hexokinase (47), and phosphofructokinase (48). A search for homology between yeast and E. coli carbamyl phosphate synthetase and the known adenine-nucleotide binding sites of these enzymes revealed two particularly striking regions of primary sequence homology. The first of these occurs in phosphoglycerate kinase. Crystallographic studies of phosphoglycerate kinase (46) indicate that the adenine nucleotide interacts with two con- stellations of amino acids located between residues 212-238 and residues 313-343. A sequence homologous to the first part of the binding site of phosphoglycerate kinase (residues 212- 238) was found in a highly conserved region of the NH2- terminal halves of the E. coli and yeast large subunits. The homology between the sequences of carbamyl phosphate synthetase and phosphoglycerate kinase is particularly evident around Lys-219 of phosphoglycerate kinase (Fig. 8A). Lys- 219 is thought to interact with the a-phosphate of ATP (46). The proposed binding site of phosphoglycerate kinase has an alternating ,!-sheet, a-helix configuration (46) characteristic of the Rossman fold (52). Based on secondary structure predictions (53), alternating a/@ structures are also present in the carbamyl phosphate synthetase sequence. I t is of interest that a similar sequence is also present in glutamate dehydrogenase. The latter sequence (Fig. 8 A ) includes reactive residue Lys-126 of Domain 1, which has been predicted by Wootton

5 0 1 0 0

CPS PKRTDIKSILILGAGPIVIGQACEFDYSGARACKALREEGYRVILVNSNPATIMTDPEMDAT YIEPIHWEWRKIIEKERP DAVLPTMGGQTALNCALE / I I I I I I I l l / I

FL @ - s u b u n i t MATGKIVQVIGAW DVEFPQDAWRWD ALEVQNG

1 5 0

LERQGVLEEFGVTMIGATADAIDKAED RRRFDVAMKKIGLETARSGIAHTMEEALAVAADVGFPCII RPSFTMGGSGGGIAYNREEFEEICARGLDL I I I l l I I I I I I I I I / / I / I

NERL VLEVQQQLGGG~VRTIAMGSSDGLRRGLDVKDLEHPIEVPVGKATLGRIMNVLGEPVDMKG~IGEEERWAIHRAAPSYEELSNSQELLETGIKVIDL 5 0 1 0 0

2 0 0 2 5 0

SPTKELLIDESLIGWKEYEMEV VRDKNDNCIIVCSIENFDAMGIHTGDSITVAPAQTLTDKEYQIMRNASMAVLREIGVETGGSNVQFAVNPKNGRLIVIE I 1 I I I I I 1 / I I

MCPFAKGGKVGLFGFAGVGKTVNMMELIRNIAIEHSGYSVFA GVGERTREGNDFYHEMTDSNVIDKVSLWGQMNQPPGNRLRVALTGLTMAEKFRDEGRD 1 5 0 2 0 0

3 0 0 3 5 0

MNPRVSRSSALASKATGFPIAKVAAKLAVGY TLDELMNDITGGRTPASFEPSID YWTKIPWNFEK FAG ANDRLTTQMKSVGEVMAIGRT I I I I I I I I I I I * / I I I / I I I I I 1 I

VLLFVDNIYRYTL$GTEVSALLGNSAVGYOPTLAEEMGVLQE RITSTKTGSITSVEAWVP4IDDLTDPSPATTFAHLDATWLSRQIASLGIYPAVDPL 2 5 0 3 0 0

4 0 0 q 5 0

OQESLQKALRGLEVGATGFDPKVSLDDPEALTKIRRELKDAGADRIWYIADAFRGLSVDG VFNLTNIDRWFLVQIEELVREEKVAEVGITGLNADFL I I I I l l * I I l l 1 I 1 I I I I I I I I 1 / 1 1 I I

DSTSRQ LDPLWGQEHYD TARGVQSILORYQELKD IIAILGMDELSEEDKLWARRKIQR FQSQPFF VAEV FTGSPGKYV 3 5 0 4 0 0

5 0 0 5 5 0

RQLK RKGFADAI;LAKLAGVREAEIIIKLRD(LYDLHPVYKRVDTCAAEFATDTAYMYSTYEEE~EA I I I I I I I I 1 I I 1 I

SLKDTIRGFK GIMEGE YDHLPEQAFYMVGSIEEAVEKAKKL.CO0H 4 5 0

FIG. 9. Alignment of amino acid sequences of NHz-terminal half of E. coli carbamyl phosphate synthetase and the &subunit of E. coli F,-ATPase. The sequences have been aligned for maximal homology. Identical amino acids are indicated by the uertical lines. Gaps in the sequence represent postulated insertions or deletions. Computer alignment (26) of the sequences gave an align score of 2.87; probability, 0.13 X lo-'. Residues with asterisks are discussed in the text.


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(54) to have a structure corresponding to the Rossman fold. Although the carboxyl-terminal halves of E. coli and yeast carbamyl phosphate synthetase have similar sequences, the homologies are significantly lower.

The second region of interest was also found in the NHZ- terminal halves of the yeast and E. coli proteins (Fig. 8B). Residues 152-210 of the E. coli large subunit and residues 173-229 of the yeast large subunit show a significant similar- ity in both amino acid homologies and secondary structure to a region of adenylate kinase shown to bind M e complexes of 1,N'"etheno-analogues of ADP and ATP (55). This region of adenylate kinase encompasses residues 1-44 and 32-40 that have been found to bind the two adenine nucleotide analogues (55). The glycines in this part of adenylate kinase have been shown to form the loop juxtaposing the @A and aB structures of the Rossman fold (44). From NMR data, His-36 is known to interact with H2 of the adenine ring of MgATP (46, 56). Histidine residues are also present in the homologous carboxyl-terminal sequences of E. coli and yeast, however, they are displaced by eleven and five residues, respectively.

Amino acid sequences homologous to the glycine-rich adenine nucleotide binding region of adenylate kinase have also been found in the @-subunit of E. coli F,-ATPase by Kanazawa et al. (51) and Walker et al. (57) (Fig. 8B). This observation prompted us to search for more extensive sequence homologies between the large subunit of carbamyl phosphate synthetase and the above enzymes. Both computer (26) and manual alignments of the sequences uncovered suggestive homologies between the @-subunit of E. coli F,-ATPase and the NH2- terminal half of the E. coli carbamyl phosphate synthetase large subunit. As shown in Fig. 9, the two sequences exhibited 23% identical amino acids with an average 5 insertions/ deletions/100 residues. The similarities span almost the entire length of the @-subunit polypeptide, but are especially evident between residues 313-422 of carbamyl phosphate synthetase and residues 250-372 of the @-subunit. This region includes the Tyr-354 and Lys-286 of the @-subunit which react cova- lently with analogues of ATP (57, 58). Kanazawa et al. (51) have also found that this region of the P-subunit conforms to a Rossman fold by secondary structure predictions. The homologous sequence in carbamyl phosphate synthetase also includes the region of homology to phosphoglycerate kinase (residues 302-352).

The number of identities between the @-subunit of F, and the carboxyl half of the large subunit is less significant, probably because of the divergence in the carboxyl half of the protein. Comparison of the sequences of the @-subunit of E. coli F, and yeast carbamyl phosphate synthetase indicates similarly weak homology. The divergence between the 8- subunit of F1 and yeast carbamyl phosphate synthetase argues against the idea of functional convergence of the amino acid sequences at the nucleotide binding sites. Taken together, the data presented above suggest a distant relatedness of the @- subunit of F,-ATPase and carbamyl phosphate synthetase in E. coli.

DISCUSSION

With the exception of S. cereuisiae (lo), eukaryotic arginine-specific carbamyl phosphate synthetases function in the mitochondrial compartment. The present studies were under- taken with the view of establishing whether this enzyme is structurally related to the prokaryotic carbamyl phosphate synthetase which functions both in the arginine and pyrimidine biosynthetic pathways, We have used a recombinant plasmid to sequence the yeast CPA2 gene coding for the large subunit of arginine-specific carbamyl phosphate synthetase. The protein has been shown to be encoded by a 3354-nuch-

tide long reading frame. The calculated molecular weight of the encoded polypeptide is in good agreement with the reported molecular weight of the large subunit of yeast carbamyl phosphate synthetase. The amino acid sequence of the polypeptide derived from the gene sequence has been found to be highly homologous with the amino acid sequence of the large subunit of E. coli carbamyl phosphate synthetase. Of 1072 amino acid residues, 426 are identical and 264 represent conservative substitutions. These results clearly show the two enzymes to be closely related. The assignment of this sequence as the structural gene of the large subunit of carbamyl phosphate synthetase is further supported by the following evidence. l) Transcription- and translation-related sequences common to other recently sequenced yeast genes have been found in regions both upstream and downstream of the reading frame. 2) RNA transcripts approximately 450 nucleotides longer than the coding sequence have been detected in North- ern blots of yeast total RNA. 3) The results of 5' mapping indicate two major transcriptional start sites located 37 and 65 nucleotides upstream of the assigned ATG initiation codon.

The NH,- and carboxyl-terminal halves of yeast carbamyl phosphate synthetase exhibit a significant homology in their amino acid sequences. We have recently reported (15) a similar internal homology in the sequence of the carB gene of E. coli and have concluded that the carB gene was formed from an ancestral gene half the size of carB. The same internal homology in the yeast CPA2 gene suggests that the duplication occurred before the divergence of prokaryotes and eukaryotes in an ancestor common to bacteria and fungi. These conclusions are corroborated by the cross-homologies of the two halves in the two species. Our data further suggest that the tandemly duplicated structure is likely to be a general property of the genes coding for carbamyl phosphate synthetase in prokaryotes and eukaryotes.

There is increasing evidence suggesting gene duplication to be a major mechanism in the evolution of new protein functions (59-63). Accordingly, it is thought that one of the duplicated genes maintains the original function, while the second copy evolves, accumulating mutations that allow it to acquire a new function (61,63). Although the carbamyl phosphate synthetase gene sequences of yeast and E. coli appear to have diverged independently since the initial duplication, the extensive amino acid sequence homology over almost the entire length of the large subunit suggests a considerable constraint on the primary sequence. This is especially clear between the NH,-terminal halves of the two proteins and could indicate that this region has the functional domains of the ancestral protein.

An interesting aspect of the catalytic mechanism of all known arginine-specific carbamyl phosphate synthetases is the utilization of 2 mol of ATP/mol of carbamyl phosphate synthesized. Only one phosphoryl group, however, is transferred during the reaction (40, 41). Since there is evidence that E. coli carbamyl phosphate synthetase binds two ATPs (42, 43), we have examined both the prokaryotic and eukaryotic sequences for possible adenine nucleotide binding sites. To detect such sites, the sequences have been compared to other ATP utilizing enzymes. Suggestive homologies have been detected between the proposed adenine nucleotide binding sites of adenylate kinase, phosphoglycerate kinase, glutamate dehydrogenase, F,-ATPase and two different regions of carbamyl phosphate synthetase. One of the regions found in both the E. coli and yeast enzymes is located between residues 300-400. This region is homologous to both phosphoglycerate kinase and glutamate dehydrogenase and includes a number of residues that have been postulated from x-ray crystallog- raphy to interact with adenine nucleotides. Some of the


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14476 Yeast Carbamyl Phc

critical residues are also present in the comparable sequences of the carboxyl-terminal halves of the carbamyl phosphate synthetase, although the number of conserved residues is less. The second region of homology is also stronger in the NH2- terminal half of carbamyl phosphate synthetase. It includes residues 152-210 and has a sequence similar to the glycine- rich segment of adenylate kinase known to be involved in nucleotide binding. A homologous sequence has also been reported by Kanazawa et al. (51) and Walker et al. (57) to be present in the @-subunit of F,-ATPase. Further studies are required to clarify the significance of the observed primary sequence homologies and the possible role of the putative adenine nucleotide binding domains in the catalytic mechanism of carbamyl phosphate synthetase. Also, the question is raised whether both halves of the enzyme are capable of carbamyl phosphate synthesis; or whether one half catalyzes phosphoryl transfer while the second half performs another function needed in the energy requirements of carbamyl phosphate synthesis.

To determine possible relationships between carbamyl phosphate synthetase and other adenine nucleotide utilizing enzymes, we have compared the sequences of carbamyl phosphate synthetase to those of other known kinases. A search that included the known sequences of kinases and related enzymes revealed a suggestive homology of carbamyl phosphate synthetase to the @-subunit of F,-ATPase. This homology was most evident between E. coli F1 and the NH,-terminal half of carbamyl phosphate synthetase. Of 459 residues, the two proteins showed 105 identities and 37 conservative replacements. The calculated align score of =3 implies a rela- tionship of the two proteins. The derivation of carbamyl phosphate synthetase and the (3-subunit of the proton trans- locating ATPase from a common ancestor is an intriguing possibility. This question may be clarified when additional data become available on the sequences of other H+-ATPases, particularly those of Streptococcus (64) and Clostridium (65) that have more primitive functions than those of E. coli or of mitochondria.

Acknowledgments-We are indebted to Dr. Lois T. Hunt of the National Biomedical Research Foundation and Roy Smith for the computer analyses of the sequences.

REFERENCES

1. Pierard, A., Grenson, M., Glansdorff, N., and Wiame, J . M. (1973) in The Enzymes of Glutamine Metabolism (Pruisner, S., and

2. Makoff, A. J., and Radford, A. (1978) Microbiol. Reu. 42, 307- Stadtman, E. R., eds) pp. 483-503, Academic Press, New York

328 3. Anderson, P. M., and Meister, A. (1966) Biochemistry 5, 3164-

3169 4. Trotta, P. P., Pinkus, L. M., Haschemeyer, R. H., and Meister,

A. (1974) J. Bid. Chem. 249,492-499 5. Lacroute, F., Pierard, A,, Grenson, M., and Wiame, J . M. (1965)

J . Gen. Microbiol. 40, 127-142 6. Davis, R. H. (1967) in Organizational Biosynthesis (Vogel, H. J.,

Lampen, J. O., and Bryson, V., eds) pp. 303-322, Academic Press, New York

7. Jones, M. E. (1980) Annu. Reu. Biochem. 49, 253-279 8. Williams, L. G., Bernhardt, S., and Davis, R. H. (1970) Biochem-

9. Nam. M.. LaPorte, J., Penverne, B., and Herve, G. (1982) J. Cell istry 9,4329-4335

L%l. 92, 790-794 10. Urrestarazu. L. A.. Vissers. S.. and Wiame. J . M. (1977) Eur. J .

Biochem. 79, 473-481 , .

11. Pierard, A,, and Schroter, B. (1978) J. Bacteriol. 134, 167-176 12. Lusty, C. J., and Lu, J . (1982) Proc. Natl. Acad. Sci. U. S. A. 79,

2240-2244 13. Glansdorff, N., Dambly, C., Palchaudhuri, S., Crabeel, M., Pier-

ard, A,, and Halleux, P. (1976) J. Bacteriol. 127, 302-308 14. Crabeel, M., Charlier, D., Weyens, G., Feller, A., Pierard, A., and

wphate Synthetase

Glansdorff, N. (1980) J. Bacteriol. 143, 921-925 15. Nyunoya, H., and Lusty, C. J. (1983) Proc. Natl. Acad. Sci. U. S.

16. Broach, J. R., Strathern, J . N., and Hicks, J. B. (1979) Gene 8,

17. Nasmyth, K. A., and Reed, S. I. (1980) Proc. Natl. Acad. Sci. U.

18. Clewell, D. B., and Helinski, D. R. (1969) Proc. Natl. Acad. Sci.

A. 80,4629-4633

121-133

S. A. 77, 2119-2123

I J . S. A. 62. 1159-1166 19. Maxam, A. M., and Gilbert, W. (1980) Methods Enzymol. 65,

~ ~ ~~ ~~

499-560 20. Schimke, R. T., Palacios, R., Sullivan, D., Kiely, M. L., Gonzales,

21. Aviv, H., and Leder, P. (1972) Proc. Natl. Acad. Sci. U. S. A. 69,

22. Bailey, J . M., and Davidson, N. (1976) Anal. Biochem. 70, 75-85 23. Thomas, P. S. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 5201-

24. Jeffreys, A. J., and Flavell, R. A. (1977) Cell 12, 429-439 25. Berk, A. J., and Sharp, P. A. (1977) Cell 12, 721-732 26. Dayhoff, M. O., Barker, W. C., and Hunt, L. T. (1983) Methods

27. Maizel, J. V., and Lenk, R. P. (1981) Proc. Natl. Acad. Sci. U. S.

28. Staden, R. (1982) Nucleic Acids Res. 10, 2951-2961 29. Pustell, J., and Kafatos, F. C. (1982) Nucleic Acids Res. 10,4765-

30. Smith, M., Leung, D. W., Gillam, S., Astell, C. R., Montgomery,

31. Holland, M. J., Holland, J. P., Thill, G. P., and Jackson, K. A.

C., and Taylor, J. M. (1974) Methods Enzymol. 30, 631-648

1408-1412

5205

Enzymol. 91, 524-545

A. 78, 7665-7669

4782

D. L., and Hall, B. D. (1979) Cell 16,753-761

(1981) J. Biol. Chem. 256. 1385-1395 32. Bennetzen, J. L., and Hall,' B. D. (1982) J. Biol. Chem. 257,

33. Zalkin, H., and Yanofsky, C. (1982) J. Biol. Chem. 257, 1491-

~~~~~I ~ ~

3018-3025

1500 34. Dobson, M. J., Tuite, M. F., Roberts, N. A,, Kingsman, A. J.,

Kingsman, S. M., Perkins, R. E., Conroy, S. C., Dunbar, B., and Fothergill, L. A. (1982) Nucleic Acids Res. 10, 2625-2637

35. Montgomery, D. L., Leung, D. W., Smith, M., Shalit, P., Faye, G., and Hall, B. D. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 541-545

36. McLaughlin, C. S., Warner, J. R., Edmonds, M., Nakazato, H., and Vaughan, M. H. (1973) J. Biol. Chem. 248, 1466-1471

37. Bennetzen, J. L., and Hall, B. D. (1982) J. Biol. Chem. 257,

38. Ikemura, T . (1982) J. Mol. Biol. 158, 573-597 39. Trotta, P. P., Burt, M. E., Haschemeyer, R. H., and Meister, A.

40. Anderson, P. M., and Meister, A. (1965) Biochemistry 4, 2803-

41. Jones, M. E. (1965) Annu. Reu. Biochem. 34, 381-418 42. Powers, S. G., and Meister, A. (1978) J. Biol. Chem. 253, 800-

43. Boettcher, B. R., and Meister, A. (1980) J. Biol. Chem. 255,

44. Pai, E. F., Sachsenheimer, W., Schirmer, R. H., and Schulz, G.

45. Smith, G. M., and Mildvan, A. S. (1982) Biochemistry 24,6119- 6123

46. Banks, R. D., Blake, C. C. F., Evans, P. R., Haser, R., Rice, D. W., Hardy, G. W., Merrett, M., and Phillips, A. W. (1979) Nature (Lond.) 279, 773-777

47. Steitz, T. A,, Fletterick, R. J., Anderson, W. F., and Anderson, C. M. (1976) J . Mol. Biol. 104, 197-222

48. Evans, P. R., and Hudson, P. J . (1979) Nature (Lond.) 279,500- 504

49. Moon, K., Piszkiewicz, D., and Smith, E. L. (1972) Proc. Natl. Acad. Sci. U. S. A. 69, 1380-1383

50. Heil, A,, Muller, G., Noda, L., Pinder, T., Schirmer, H., Schirmer, I., and von Zabern, I. (1974) Eur. J . Biochem. 43, 131-144

51. Kanazawa, H., Kayano, T., Kiyasu, T., and Futai, M. (1982) Biochem. Biophys. Res. Commun. 105, 1257-1264

52. Rossman, M. G., Liljas, A., Branden, C. I., and Banaszak, L. J . (1975) in The Enzymes (Boyer, P. D., ed) Vol. 11, Part A, pp. 61-102, Academic Press, New York

53. Chou, P. Y., and Fasman, G. D. (1974) Biochemistry 13, 211- 222; 222-245

54. Wootton, J . C. (1974) Nature (Lond.) 252, 542-546

3026-3031

(1971) Proc. Natl. Acad. Sci. U. S. A. 68, 2599-2603

2809

803

7129-7133

E. (1977) J . Mol. Biol. 114, 37-45


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


55. Hamada, M., Palmieri, R. H., Russell, G. A., and Kuby, S. A.

56. McDonald, G. G., Cohn, M., and Noda, L. (1975) J. Biol. Chem.

57. Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. J. (1982)

58. Esch, F. S., and Allison, W. S. (1978) J. Biol. Chern. 2 5 3 , 6100-

59. Watts, R. L., and Watts, D. C. (1968) J. Theor. Biol. 20, 227-

(1979) Arch. Biochem. Biophys. 1 9 5 , 155-177

250,6947-6954

Eur. Mol. Biol. Org. J. 1, 945-951

6106

245

60. Doolittle, R. F. (1981) Science (Wash. D. C.) 214, 149-159 61. Wilson, A. C., Carlson, S. S., and White, T. J. (1977) Annu. Reu.

62. Anderson, R. P., and Roth, J. R. (1977) Annu. Reu. Microbiol.

63. Lewis, E. B. (1951) Cold Spring Harbor Symp. Quant. Biol. 1 6 ,

64. Heefner, D. L. (1982) Mol. Cell. Biochem. 44,81-106 65. Clarke, D. J., Fuller, F. M., and Morris, J. G. (1979) Eur. J .

Biochem. 4 6 , 573-639

3 1,473-505

159-174

Biochem. 98, 597-612


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

C J Lusty, E E Widgren, K E Broglie and H Nyunoyato Escherichia coli carbamyl phosphate synthetase.

Yeast carbamyl phosphate synthetase. Structure of the yeast gene and homology

1983, 258:14466-14477.J. Biol. Chem.

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