Nucleic Acids Research Seqneoce for hnman argininosucdnate syntbetase cDNA


The nucleotide sequence for human arglnlnosucclnate synthetase cDNA was determined by analysis of six clones isolated from a single experiment. The sequence covered 1623 nucleotides Including 76 bases of poly(A) and contained a 1236 nucleotide open reading frame encoding a protein of 46,434 daltons. In one cDNA isolate, a cloning artifact or perhaps RHA polymerase error involving addition of an A in a region of six A's within the coding sequence was documented. Single base variations in the 3' untranslated region were examined in detail since detection of DNA polymorphisms in the cDNAs could imply over-expression of both alleles at the active locus in canavanineresistant cells, i.e. a trans-acting mechanism for enzyme overproduction. However, the sequence from five cDNAs suggested some single base artifacts, and DNA polymorphism remains uncertain. The occurrence of three tandem arginine codone in the 5' untranslated region of the cDNA suggested the possibility of an interaction of arginyl-tRNA with mRNA to regulate RNA processing or half-life as a mechanism for arginine-mediated repression. INTR0DUCTI0H Argininosuccinate synthetase functions in the urea cycle to convert citrulline and aspartate to argininosuccinate. A deficiency of this enzymic activity in humans is recognized as the disorder citrullinemia, which is inherited in an autosomal recessive manner (1). In some cultured cell lines, argininosuccinate synthetase is subject to metabolite regulation by arginine (2). Variant cultured human cell lines resistant to the arginine analog canavanine have an argininosuccinate synthetase specific activity which is 180-fold higher than that of parental cells. This canavanine-resistant phenotype is stable and the enzymic activity is not subject to an arginine-mediated regulation (3,4). The steady state level of this mRNA correlates closely with the specific activity of the enzyme in the cell extract, indicating regulation at a pretranslational level (4,5). There is no evidence for gene amplification in canavanine-resistant cells (5). There are multiple processed argininosuccinate synthetase pseudogenes (6). However, there is abundant 

Nucleic Acids Research

evidence that none of these pseudogenes is expressed, and all of the available data are consistent with the presence of a single active gene (6)(7)(8). 
We report here the DNA sequence of several cDNA clones for human argininosuccinate synthetase obtained from a single cloning experiment. 
The sequence provides data relevant to possible mechanisms of canavanlne resistance and arginlne-medlated regulation. 

METHODS

The isolation of the cDNA clones labeled pASl, pAS2, and pAS3 from a recombinant cDNA library constructed from pBR322 and poly(A) + RNA from canavanine-resistant RPMI-2650 cells was described previously (5). The cDKA clones pAS4, pAS9 and pAS12 were isolated by rescreening that library with nick-translated pASl. All isolates were from a single cloning experiment using mRHA from one cell harvest. 

Sequence Analysis

Restriction maps of all Isolates were prepared. Restriction fragments were isolated, labeled at 5' ends, and sequenced by the method of Max am and Gilbert (9). Again assuming a single active locus, these three combinations indicated at 

RESULTS


Sequencing Strategy and Hucleic Acid Sequence


AFigure 1 .

 1 Figure 1. The sequencing strategy for human arginlnosucclnate synthetase cDNAs. The uppermost line provides a restriction map for the entire cDNA sequence. The sequence numbering proceeds in a 5' to 3' direction. Shown below and relative to this restriction map are the six cDNA inserts studied, each presented together with its own sequencing strategy. The solid, heavy lines represent protein coding regions, while the open, heavy lines represent untranslated regions. The cDNA fragments were labeled at the 5' ends, and the direction and extent of each sequence is indicated directly below its respective cDNA map. 


Figure 2 .Figure 3 .

 23 Figure 2. The nucleotlde and amlno acid sequences of human arginlnosucclnate synthetase cDNA. Three tandem arginine codons 5' to the initiator ATG are overlined. Arrows indicate the locations of the observed nucleotide variations. 


L T HE 'I-V3MBVQCDTEPTDATGFI«I!I CClCT(TrCICTCIACAATCAO(M<»rrGCTGA«ATCAACCTC<UGCCTGATTATCACCCAACTGATGCCAiXGOCTrCATC^1 180

  
	80	290	300	310	320	330	3*0	350	360
	PCIARIQVEIAQREGAKTVSHCATGKCHDQ
	CCCT<K^TCCODCGCAAACAACTCGAAATCGCCCAGCCCCAGCCCCCCA/UrrATCTCTCCCACGCO3^3								
	70	380	390	400	410	*20	430	440	450
	VIFELSCTSLAPQIKVIAPWRHPEFTHRFI
	CTCCOCTTTCACCTCACCTGCTACTCACTCCCCCCCCACATAAACCTCATrtMrrCCCTGC^4								
	60	470	480	490	500	510	520	530	540
	CRHDLMEYAIQHCIPIPVTPIHFVSHDBHL
	aXXCCAAIGACCTCATCGACTAO3CAAA<XAACACCGCATTCCCATCCCGGTCACTCCCAACAACCCC5								
	50	560	570	580	590	600	610	620	630
	HHISYEAGILEIPISQAPPGLTTITQDPAI
	ATGCACATCACCTACCACGCTGC^TCCrGCACAACCCCAACAACakACCCCCTCCACCTCTCTACAC^6								
	40	650	660	670	680	690	700	710	720
	APHTPDILEIEPrrCVPVrVTHVIDCTTHQ
	CKXCCCAACACaCTCACATTCTCGA£ATCCAGTTCAAAAAAC(»rrCCCTCTC				
	730	740	750	760	770	780	790	800	810
	TSLELFMTLBE7AGIHCVGIIDIVEIRFIC
	ACCTCCTTCCACCTCTTCATCTACCTCAACCAACTCCCCCCCAACCATCCCCTCGCCCCTATTGAC^8								
	20	830	840	830	860	870	880	890	900
	HtSROITETPAGTILTHAHLDlEAFTHDRE
	ATCAACTCCaMaTrATCTACCACACCCCACCAGCCACCATCCTTTACCATCCTCATTTAC^9								
	10	920	930	940	950	960	970	980	990
	VRIIIQGLGLKFAELVITCLRPSPECEFVR
	GTCCCCAAAATCAAACAAGCCCTGGCCTTGAAAITTCCroACCTCCTCTATACCGCTrTACCCCCTACCCCTG1								
	000	1010	1020	1030	1040	1030	1060	1070	1080
	HCIAKSQERVECIVQVSVLtOQVTILGRES
	CACTCCATCCCCAACTCCCACCACCCACTGC^AGCGAAAGrW^GCTCTCCCTarCAAara1								
	09O	1100	1110	1120	1130	11*0	1150	1160	1170
		1190	1200	1210	1220	1230	12*0	1250	1260
	LtlKETHRLQSrvTAI				
	CTCACCCTCAAGOAATAICATCGTCTCCAGAGCAAGCTCACn»IAAArACAATGA<Ml		
	1270	1280	1290	1300	1310	1320	1330		
	TCCiCAICCCCCAACTACACCCCCTAAnClTCll^TAATTrCTAATTCTCACUiriClC			
	1360	1370	1380	1390	1400	1410	1420	1430	1*40
	CCCCCiCCI TTCTTCCCTGCTCCCCCrauCCCT«aUACCTTCT^1								
	450	1460	1470	1480	1490	1500	1310	1520	1530
	AATCACAATTAAAACAGAAAAAAAAAAAAA [Ax] ]						
	1540	1550	1560						

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ACKNOWLEDGEMENTS


This work was supported by National Institutes of Health Research

Fellowship Award GM07466-02 and grant GM 27593. We thank Drs. Svend 0. 

Freytag and Jeffrey Rosen for constructive criticism of this manuscript and

Lynn Loewenatein for help in the preparation of this manuscript. 
•To whom, correspondence should be addressed
Hans-Georg O Bock, Tsung-Sheng Su, William E O'brien, Arthur L Beaudet
Nucleic Acids Research Seqneoce for hnman argininosucdnate syntbetase cDNA