Structural and functional analysis of the first intron of the human α2(I) collagen-encoding gene

Gene, 89 (1990) 239-244 Elsevier

239

GENE 03487

S t r u c t u r a l a n d f u n c t i o n a l analysis o f the first intron o f t h e h u m a n ~2(I) collagen-encoding gene (Nucleotide sequencing; transcriptional regulation; gene expression; recombinant DNA; gene evolution; transfection)

Anne L. Sherwood*, Ralph E. Bottenus, Mark IL Murtzen and Paul Borns~ein Department of Biochemistry, Univers~ffof Washington, Seattle, WA 98193 (U.S.A.) Received by J. Piatigorsky: ! ! October 1989 Revised: 17 December 1989 Accepted: 19 December 1989

SUMMARY

The nucleotide (nt) sequence of the first intron of the human u2(I) collagen-encoding gene (COLIA2) has been determined from its 5' terminus (nt 207) to nt 2045 with respect to the transcription start point. Although the first intron contains elements known to function in transcriptional regulation of other genes (two APl-binding sites and an alternating GT stretch), comparison of this sequence with that of the mouse COLIA2 first intron revealed a low degree of nt sequence identity and very few common DNA-protein binding motifs. In keeping with this structural analysis, the human intron was found to inhibit COLIA2 promoter activity in transfection experiments, whereas a strong enhancer was reported to be present in the first intron of mouse COLIA2 [Rossi and deCrombrugghe, Proc. Natl. Acad. Sci. USA 84 (1987) 5590-5594]. We conclude that the high degree ofnt sequence conservation existing in the promoter and first exons of human, mouse and chicken COLIA2 does not extend to the first introns of these genes but that the promoter activity of COLIA2 is strongly influenced by the presence of the first intron.

INTRODUCTION

A number of potential regulatory elements have been identified in the first intron of several collagen-encoding genes, including human COL1AI (Bornstein et al., 1987, 1988a; Bornstein and McKay, 1988; Rossouw et al., 1987), Correspondence to: Dr. P. Bornstein, Dept. of Biochemistry SJ-70, University of Washington, Seattle, WA 98195 (U.S.A.) Tel. (206)543-1789; Fax (206)545-1792. * Present address: Pacific NW Research Foundation, Dept. of Biochemical Oncology, 720 Broadway, Seattle, WA 98122 (U.S.A.) Tel. (206) 726-! 246. Abbreviations: ~2(!), ~2(1) collagen; BOH,gene encoding bGH; bp, base pair(s); CAT, chloramphenicol acetyl transferase; COLIA2, gene encoding ,,2(1); bGH, bovine growth hormone; CTF, chick tendon fibroblasts; kb, kilobase(s) or 1000 bp; LTR, long terminal repeat; nt, nueleotide(s); oligo, oligodeoxyribonucleodde; RSV, Rons sarcoma virus; ss, single strand(ed); TPA, 12-O-tetradecanoylpborbol-13-acetate; tsp, transcription start points. 0378-1119/90/S03.50 © 1990 ElsevierSciencePubh~hersB.V.(BiomedicalDivision)

mouse COLIAI (Rippe etal., 1989), mouse COLIA2 (Rossi and deCrombrugghe, 1087), rat COL2AI (Horton etal., 1987) and mouse COL4AI (Killen etal., 1988). Regions containing strong transcriptional enhancers were reported in the first intron of mouse COLIA2 (Rossi and deCrombrugghe, 1987) and rat COL2AI (Horton et al., 1987). Blocks of regulatory sequences capable of exerting both positive and negative effects on promoter activity were described in the first intron of mouse (Rippe et al., 1989) and of human (Bornstein etal., 1987; Bornstein and McKay, 1987; Rossouw et al., 1987) COLIAI. Some of these intronic segments were shown to contain consensus sequences for known transcription factors (Bornstein et al., 1987; Rossouw et ai., 1987), but the precise role of such intronic elements in transcriptional regulation of collagen genes is not yet clear. The structure ofhuman COLIA2 has been studied by a numberofinvestigators(Myerset al., 1983; Bernardet al., 1983; Dickson et al., 1985; De Wet et al., 1987; Lee et al.,

240

1988). Studies have also been conducted on the role of the first intron in regulation of mouse COL1A2 expression (Rossi and deCrombrugghe, 1987), and on functional elements in the mouse COL1A2 promoter (Karsenty et al., 1988). However, to date no work has been reported on the regulation of expression of human COLIA2. We have sequenced 1.8 kb of the first intron of human COLIA2 from the first exon/intron junction at nt 207-2045 with respect to the start of transcription. In this paper we present a structural analysis of this region and a comparison of mouse and human COLIA2 intronic sequences in order to identify conserved regions that might be important in the regulation of the gene. We also report the results of transfection experiments demonstrating that, in contrast to the mouse intron, the inclusion of intronic sequences in human COLIA2 promoter-bovine growth hormone (bGH) constructs inhibits promoter activity.

MATERIALS AND METHODS

Chick tendon fibroblasts (CTF) were transfected as previously described (Gorman, 1985; Bornstein eta]., 1987) using the calcium phosphate coprecipitation technique for the introduction of DNA into cells. Efficiency of transfection was monitored by cotransfection with a plasmid containing the lacZ gene driven by the RSV-LTR (Bornstein and McKay, 1988) or an RSV-LTR luc plasm'~d (De Wet et ai,, 1987). in one set of experiments, transfection efficiency was assessed by cotransfection with a metallothionein I-human growth hormone plasmid (Selden et al., 1986). Total RNA was isolated from transfected cells and analyzed for bGH messenger RNA by an RNase pro. tection assay with a 32P-labeled antisense riboprobe as previ6usly described (Bornstein et al., 1988b).

RESULTS AND DISCUSSION

(a) Sequence analysis The DNA sequence of a portion of the first exon from an Sphl site at nt 56 through most of the first intron to an EcoRl site at nt 2040 was determined. A restriction map of this region and of ~ 3.7 kb of a 5'-flanking sequence is shown in Fig. 1 and the sequence of the intron plus a portion of the first exon is presented in Fig. 2. The sequence of the first exon and the first 76 bp of the first intron had been reported previously (Dickson eta]., 1985). The sequence in Fig. 2 differs from the published sequence in that nt 70 is a T instead of a G, a G is inserted at position 96 and a T at position 117. All three changes increase the similarity of the human and mouse genes (Rossi and deCrombrugghe, 1987).

Xb Xh B Ps

?

,,

V

?

Fig. 1. Restriction map of human COLIA2 first ~Ltron and 5'-flanking region. The sequence for the 5'-flanking region has been determined to nt -335 by Dickson et al. (1985). The untranslated region of the first exon is represented by the open rectangle and the translated region by the closed rectangle. The intron is represented by the parallel lines. The positions of restriction sites in the Y-flanking sequence are approximate. B, BglIl; E, EcoRI; H, Hindlll; Ps, Pstl; Pv, P~ulI; Xb, XbaI; Xh, Xhol.

Sequence analysis revealed several consensus motifs for trans-acting protein factors. At nt 192-205, just 5' to the intron/exon junction, is a sequence (TAGCAACATGCCAA) which closely resembles the recognition site for the adenovirus replication protein, nuclear factor 1 (NF1) (Jones et al., 1987). The consensus recognition sequence for NFI has been established as TGGN(6-7)GCCAA (Jones et al., 1987; Oikarinen et al., 1987). A potential AP 1 binding site (TGAGTCT) is present on the top strand at nt 1112 and another is found on the bottom strand at nt 1085. AP1 is reported to be ~ DNA-binding protein that interacts with enhancer elements and is inducible by TPA (Lee etal., 1987). A consensus motif (TGA_CrFCT) has been reported in a number of TPAC inducible genes (Lee et al., 1987; Angel et al., 1987). The pentanucleotide (CCCTG), which is highly repeated (twelve times) in the mouse COLIA2 first intron on both DNA strands (Rossi and deCrombrugghe, 1987) is present ~ive times in the human intron and once in the first exon. Within the intron, it appears as a direct repeat at nt 264 and 269. This motif is also present at nt 759, nt 766 and 870 in the intron and at nt 92 in the first exon. Finally, a region containing an alternating GT stretch is found at nt 1421-1460. Alternating dT and dG residues are highly repeated in the human and other genomes, with estimates of(dT-dG)n in the human genome ranging as high as 105, assuming the average size o f n is 25 (Hamada and Kakunaga, 1982; Hall and Cowan, 1985). Alternating purine/pyrimidine sequences have the potential to form Z-DNA, a left-handed conformation of the double helix (Hamada et al., 1984; Rich et al., 1984). Z-DNA has been found in association with a number of known enhancer and regulatory sequences and has been proposed to function in transcriptional control, perhaps through an effect on chromatin structure (Nordheim and Rich, 1983; Rich et al., 1984). (b) Comparison of the human and mouse COLIA2 first intron A sequence comparison was made between the human and mouse (Rossi and deCrombrugghe, 1987) COLIA2 ru.st introns and a dot matrix diagram is presented in Fig. 3. When the first 1390 nt in the human intron were aligned

241 GCATGCCCG CGCnCGnOAG GTGAT&C~J~C CGCCGGTGAC CCAGGGGC'I~2

100

TGCGACACAI% GGI%GTCTGCA T G T C T ~ G T G CTI%GAC,ATGC T ~ G ~ E T T G T GGI%T,~CGCGG ACTTTGTTGC TGCTTGCAGT ~ C C T T A T G C ~ A G C A I % ~ T

200

GOCRATGTAA

GTGCCTTCAG

CTTGTTTGGG

GGAGACTGGG

TAGAGAGGTT

AGATGGGAGO

GCACCCTGCC

CTGAAAAGGA

AAACCTGTAA

CCTGAATTCC

3C0

AGGTACACTT

GGAGGGCAGA

CTCTCAGGCA

TGTGGGAAAA

CGCCGGAATT GATAAGAAAC

ATGGAAATTA

CTTTAAAAAA

TGAAAACATA

AAAGCCTTGC

~ 00

CAAAAGTTAG

GGAACTTTTC

CTCTAAGTTC

AGAOTGAGAC

AGTTAACTCG

T C A G C T T A G T AACCCCCAAA G G G A G C G G A A

GGTCTTTTTC

500

GTCTGGCTCC

CCTAAGGATG

AGATATTAAC

GACCAATGTG

GTGGAGGAAG

TCAAGGGCCT GCACCCCACA

GGCCCCATAA

CCGCACTGAT

GTCCACCTTG

TAAAACTTGA

600

GGCCTGCGTT

AGAAAGCCCT

TCAACTGAOT

AATGTAAAAC

TCACCTCCTA AGAGCTTTTA

TCTTCTGGGC ATTGTAAGGC

TTGTCCGGAG

GAGGAGGATG

7OO

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GCCCTCTGGA

GGAAGGAAAA TGAAAGTACA

GGGGACAGGG

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AATTAAAGCT

800

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TATACAGATT

AATGATCAGC

ATTTCTGGTT

GGAGCCTTTC

GTCAGTGAAC

CCTGGAAAGA AGAATGGATG

CTACTTGGAG

900

TGGGTACATT

CTGAAAAGTA

ATATAAGTGT

CTCAATTCAC

TTTCTAGTCA TGGAAATGGT

A A C A T T T T T T AACTCAAATC

TGCTCTAAAT

TTTGTTTGAG

i000 ii00

CCAGTGGCTA

CCTGAGAATT

ACCCCTTTGA

CATGTTCCCA

GTGATAAGCA

AACATTATGA ACGCAGCAAG

T T G A G A A A T A TCAACATTGA

GATG~-~'~C--~GAGACCGG

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/~-G~C-~GA

CACCAATTTG

CTGCGTGACT

TTGGGCAAGT

T T T C T A A A A T GTGAGACAGA

GATTAAAGGG

ACCCCAAGGC

1200

CACTTTCCAG

CTCTAGGTTC

CATGGCCAGA

CTTTCATGTC

AACAGAGAAT GAAGAAGATC

AGTCCGTTTT

CATCTTGAAA

ATGGCTGCCA

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1300

CAAACGGCCT

CAAAGATATT

GACTAGATGG

GGGATGGTAT

TGTCTGACCA

CACCCAGTAC

TCCAAAAAGT

TGTTCCACCC ACACAGCACG

GTGTCTACCA

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1400

TCTAATGCAT

TTGTGTGCTT

GTGTGTGTGT

GTGTGTGTGT

GTGTCTGTGT

GTCTGTGTGT

CTCTTCCCCC

TTCATTCACT

TTTAGTATAC

ATACTGTGGA

1500

TACTAAGGAG

TAATTGCAGT

GAACAAATTC

ACATTACCGA

GTTCATATTT

TTAATGAGAT

CTTGAGAGTG

GG~GGAAAGA

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AGAGAATAAA

1600

ATGAAGGCAG

ACTTAGGGAA

ATTTGAAGGT

ACAAAGGCAA

CTTACCTTCT

GATCAACAGC

CAACCACAGT

C~GGAATAAA

TGTTATCAAA

CACACATTCT

1700

TCAAAATGGT

CCGTGTCTGA

OTAATTAAAA

GGCAAATTTC

CAAAATCATA

AGGACTTCCG

TTAATCAACT

CAGGCATAAT

TATTCTTCCT

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1800

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TAACAGTAAT

TCTCGTAAAT

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CACCCTCCTA AATTGTGACT

1900

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ATGTTCTCAC

TTCA~%ATAAC G C A C T T C T T G

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TTCATCCTAC

TGCAAGCTTG GCCACACTTG

2000

TATCCTGTAT

TAACCTATAA

TTTTTGTACC

AATTC

GTAGGAGAAG

2045

Fig. 2. The nt sequence ofthe human COLIA2 first intron and a portion of the first exon spanning a region from nt 53-2045. Exonic sequence is shown in bold print. A potential NFI binding site, TAGCAACATGCCAA (underlined), is present at nt 192; potential API binding sites, TGAGTCT (boxed), are present at nt 1085 (bottom strand) and I 112; and a G + T-rich tract (doubly underlined) is located at nt 1421 (GenBank accession number M31886). DNA sequence analysis was performed using ss DNA and complementary oligos to form specific cleavage and ligation substrates (Dale et al., 1985; Cyclone ! Bicsystem, International Biotechnologies, Inc.). A specific oligo was hybridized to recombinant MI3 vector at the 3' end of the cloned insert. The annealed hybrid was digested with either Hindlll or EcoRI, depending on the specific oligo used, to open the vector• The linearized molecules were then digested using the exonuclease of T4 DNA polymerase to generate an overlapping set of subdanes. Aliquuts were removed at 5 rain intervals and rapidly heat inactivated. Aliquots from all time points were pooled and the deleted ss chains were tailed with dATP (M 13topiS) or dGTP (M 13mp19). Fresh oligo primer was annealed to the deleted products to join the two ends of the molecule and T4 DNA figase was added to seal the remaining nick• Competent Escherichia coil TGI were transformed, individual recombinant M13 plaques were obtained and ss DNA template was made by standard methods. Samples were then analyzed by electrophoresis on a 0.7% agarose gel to verify that a sui:able range of different size deletions had been generated• Snbclones were sequenced by the chain termination method of Sanger et al. (1977). Sequenase (U.S. Biochemical Corp.) was used with 3SS-ATP for DNA sequencing and labeled nucleic acids were electrophoresed on 6Yo polyacrylemide gels in an electrolyte gradient (Lurquin, 1988). Both strands were sequenced independently. The positions of the start of translation and the first exon/intron boundary were obtained from Dickson et al. (1985). DNA sequence analyses were performed using Genepro programs (Riverside Scientific, Seattle, WA).

with the first 1320 nt in the mouse intron, an overall identity of41% was found. There appear to be no areas ofextensive 1200 Od oJ U~

I000 800 I

O

~;

600

c o

400

H

200

.r" o."

J

0

z6o 46o' e6o 'e o ,obo Intron I , Hurnon a2 (I)

Fig. 3. Dot-matrlx analysis comparing the ~aman and mouse COLIA2 first introns. Human intronic sequences e~rtend from the exon/intron junction at nt 207-1600 and mouse intronic sequences extend from the exon/intron junction at nt 209-1529 with respect to the tap. A dot is reported whenever there are five matches out of 10 nt.

sequence identity between the two introns. Indeed, when the entire human first intron was aligned with only the region of the mouse first intron containing strong enhancer activity, nt 748-1040, (Rossi and deCrombrugghe, 1987) 35% identity was found. The portion of the human intron that best aligned with the mouse enhancer region spanned nt 235-673. These findings are in contrast with the extensive sequence identity between the first introns of human and rat COLIAI (Bornstein and Sage, 1989) and between the 5'-flanking sequences and first exons of human and mouse COLIA2 (Dickson et al., 1985). It has been reported that little sequence similarity was observed when the sequence of the first intron of mouse a2(1) (Rossi and deCrombrugghe, lq87) was compared with the sequence of the first intron of the corresponding chicken gene (Boedtkez et :11., 1985). Our results strengthen the observation that a 3eneral lack of conservation exists in the first intrcn of COLIA2 among different species. It is interesting that several areas of homology were found when the mouse first intron was compared with avian COLIA2 second and third introns (Rossi and deCrombrugghe,

242

1987). This observation suggests that key regulatory regions have been conserved, but are located in different regions within the gene of each species. The contrast in the types of consensus motifs for DNA binding factors that are present provides further evidence for functional differences between the human and mouse first introns. Rossi and deCrombrugghe (1987) reported the presence of two enhancer 'core' elements in the mouse intron, while none are present in the human intron on either strand. Two AP! consensus sequences and a GT stretch were found in the human intron, but the corresponding motifs were not present in that of the mouse. Finally, the highly repeated pentanucleotide (CCCTG) reported in the mouse intron is present only five times in the human. (c) Effect of the human COLIA2 first intron on promoter activity The structures of plasmids used in transfection analyses are shown in Fig. 4. In transiently transfected CTF, plasraids containing the COL,IA2 promoter alone were transcribed several-fold more effectively than plasmids containing the first intron in either orientation. The insertion of the 1.8-kb intronic fragment in the ( + ) orientation, in the appropriate position 3' to the promoter and first exon, resulted in a fourfold reduction in promoter activity, while the same fragment in the ( - ) orientation caused a less than twofold reduction, as measured by relative levels of bGH mRNA (Fig. 5). A typical experiment using the RNase protection assay for bGH mRNA is shown in Fig. 6. Preliminary experiments in which the same plasmids were transfected into NIH 3T3 cells yielded consistent results (data not shown). These findings differ from those reported by Rossi and deCrombrugghe (1987) for the mouse COLIA2 gene. These workers reported that a segment of the first intron in this gene markedly stimulated CAT activity when cloned either 5' or 3' to the COLIA2 pro. moter in collagen-CAT constructs. This difference may not be surprising in view of the marked sequence divergence between the first introns of COLIA2 in the two species (Fig. 3). It should be noted that the mouse promoter used by Rossi and deCrombrugghe (1987) in their studies extended to about 2.0 kb, while the human promoter used in our investigations was about 0.7 kb in length. However, this is unlikely to be the basis for the difference in these two studies since our preliminary experiments indicate that extending the 5'-fianking sequence to -3.7 kb does not significantly affect the activity of the human COLIA2 promoter (data not shown). (d) Conclusions We present here a structural and functional characterization of the first intron of human COLIA2. (1) Sequence analysis reveals the presence of several

(_~ E94) ,

_ E (N/B) CO ~ X b ~

(20E40| 3>

H E

Fig,4. StructureofpCOL~2-hGH and itsderivatives.The startingplasmids for this construction were pCOLI3 which contains a 4.0-kb flagment of human COLIA2 extending from an EcoRI site about 3.7 kb upstream of the ~p to an E¢oRI site at nt 294 in the first intron (88 bp from the intron/exon junction) and pCOLI6 which contains a 1.8-kb fragment encompassing the COLIA2 first intrun extcndi.n8 from the EcoRl site at nt 294 to the next EcoRI site at nt 2040. The latter fragment contains almost the entire first intron of COLIA2 (de Wet et at., 1987~ A 1.0-kb fragment extending from Xbal ( ~ -700) to EcoRI (nt 294) (see Fig. I) was excised from pCOLI3 and cloned into pUCI8 to yield pCOL~2. A 2,1 kb BaII-EcoRI fragment of BOll, containing cxons 2-5, was cloned by blunt and sticky end ligation into the polylinker ofplCl9R (containing two £¢olu sites), excised with E¢oRI and inserted into the unique EcoRI site in pCOL~2 to yield pCOL:t2-bGH. The correct orienration of b o l l was established by restriction analysis. Finally, the 1.8.kb intronlc fragment was excised l~om pCOLI6 with EcoRl and inserted in both orientatlons into pCOL~2.bGH, which had been partially dlguted with £¢oRI, to yield pCOL~2(294-2040)bGH and pCOL~2(2040-294)boll, In the latter two plasmids, the junction l~tw~en CO&IA2 and BGH occurs in an intron; therefore, splicing between the collapn first exon and bOH second exon would be expected to occur normally. All plasmids were grown in E. co//, DHS~ or BSJ72, and purified by bandinR twice in a CsCI gradient. Only plasmid preparations containing highly supercoiled DNA, as determined by agarose gel electrophoresis, were used in transfeedons, Numbers are relative to the start of transcriptiun (bent arrow) in the collagen gent as l. COL, collagen promoter; X, Xbal; E, EcoRI; N, Nml; B, Bail.

consensus motifs for DNA-binding factors that may play a role in regulating expression of this gene. (2) The degree of sequence identity is limited to 41% when the first introns of human and mouse COLIA2 are compared. This identity is reduced to 35% when the region of the mouse intron reported to have enhancer activity is specifically examined. These findings contrast with the considerable sequence similarity among human, mouse and chicken COLIA2 promoter and first exon sequences (Dickson et at., 1985, Rossi and deCrombrugghe, 1987; Schmidt et at., 1984; Vogeli et at., 1981). The human intron

243 Plosmid

Promoter

oCOLo2-bGH

Inlron

x~m~

G. b ._~H ~

bGH mRNA 402 ± 73 {12)

is more in keeping with that reported by Rippe et al. (1989) for mouse COLIAI. (4) T h e possibility exists that functional elements similar

pCOLa2(294-2040)bGH X ~ m ~ ~ ) ~

10

pCOLa2(2040-294)bGH Xi l l m "[e,rl [t~ = m ~.

(9)

2 75 + 53 (14)

FiR. 5. Diagrammatic representation of plasmid pCOL=2-bGH and its derivative and tabulation of relative transcriptional activity in Ci'F. All values for bGH mRNA represent mean and S.E., normalized to pCOLa2(294-2040)bGH, with the number of independent determinations g/yen in parentheses. Data for pCOLa2-bGH, pCOL'`2(294-2040)bGH and pCOL=2(2040-294)bGH were obtained using 3, 2 and 3 different clones, respectively, ofthc same construct. The heavy line designates a 5'-flankin$ sequence starting at the Xbal (Xb) site at nt ,,, -700. The bent arrow indicates the tsp. The untranslated region of the first exon is represented by the open rectangle and the translated region by the closed rectangle. The orientation of the intron is indicated by the arrow. The B O H cassette is represented schematicallyby four rectangies.

201 --

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4

5

6

Fig. 6. Autoredtogram of bGH mRNA analyzed by solution hybridt~atinn. RNA from CTF, transiently transfected with COL IA2-BGH fu, ion Cane plasmids°was hybridized with a 3=P-labeledanti-senseI ~ H riboprobe. Alter RNase digastion, material precipitated with ethanol was analyzed by alectrophorasisin a 6% polyacrylamide.ureadenaturing gel. A major protected band of about 170 nt is indicated by the arrow and a minor band orabout 130 nt by the arrowhead. Lanes: I° Mspl digest of pBR322 end-labeled with ]'P and 1"4 kinase; 2 and 3, pColh%bGH; 4, pCola2(2040-294)bGH; 5, pCoI'`2(294-21M0)bGH;6, bGH riboprobe. The relative bGH mRNA levels in this experiment, corrected for quantity of RNA hybridized and efficiencyof transfectinn are: lane 2, 6.0; lane 3, 7.4, lane 4, 1.8 and lane 5, 1.0.

contains two potential API binding sites and a long G + T rich stretch both of which are lacking in the mouse intron. The human intron does not contain a number o~"motifs found in the mouse intron, including two enhancer 'core' elements. (3) Based on transfection studies, we found no evidence for an enhancer in the first intron of human COLIA2, in contrast to that reported for the first intron of the corresponding gene in the mouse (Rossi and deCrombrugghe, 1987). Instead, there was a fourfold inhibition of human COLIA2 promoter activity by the first intron. This finding

to those identified in the first intron of mouse COLL,42 may be~located elsewhere in human COLIA2. ACKNOWLEDGEMENTS

We thank Dr. Russel E. Kaufman (Duke University Medical Center, NC) for the generous gift of pCOLI3 and p C O L I 6 plasmids, Helena Sage for a critical reading and Kathleen Doehring for preparation o f the manuscript. This work was supported by grants AR11248 and DE08229 from the National Institutes o f Health.

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Oorman, C.: High alfician~ gone Uensfer into mammalian cells. In

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