214

Biochimica et Biophysica Acta, 1132 (1992) 214-218 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.00

Short Sequence-Paper

BBAEXP 90393

Isolation and characterization of the rat gene encoding ornithine aminotransferase James D. Shull

a

Karen L. Pennington a, Henry C. Pitot and Brian L. Schulte a

b

Victoria S. Boryca a

"Eppley Institute for Research in Cancer and Allied Diseases, and The Department of Biochemistry and Molecular Biology, Unic'ersity of Nebraska Medical Center, Omaha, NE (USA) and b McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI (USA) (Received 8 May 1992)

Key words: Ornithine aminotransferase; Nucleotide sequence; Gene structure

Herein we describe the isolation and characterization of the rat gene encoding ornithine aminotransferase (rOAT). Six unique genomic clones were characterized and assigned to two nonoverlapping contigs representing approx. 33 kb of the rat genome. The 5' contig contains the rOAT promoter, exons 1 and 3, and a portion of exon 4; an exon corresponding to exon 2 of the human OAT gene (hOAT) was not identified. The rOAT promoter contains several putative regulatory elements in positions similar to hOAT. The 3' contig contains exons 7 through 11 in their entirety. Data presented and discussed herein suggest that approx. 3.0 kb of uncloned genomic DNA, containing the remainder of exon 4 and all of exons 5 and 6, separate the two contigs. Together, these data suggest that rOAT extends over approx. 20 kb and is organized into at least 10 exons, thereby closely resembling hOAT in size and exon/intron organization. Isolation of rOAT provides an important tool for examining the molecular mechanisms through which estrogen and thyroid hormone regulate transcription of this gene in a cell-type specific manner.

Ornithine aminotransferase (OAT, [EC 2.6.1.13]) catalyzes the interconversion of L-ornithine and L-Atpyrroline-5-carboxylic acid, and thereby functions in the metabolic pathways leading to proline and arginine. The human gene ( h O A T ) encoding O A T has been isolated, characterized [1,2], and m a p p e d to the long arm of chromosome 10 [1,3,4]. Defects in this gene are associated with the development of gyrate atrophy, an autosomal recessive genetic disease characterized by chronic hyperornithinemia, a progressive degeneration of the chorioretina, and ensuing blindness [5-12]. The O A T gene is transcriptionally regulated by estrogens and thyroid hormones in the rat kidney [13] and human retinoblastoma cell lines [14], and translationally regulated by dietary factors, acting through glucagon and 3',5'-cyclic adenosine monophosphate, in the rat liver [15,16]. Therefore, this gene would appear to be an important model for the study of hormone action at the molecular level.

Correspondence to: J.D. Shull, Eppley Institute for Research in Cancer, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, NE 68198-6805, USA.

As a prelude to investigating the molecular mechanisms underlying the cell-type specific regulation of O A T gene transcription, we used a near full length O A T cDNA, prepared from rat liver m R N A [17], to isolate related sequences from a rat genomic D N A library. 21 unique clones were isolated, characterized by restriction endonuclease mapping, and the regions of homology with the O A T c D N A were identified by the Southern blot procedure. From these data, the clones were assigned to five distinct contigs representing approx. 140 kilobase pairs (kb) of the rat genome. Nucleotide sequence analyses of these contigs indicated that they correspond to two regions of a single functional O A T gene (this report) and three processed pseudogenes [18]. The organization of the expressed rat O A T ( r O A T ) gene is illustrated in Fig. 1. The 5' and 3' regions of this gene were derived from cloned members of two distinct contigs. The organization of the uncloned central region of r O A T is tentative and was derived from the known nucleotide sequence of the rat O A T c D N A [17], data from a series of genomic Southern blots [18] (and data not shown), and the organization of h O A T [1,2]. The nucleotide sequences of the cloned exons, as

215 tion correspond to two of four start sites utilized in hOAT [1,2]. This major start site was localized 336 bp upstream of the first of five BamHI sites contained within rOAT (Fig. 1). Upstream of this start site were observed a number of putative promoter elements, including an atypical T A T A A box, two Spl binding sites, and two CCAAT motifs, one of which is on the noncoding strand. Similar elements are located in approximately the same relative positions upstream of the human OAT gene [1,2,19]. When a restriction fragment containing these putative promoter elements was cloned into the plasmid pBLCAT3 [20], which contains the bacterial chloramphenicol acetyltransferase (CAT) gene but no promoter, expression of the reporter gene was observed following introduction of the r O A T / C A T plasmid into a variety of rat and human cell lines indicating that these elements comprise a functional promoter (unpublished data). An exon corresponding to exon 2 of hOAT has not, as yet, been identified in rOAT. Exon 2 of hOAT was originally identified based upon sequence relatedness to processed OAT pseudogenes located on the X chromosome [1]. The presence or absence of exon 2 in transcripts arising from hOAT appears to be due to alternative m R N A splicing [14], and may play a role in

well as limited regions of the flanking introns, are illustrated in Fig. 2. The structure of the 5' contig was derived from 2 distinct overlapping genomic clones. Exon 1 was localized by synthesizing an oligodeoxyribonucleotide corresponding to the 5' end of the rat OAT cDNA and using it to prime sequencing reactions in which various subcloned restriction fragments from the 5' contig were included as templates. Although exons 1 of rOAT (Fig. 2) and hOAT [1,2] differ substantially in terms of nucleotide sequence (53.8% identity), both are 52 base pairs (bp) in length and share a common 3' splice junction. Comparison of these rat genomic sequences with those of the previously described rat OAT cDNA [17] revealed that this cDNA lacks 38 bp at its 5' end. In addition, rOAT was observed to contain a deoxycytosine residue at position 46 (Fig. 2) that was not found in the cDNA. This deoxycytosine residue is also contained within the OAT-1 pseudogene [18] and hOAT [1,2]. Other than these differences, the sequences of rOAT and the cDNA were identical. Two transcription start sites were identified by primer extension analysis (data not shown); a major site at position 1 and a minor site at position 2, as illustrated in Fig. 2. These sites of transcription initia-

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i 3000 bp I Fig. 1. Structure of region of the rat genome containing the ornithine aminotransferase gene. The structures of the 5' and 3' contigs were derived from two and four unique overlapping clones, respectively. The putative structure of the uncloned region of the OAT gene domain separating these contigs was derived from the nucleotide sequence of the rat OAT cDNA [17], Southern blot analyses of genomic DNA digested with the indicated restriction endonucleases [18], and the organization of hOAT [1,2], and is enclosed by the shaded box. The top line represents the OAT gene domain, with the locations of the exons designated by the filled boxes. An exon corresponding to exon 2 of hOAT has not been identified in rOAT gene. The location of exon 6 (hatched box) could not be determined from existing data. The locations of the sites of cleavage by HindlII, BamHI, and EcoRI, are indicated on the lower lines, as are the approximate sizes (in bp) of the fragments generated by these enzymes. The order of the 3100 bp and 1700 bp EcoRI fragments was not unambiguously determined. The numbers within boxes refer to restriction sites located within exons and indicate the positions of these sites relative to the start site of transcription, disregarding introns. Methods: construction and screening of the rat genomic D N A library were described previously [18]. The OAT-related clones were propagated and the DNAs were isolated using standard procedures. Following digestion with various restriction endonucleases, the resulting DNA fragments were separated by electrophoresis in 1.0% agarose gels, visualized by staining with ethidium bromide, and transferred to Gene Screen Plus (New England Nuclear). The blots were prehybridized, hybridized using nick translated OAT cDNA or cloned subfragments of this cDNA as probes, and washed using standard, high stringency, conditions as described previously [8,18].

216

Exon

1

Exon

6

609

Exon

Exon

ccatagccaa tgaactgaga

gtcgagggtg

agcctcttat

gattggtgtg

cggccctccg

gaacgggcgg 1 ATCGGGCTTC

gggcgggaca

gacacgtcgt

cttaaacagc ctgacgcgcc gggctgccac

GTGGCTGAAA

TACAGTCAGT

GTCGCTTAGC

GGACGCTGTC

tgcgcgccgg

ggctgggccg

gagctacccg

gactctcggt

gttccggagt ctccaccggg

tttagattta

tttgtttact

ttgagtgttt

tgcctacctg tatgtatgca ccatgtgggt

ggatcccgac

tgtagcggaa

gggaaagctc

ggcagttact

tggtgagcag aacagcgaag

gctgggagtc

aaacgcagag

ctctgggaaa

gcagcaagta

ttccagactg

ttgaactctg

gaaaaggttc

ccactctcaa

cttactttgg

53 acagGTCTCT

tctcccaccc

ctcataatta

aaagaagcta

ATTCCTGAAG

AACTAGCAAG

TCTGCAGACT

ttaaaactcc

tgtttctttt

attctcaaca ggcagttgcc 737 BamHI748 gtagCGTGCT CTTCAGGATC

aaacaaatag

CTGCACTTCC

gcaccttctc 89 GACCCACACA ATGCTTTCCA

CCAATGTTGC

TGCTTTCATG CCTGACGGGA

TTGGAA TCGGACGCTG

TCTGCAGTCT

CCAGTTCCAC

AGATCCGACC

AGTTATGATG

CTTCATGCCA

GGCTTTG~A

CCATCCCATA

TAACGATCTG

CCTGCGCTAG

52

ACTTTTGGGG GCTTTGGACC

736

AGgtactacg Exon

3

AG

7

ctaagccgtc

GTTGCCGCTC

TGCGTCGAGG

ACTCCGCACC

TCAGTCGCCT

CTGCCACATC

TGTTGCAACG

GTGGAGCCCA

TCCAGGGTGA

ATCCAGGATA

AGCAAGGCCC

ACCATCCTCT

GAGTACATTT

ATCTAAATAT

GTTCGAGAAC

TTTGCACCCG

gtcccactca

ctttgcagtg

acgccaggct

GGTGCACACA

ATTACCATCC

TTTACCTGTA

GCCCTGGAGA

TTGAACGGGA 287 GAGGAAAAGg

AGCGGGTGTT 859 GCACCAGgtt

ATCGTTCCAG

AAGAAGACAG

tatgttttgg

cgcctgcact

tgtttggact

ctgctgacag

gctagatgat

ggattccaag

gcttgaggaa

gttagtccac

ccccacattt

ggtttgaaat

gcattgtcag

aattggttgt

tttgtacttt

catgctaatg

cttgcacctt

ctgaggaaca

cgctaatgct

tgcaccctct

gaggaacatg

ctcacgttcc

aagccttcac

tgaattgtct

gttgcagata

gggctccttt

tgaggttttc

ctaaatgctt

gcaccttctg

aggaaca

ttaagtgact

aaggcacctt

qtgcccatca

gtttcgccgt

ggagccttta

gatggg

taccattgat

tgtgtgggta

cggaaaacat

ctttgtagat

cacagggtgt

gtcagaatga

tatactacta

ttaaattact

taagtagtca

gataaaatat

agcttaatat

ttctttctaa

ctcttagtgg

gaagattgaa

gtctttgttt

catcgatgca

tgagaatgac

atcaaagtca

gtaggcttgc

accaaaccat

tttaagcata

gaataataat

atcaatatca

agttgtgccg

cctgttgtct

attgtggatt 860 ctcccagGTC

CTGTTTATTG

CTGATGAAAT

ACACACAGGA

gtgtttagag

gcttaagaaa

gtggcataca

agactgtcat

TTGGCTAGAA

CTGGTCGATG

GCTAGCTGTG

TGACATAGTT

TATGTGGGAT

GTGGAAGGCA

GGCAGTACTT

CTTCTTGGGA

AGGCCCTTTC

TGGTGGCTTA

GATCATGAGA 988 TATCCCgtaa

ATGTCAGACC

ccctctactt

tcatcttcat 288 cagGCATTTA

tttgggtctt

tctttcccct

gtcaatgccc

tgatttttat

CGACTTCCTG

AGTGCTTATG

GTGCTGTCAG

CCAAGGACAC

TGCCACCCAA

AGATCATAGA

tttctcctct

gctgactgtg

actgggatgc

c

AGCCATGAAG

AGTCAGGTGG

CGGGCTTTCT

ATAACAATGT

TATGAGGAGT

ACAAGCTGAC HindIII473 ACATCACCAA

ATTAACATCT

CCTTGGTGAA

GCTTTTCAAC

TACAACAAAG

TTCTTCCTAT

Exon

4

8

379 Exon

9 tggttgaagc

cttacagagt

tttagtacca

gcattataga

gctgagaaaa

tgaggagcaa

catgaagttt

ttgtctttca

gGTGTCTGCA

Exon

agtatctgtg

tccctggtgt

ATGATGACAT

AATGCTGACC

ATTAAACCAG

GGCGGCAACC

CCGAATTGCT

ATTGCGGCTC

GCGAGCACGG 1102 TTGAGgtgaa

CTCCACGTAT

CACTAGGCTG

aacggatatg

gctgaatgat

agcatcttta

agatgtgttg

aagttatttt

989

512 GAATACAG 5

513 GA GTGGAGGCCG

HindIII539 GAGAGACTGC GTGTAAGCTT

GCTCGCCGTT

ACAAAGCAAA

GCTG

GGGGCTACAC

GTGCTGTGTG

6O8 CGTGAAAGGC

ATCCAGAAAT

GATCGTTTTT

Exon

10

acatgggttg c t c t a g g a t a

ctcattggta

taatttttta

1103 aaaaacagGT

TTTAGAAGAA

GAGCATCTTG

CTGAAAACGC

AGACAAGATG

GGTGCCATCT

TGAGAAAGGA

GCTCATGAAG

ATGTTGTGAC 1247 CGAGAAACCA AAGgtagtat

TGCTGTGAGA

GGGAAAGGGT

TGCTAAATGC

CATCGTCATC

cacagcacac

tgtgctgagg

gccctttcaa

qgcgccgtgg

gtaaggcagt g c c t a a c a g t

tgattaga

ACGCTTGGAA

CTGCCATCTG

Exon

II

ttcatactag t c t c t g g g t t

tatactgtga

ccattttctt

1248 gtagATTGTG

GGTGTGCCTG

CGACTCCGAG

ATAATGGGCT

TCTGGCCAAG

CCAACCCATG

GTGATATCAT

CCGGCTTGCC

CCTCCACTTG

GGATGAGATC

CGGGAGTCAG

TGGAGATCAT

CAACAAGACC

ATCTTGTCCT

TGATCAAAGA 1408 TCTGAGAGTA

GGAACTCTGG

GGAGCCATCT

TCAGATGGGG

CTCTTGTGAA

ACTCTGCTTG

GGATGGGCAG

ATTCGGCTTG

TCTGTCTCCT

AAAAGACAAT

TTTTTGAATA

TGTATTATAT

ATTTCAGTTG

ATGCATAGTG

GAGTGACACC

TAGGAACCTG

CAGGTGGCTG

CGTGACACAA

GAGTGAGAGC

GAGAGGCATC

TCTTTGTTAA

AGTTTGACTG

TGTGGGAGCT

TTCTAAGGAG

AAACAGATCT

ATCTGCATAC

AGCCTGCAGA

GTCCTGCCGT

AATTTACATG

TGTCTTTACA

TCTTCCTTGC

TGGCATGAAT

GTTTTGTATT

TGGAAGT~'~T

TTTCTGAGAT

ACTACATAAG

AAACTGTTGA

ATCATTATAA

TCAATGAATG

GTAAGATGAT

TGAGGGTTGA

GCATATGTAA

AATACTAGTT

TAAAGTACAC

TTTGCATTGG

CCAACAGCAG

AATGTATTAT

ATAGTTTCTG

AGAATTCATT

ACCAAATTAT

ACTTTAAGTG

ATTTGTAAAA

CATTTATTTT

CAGTATTTCT

CTTGATTGCA 1977 CTTT

EcoRI1885 HindIII1961

TTGAATAAAG

CTTCGTTTTT

Fig. 2. Nucleotide sequence of the rat gene encoding ornithine aminotransferase. The 5' flanking domain and introns are presented as lower case letters and the cloned exons are presented as upper case letter. An exon corresponding to exon 2 of h O A T has not been identified in rOAT. The sequences of the uncloned exons (part of 4 and all of 5 and 6) are presented as italicized upper case letters, and are derived from the rat O A T c D N A [17]. The putative organization of these uncloned exons is derived from the structure of h O A T [1,2]. Nucleotides contained within the exons are numbered relative to the major site of transcription initiation. Position 89 corresponds to the first nucleotide of the translation initiation codon, position 1408 corresponds to the last nucleotide of the translation stop codon, and position 1977 corresponds to the nucleotide preceding the site of polyadenylation [17]. Methods: Select restriction fragments from the genomic clones were recloned into M13 and pUC vectors for D N A sequence analysis by the method of Sanger [22]. Oligodeoxyribonucleotides for use as sequencing primers were synthesized on an Applied Biosystems Model 380B D N A synthesizer. D N A sequence data were stored and analyzed using the SAM and EuGene programs (Molecular Biology Information Resource, Baylor College of Medicine). The sequences of the exons and flanking sequences have been assigned the following GenBank accession numbers: exon 1, M93294; exon 3, M93295; exon 4, M93296; exon 7, M93297; exon 8, M93298; exon 9, M93299; exon 10, M93300; and exon 11, M93301.

217 the regulation of OAT gene expression at the translational level [21]. The exons of rOAT were numbered as illustrated in Figs. 1 and 2 in order to maintain consistency with the numbering of exons in hOAT. Exon 3 of rOAT was localized 113 bp to the 3' side of the third BamHI sites contained within this gene (Fig. 1). It was determined that exon 3 of rOAT is 235 bp in length and contains the translation initiation codon at position 89 (Fig. 2). In hOAT, exon 3 is 228 bp in length; nonetheless, following alignment of the rOAT and hOAT sequences to maximize identity, the locations of the 5' and 3' splice junctions were observed to be conserved (data not shown). The first 92 bp of exon 4 of rOAT were localized at the 3' terminus of the 5' contig (Figs. 1 and 2). If the e x o n / i n t r o n organization of rOAT is identical to that of hOAT, then exon 4 of rOAT would be 225 bp in length and would contain the HindIII site located at position 473. The HindIII fragment extending from the 5' contig into the uncloned region of rOAT would then be approx. 600 bp in length (Fig. 1). Supporting this prediction is the observation of a 600 bp HindIII fragment, upon Southern blot analysis of rat genomic DNA using the OAT cDNA as the probe [18], that is not derived from other regions of rOAT (Fig. 1) or either of the three OAT pseudogenes [18]. If rOAT and hOAT are identical with respect to exon/intron organization, then exon 5 of rOAT would be 96 bp in length and would contain the HindIII site located at position 539, whereas exon 6 would be 128 bp in length. The only HindIII fragment observed upon Southern blot analysis of rat genomic D N A that could not be unambiguously assigned to one of the OAT pseudogenes or the cloned regions of rOAT is approx. 1100 bp in length [18]. It was assumed that this HindIII fragment is located in rOAT adjacent to the 600 bp HindIII fragment (Fig. 1). If this assumption is correct, then this fragment would contain 39 bp of exon 4 at its 5' terminus and 27 bp of exon 5 at its 3' terminus. The apparent lack of other OAT-related HindIII fragments in the rat genome [18] suggests that the remaining 69 bp of exon 5 and all of exon 6 are contained within an approx. 9000 bp HindIII fragment that extends from the uncloned region into the 3' contig. From the available data, the location of exon 6 within rOAT could not be established. This proposed organization of rOAT suggests that the uncloned region extends over approx. 3 kb (Fig. 1). Supporting this proposed organization of rOAT are genomic Southern blot data suggesting that exons 4, 5, and 6 of rOAT reside within a BamHI fragment of approx. 6500 bp and within EcoRI fragments of approx. 3100 and 1700 bp [18]. Should rOAT contain additional undetected intron sequences within this uncloned region, it would be unlikely that the putative HindIII, BamHI, and EcoRI maps would align so precisely. As our research interests lie primar-

ily in the regulation of rOAT expression, it was decided not to isolate and characterize the uncloned region of rOAT. The structure of the 3' contig was derived from 4 distinct overlapping clones and represents approx. 19.5 kb of the rat genome. Exons 7, 10, and 11 of rOAT were localized within this contig by Southern blot analyses (data not shown). The distances separating each of the five exons within this contig from the others were determined through the use of the polymerase chain reaction coupled with exon specific primers and the cloned DNA template (data not shown). Exon 7 is 123 bp in length and contains the BamHI site at position 748 (Figs. 1 and 2). Exons 8, 9, and 10 are 129 bp, 114 bp, and 145 bp in length, respectively. The corresponding four exons in hOAT are identical with respect to size and splice junctions [1,2]. Exon 11 of rOAT is 730 bp in length and contains the EcoRI and HindIII sites located at positions 1885 and 1961, respectively. In hOAT this exon is 793 bp in length due to the presence of additional nucleotides in the region of the exon encoding the 3' untranslated portion of the mRNAs. In summary, the data presented above illustrate the probable structure of a 36 kb region of the rat genome containing rOAT. This gene appears to extend over approx. 20 kb from the site of transcription initiation to the site of polyadenylation, and is organized into at least 10 exons. The promoter has been localized and approx. 5 kb of the 5' flanking region have been cloned. The regulation of expression of this gene can now be examined in vivo, in culture cells, and in various in vitro systems, thereby allowing the physiological significance of observed molecular regulatory mechanisms to be assessed. This research was supported by grants HD24189, CA36727, CA07175 and CA22484 from the National Institutes of Health, grants JFRA-227, SIG-16, and IN165A from the American Cancer Society, and grants 90-39 and 91-02 from the Nebraska Department of Health. The authors wish to thank Drs. Tom Beck and Charles Kasper for providing the genomic DNA library, and Dr. Tony Hollingsworth for his comments on the manuscript. References 1 Mitchell, G.A., Looney, J.E., Brody, L.C., Steel, G., Suchanek, M., Engelhardt, J.F., WiUard, H.F. and Valle, D. (1988) J. Biol. Chem. 263, 14288-14295. 2 Zintz, C.B. and Inana, G. (1990) Exp. Eye Res. 50, 759-770. 3 Barrett, D.J., Bateman, J.B., Sparkes, R.S, Mohandas, T., Klisak, I. and Inana, G. (1987) Invest. Ophthalmol. Vis. Sci. 28, 10371042. 40'Donnell, J.J., Vannas-Sulonen, K., Shows, T.B. and Cox, D.R. (1988) Am. J. Hum. Genet. 43, 922-928. 5 Ramesh, V., McClatehey, A.I., Ramesh, N., Benoit, L.A., Berson, E.L., Shih, V.E. and Gusella, J.F. (1988) Proc. Natl. Acad. Sci. USA 85, 3777-3780.

218 6 Mitchell, G.A., Brody, L.C., Looney, J., Steel, G., Suchanek, M., Dowling, C., Der Kaloustian, V., Kaiser-Kupfer, M. and Valle, D. (1988) J. Clin. Invest. 81, 630-633. 7 Inana, G., Hotta, Y., Zintz, C., Takki, K., Weleber, R.G., Kennaway, N.G., Nakayasu, K., Nakajima, A. and Shiono, T. (1988) Invest. Ophthalmol. Vis. Sci. 29, 1001-1005. 8 Shull, J.D. and Pitot, H.C. (1989) In Vitro Cell. Dev. Biol. 25, 971-976. 9 Inana, G., Chambers, C., Hotta, Y., Inouye, L., Filpula, D., Pulford, S. and Shiono, T. (1989)J. Biol. Chem. 264, 17432-17436. 10 Mitchell, G.A., Brody, L.C., Sipila, 1., Looney, J.E., Wong, C., Engelhardt, J.F., Patel, A.S., Steel, G., Obie, C., Kaiser-Kupfer, M. and Valle, D. (1989) Proc. Natl. Acad. Sci. USA 86, 197-201. 11 Mitchell, G.A., Labuda, D., Fontaine, G., Saudubray, J.M., Bonnefont, J.P., Lyonnet, S., Brody, L.C., Steel, G., Obie, C. and Valle, D. (1991) Proc. Natl. Acad. Sci. USA 88, 815-819. 12 Ramesh, V., Gusella, J.F. and Shih, V.E. (1991) Mol. Biol. Med. 8, 81-93. 13 Mueckler, M.M., Moran, S. and Pitot, H.C. (1984) J. Biol. Chem. 259, 2302-2305.

14 Fagan, R.J., Sheffield, W.P. and Rozen, R. (1989) J. Biol. Chem. 264, 20513-20517. 15 Mueckler, M.M., Merrill, M.J. and Pitot, H.C. (t983) J. Biol. Chem. 258, 6109-6114. 16 Merrill, M.J., Mueckler, M.M. and Pitot, H.C. (1985) J. Biol. Chem. 260, 11248-11251. 17 Mueckler, M.M. and Pitot, H.C. (1985) J. Biol. Chem. 260, 12993-12997. 18 Shull, J.D., Pennington, K.E., George, S.M. and Kilibarda, K.A. (1991) Gene 104, 203-209. 19 Engelhardt, J.F., Steel, G. and Valle, D. (1991) J. Biol. Chem. 266, 752-758. 20 Luckow, B. and Schutz, G. (1987) Nucleic Acids Res. 15, 5490. 21 Fagan, R.J., Lazaris-Karatzas, A., Sonenberg, N. and Rozen, R. (1991) J. Biol. Chem. 266, 16518-16523. 22 Sanger, F., Nicklen, S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467.