Sequence of the cDNA encoding bovine uridine monophosphate synthase

Gene, 124 (1993) 307-308 0 1993 Elsevier Science Publishers

GENE

B.V. All rights reserved.

307

0378-l 119/93/$06.00

06944

Sequence of the cDNA encoding bovine uridine monophosphate (Recombinant

Stephan

DNA; phage h libraries;

Schoeber,

de novo pyrimidine

Detlef Simon and Barbara

synthesis;

RT-PCR;

RACE; sequencing

synthase*

of PCR products)

Schwenger

Institutefor Animal Breeding and Genetics, Hannover School of Veterinary Sciences, D-3000 Hannover 71, Germany Received by J.A. Engler: 21 August

1992; Accepted:

19 September

1992; Received at publishers:

17 November

1992

SUMMARY

A 1869-bp cDNA encoding bovine UMP synthase (UMPS), including the 3’-untranslated and 34 bp of the S-untranslated regions, was isolated and sequenced. The deduced amino acid sequence shows a high degree of homology to UMPS sequences reported from other species, namely for regions corresponding to the putative catalytic sites. The sequence information will be used to analyse the molecular basis of the deficiency of UMPS (DUMPS) in cattle.

In mammals the last two steps of de novo pyrimidine biosynthesis are catalysed by UMPS converting erotic acid to UMP. The bifunctional protein is comprised of two domains with an OPRTase and an ODCase activity linked by a connector peptide. A hereditary pyrimidine auxotrophism caused by DUMPS is known in cattle. Shanks et al. (1984) demonstrated a monogenic autosomal recessive pattern of inheritance. The homozygous genotype is lethal in utero (Shanks et al., 1992). In man a similar disorder is known as hereditary erotic aciduria (Suttle et al., 1989). We cloned and sequenced

the cDNA

encoding

bovine

(Suttle et al., 1988). In Fig. 1, we compared the bovine UMPS with human and Dictyostelium discoideum UMPS. A high degree of conservation is observed for regions corresponding to the putative catalytic sites described by Jacquet et al. (1988), while the central region belonging to the connector peptide is less well conserved. In order to find the molecular basis of DUMPS, the DNA and RNA of DUMPS carrier animals are being investigated). We are grateful to D.P. Suttle for providing the human UMPS cDNA probe HUSc33. This work was supported by a grant from the Deutsche Forschungsgemeinschaft.

UMPS (Fig. 2). The deduced UMPS protein consists of 480 aa. It exhibits 84% identity to the human UMPS Correspondence to: Dr. B. Schwenger, Institut fur Tierzucht und Vererbungsforschung, Tierlrztliche Hochschule Hannover, Biinteweg 17p, D-3000 Hannover 953 8582; e-mail: DBP.DE

n

s

UMPSt

I

I

so

71, Germany. Tel. (49-511) 953 8877; Fax (49-51 I) [email protected].

*On request, the authors will supply detailed the conclusions reached in this Brief Note.

experimental

evidence for

Dd

lOOas

Abbreviations: aa, amino acid(s); bp, base pair(s); cDNA, DNA complementary to RNA; DUMPS, deficiency of UMPS; kb, kilobase or 1000 bp; nt, nucleotide(s); ODCase, orotidine 5’-monophosphate decarboxylase; OPRTase, orotate phosphoribosyltransferase; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; RT, reverse transcriptase UMPS, uridine monophosphate synthase.

Fig. I. Comparison of the bovine (Bo) UMPS with human (Hu) (Suttle et al., 1988) and Dictyostelium discoideum (Dd) UMPS (Jacquet et al., 1988). Deduced aa sequences were aligned by the GAP program of the HUSAR package which introduced gaps (5 aa in total) to align the Dd sequence. Identity with the Bo UMPS is indicated by black lines. A, OPRTase;

B, connector

peptide;

C, ODCase.

308

1 35 1 95 21 155 41 215 61 275 61 335 101 395 121 455 141 515 161 575 181 635 201 695 221 755 241 615 261 675 281 935 301 995 321 1056 341 1115 361 1175 361 1235 401 1295 421 1355 441 1415 461 1475 1470 1657 1636 1715 1794

Fig. 2. The nt sequence

TTTGGAGTTTGGAGTGAACTGAGAGAGCGGGACC && GCG GCG GCT OAT MAAADALLGSLVTGLYOVQA TTT AAG TTT GGG AAC FKFGNFVLKSGLSSPVYIDL CGG GGC ATC ATA TCT RGIISRPSILNQVAEHLFQT GCC GM AAT GCA GAG AENAEINFDTVCGVPYTALP TTG GCT ACA ATT GTC LATIVCSTHEIPHLIRRKEK AAG GAT TAT GGT ACT KDYGTKRLIEGAVNPGDTCL ATC ATT GAA GAT GTT I I E D V AAA GAA GGC TTG AAG K E G L K GAC AAT CTG CAG GCC 0 N L Q A TGT ATT CTT GAG CAA C ATT

I CAG

L GAG

E AAT

Q GCT

IQENAFVAANPNDSLPSVKK GAA CCC AAA GAA CTA EPKELSFGARAELPGTHPVA GCG AAG CTT CTC AGO AKLLRLHQKKETNLCLSADV TCA GAG TCC AGA GAG SESRELLQLAOALGSRICLL AAG ATT CAT GTA GAT KIHVOILNDFTLDVHKELTT CTG GCA AAA CGC CAT L A K R H ACA GTA AAA AAG CAG T V K K Q AAT GCT CAC GCC GTG NAHAVPGSGVVKGLEEVGLP CTG CAC COG GCG TGC LHRACLLVAEMSSAGTLATG AGC TAC ACT GAG GCA SYTEAAVQHAEEHSEFVIGF ATT TCT GGC TCC CGA ISGSRVSMKPEFLHLTPGVQ TTA GAA GCA GGA GOT LEAGGDNLGQQYHSPQEVIG AAA CGA GGC TCT GAT KRGSDIIIVGRGIIASANQL GAA GCA GCC AAG ATG EAAKMYRKAAWEAYLSRLAV

GCC

CTT

TTG

GGG

TCA

TTG

GTA

ACG

GGT

CTG

TAC

OAT

GTG

CAG

TTC

GTG

CTG

AAG

CGA

CCG

AGT

ATT

ATC

AAT

TTT

TGT

TCA

AAA

CGG

GTG V GTC V CGT R CAG

GCT

AGT

GGG

CTC

TCT

TCC

CCC

GTG

TAC

ATC

GAT

CTG

CTG

AAT

CAG

OTT

GCA

GAA

ATG

TTA

TTT

CAA

ACT

GAC

ACT

GTG

TGT

GGA

GTA

CCT

TAT

ACA

GCT

TTA

CCA

ACC

CAC

GAA

ATT

CCA

ATG

CTT

ATC

AGA

AGG

AAG

GAG

AAA

CTT

ATA

GAA

GGG

GCT

GTT

AAT

CCA

GGA

GAC

ACC

TGC

TTG

TCC S ACT T GGG G AAG

AGC S GAC D ATT I AAA

GGA G GCT A CGT R ATC

TCT S GTA V CTC L AAT

AGT S GTG V CAC H GCT

OTT V CTG L TCA S GAG

TOG W GTG V GTG V ACT

GM E GAC D TGC C GTG

ACT T AGA R ACA T GAG

GCT A GAG E TTG L AGA

GAG E CAG Q TCC S GTG

GTT V GGG G ACA T AAG

CTT L GGC G GTG V AGA

CAG Q AGG R CTG L TTT

Q TTT

K GTG

K GCA

I GCT

N AAC

A CCT

E AAT

T GAT

V TCT

E CTC

R CCT

V TCT

K GTG

R AAG

F AAA

AGT

TTT

GOT

GCA

CGT

GCA

GAG

CTG

CCT

GGG

ACC

CAC

CC0

OTT

GCA

CTT

ATG

CAA

AAG

AAG

GAG

ACC

AAT

CTG

TGT

CT0

TCT

GCT

GAT

GTT

CTG

CTG

CAG

CTA

GCA

GAT

GCT

TTG

GGA

TCC

AGA

ATC

TGC

TTG

CTT

ATT

TTG

AAT

GAT

TTT

ACT

CTG

GAT

GTG

ATG

AAG

GAG

TTA

ACC

ACT

GAG E TAT Y CCC

TTT F GAA E GGC

CTA L GGT G TCA

ATT I GGG G GGA

TTT F GTC V OTT

GAA E TTT F GTG

GAC 0 AAA K AAA

COG R ATA I GGC

AAA K GCT A CTG

TTT F TCC S GAA

GCA A TOG W GAA

GAT D GCA A GTC

ATA I GAT D GGC

GGA G CTT L CTG

AAC N GTG V CCA

CTC

CTT

GTT

GCA

GAA

ATG

AGC

TCT

GCT

GGC

ACC

TTG

GCT

ACT

GGG

GCA

GTG

CAA

ATG

GCT

GAA

GAA

CAT

TCT

GAA

TTT

GTG

ATT

GGT

TTT

GTA

AGC

ATG

AAA

CCA

GAA

TTT

CTT

CAC

TTG

ACT

CCA

GGA

GTT

CAG

GAT

AAT

CTC

GGC

CAG

CAG

TAC

CAC

AGC

CCA

CAA

GAA

GTC

ATC

GGC

ATC

ATC

ATT

GTG

GGC

COG

GGC

ATA

ATA

GCA

TCA

GCT

AAT

CAG

CTG

TAC

AGA

AAA

GCT

GCT

TGG

GAA

GCT

TAC

TTG

AGT

AGA

CTT

GCT

GTT

m GCCTCTTGGATGTGCTTTTOGATGGCCCTGGAGCTAOATACATGGACTCCTMOATGCTACTGGCTTOAC~G CAGCCTTTMTCCTGCTTGGATTTTTCCATG~TCCTGTGTGGMCTGGAGTTACCGTGGTCTACAG~TATCTAAT QACTTGTGATCACTACACTGTCACTGTMTCCGTTCMGATTTCTTTACT~GACT~TGGTAGTTAGGCAGTCACACC CCGTTCTGTCAGMTGTCACATTGGACTGTOCCCCCCCACCATCTCCACATCAOACCTTTTGATTCTTTTMCTGCC GTGAGACAGAAATTTTAGGTCATAAAOAGCCTAAMCTAAAAAAAMAMAAAMAAAAAAAAAA~AAAAA

of bovine UMPS-encoding

cDNA

and the deduced

aa sequence.

The start, stop and polyadenylation

signals are underlined.

The strategy used to obtain the bovine UMPS-specific cDNA included: (a) isolation of a clone (BUS6) from a hgtl0 cDNA library from bovine liver (Clontech) using a human UMPS-specific cDNA HUSc33 (Suttle et al., 1988) as the hybridization probe; (b) sequencing of BUS6 after subcloning into pTZ19R, (c) PCR amplification of a part of the missing 5’-end from the library with primers derived from the coding sequence of BUS6 and hgtl0 vector sequences; PCR amplification for completion of the 5’-end from poly(A)‘RNA by RACE (Frohman et al., 1990); (d) direct sequencing of the complete translated region generated from RNA of an additional individual by RT-PCR after asymmetric amplification, EMBL Data Library accession No. is X65 125.

REFERENCES

Frohman, M.A.: RACE: rapid amplification of cDNA ends. In: Innis, M.A., Gelfand, D.H., Sninsky, J.J. and White, T.J. (Eds.), PCR Protocols - A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990, pp. 28-38. Jacquet, M., Guilbaud, R. and Garreau, H.: Sequence analysis of the DdPYRS-6 gene coding for UMP synthase in Dictyostelium discoideum and comparison with orotate phosphoribosyl transferases and OMP decarboxylases. Mol. Gen. Genet. 211 (1988) 44-445. Program manual for the Program Package HUSAR (Heidelberg Unix Sequence Analysis Resources). Release 2.0, 1990. German Cancer Research Center, and Center for Molecular Biology, University of Heidelberg, Heidelberg, FRG, 1990. Shanks, R.D., Dombrowski, D.B., Harpestad, G.W. and Robinson, J.L.:

Inheritance of UMP synthase in dairy cattle. J. Heredity 75 (1984) 3377340. Shanks, R.D., Popp, R.G., McCoy, CC., Nelson, D.R. and Robinson, J.L.: Identification of the homozygous recessive genotype for the deficiency of uridine monophosphate synthase in 35-day bovine embryos. J. Reprod. Fert. 94 (1992) 5-10. Suttle, D.P., Bugg, B.Y., Winkler, J.K. and Kanalas, J.J.: Molecular cloning and nucleotide sequence for the complete coding region of human UMP synthase. Proc. Nat]. Acad. Sci. USA 85 (1988) 1754-1758. Suttle, D.P., Becroft, D.M.O. and Webster, D.R.: Hereditary duria and other disorders of pyrimidine metabolism. C.R., Beaudet, A.L., Sly, W.S. and Valle, D. (Eds.), The Basis of Inherited Disease, 6th ed., Vol. 1. McGraw-Hill, 1989, pp. 1095-1126.

erotic aciIn: Striver, Metabolic New York,