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Human Molecular Genetics Pages 1491-1496  

Characterization of an intron splice enhancer that regulates alternative splicing of human GH pre-mRNA
Introduction
Results
   Location of the GH-1 IVS3 ISE
   Importance of runs of Gs within the GH-1 IVS3 ISE
   Importance of individual nucleotides within the GH-1 IVS3 ISE
Discussion
   Alternative splicing of GH-1 IVS3 is regulated by an ISE
   Tandem repeats of G3s are sufficient to comprise a cis splice element
   G3 repeats in GH-1 IVSs
   ISEs may regulate other endocrine pathways
Materials And Methods
   Site-directed mutagenesis
   Cell culture, transfections and RNA purification
   Quantitative RT-PCR analysis of mRNA
   Statistical analysis
Acknowledgements
Abbreviations
References

Characterization of an intron splice enhancer that regulates alternative splicing of human GH pre-mRNA

Characterization of an intron splice enhancer that regulates alternative splicing of human GH pre-mRNA

Eleanor M. S. McCarthy* and John A. Phillips III

Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, TN 37232-2578, USA

Received May 19, 1998; Revised and Accepted July 2, 1998

Splicing of pre-mRNA transcripts is regulated by consensus sequences at intron (intervening sequence, IVS) boundaries and the branch site. In vitro studies have shown that the small introns of some genes also require intron splice enhancers (ISE) to modulate splice site selection. An autosomal dominant form of isolated GH deficiency (IGHD-II) is caused by mutations in IVS3 of the GH-1 gene that cause exon 3 (E3) skipping, resulting in truncated hGH products that prevent secretion of normal hGH. Interestingly, some of these IGHD-II mutations perturb an ISE that is buried in IVS3. We localized this ISE by quantitating the effects of deletions within IVS3 on E3 skipping. The importance of individual nucleotides to ISE function was determined by analyzing the effects of point mutants and additional deletions. Our results show that (i) an ISE with a G2X1-4G3 motif resides in IVS3 of GH-1; (ii) both runs of Gs are required for ISE function; (iii) a single copy of the ISE regulates E3 skipping and (iv) ISE function can be modified by an adjacent AC element. Our findings reveal a new mechanism by which mutations can cause inherited human endocrine disorders and suggest that (i) ISEs may regulate splicing of transcripts of other genes and (ii) mutations of these ISEs or of the trans-acting factors that bind them may cause other genetic disorders.

INTRODUCTION

Genetic disorders can be caused by defects in transcription or translation as well as dysfunction of the final protein product. Many point mutations that cause genetic disease disrupt normal splicing of pre-mRNA, which removes intervening sequences (IVSs) and joins exons to produce mature mRNA (1). Splicing is regulated by consensus sequences at IVS boundaries (5[prime] and 3[prime] splice sites, called 5[prime]SS and 3[prime]SS) and the branch site (2).

Multiple cis elements and trans-acting factors interact to activate potential splice sites during development and regulate selection of alternative splice sites to achieve tissue-specific expression of different mRNA isoforms (2-4). These elements include purine-rich exon splice enhancers (ESEs) and A/C-rich enhancer (ACEs) elements (5). ESEs and ACEs are located within exons and they enhance the use of specific splice sites. While ESEs are purine rich, no consensus sequence describing all ESEs has been recognized. The ACEs are also difficult to recognize by simple sequence comparisons (5).

The small introns of some genes also require cis elements called intron splice enhancers (ISEs) for efficient splicing of transcripts (6-14). One example is IVS7B of chicken [beta]-tropomyosin, which contains an (A/T)GGG motif ISE (11). Multiple copies of this ISE work additively to regulate alternative splicing of [beta]-tropomyosin transcripts. A second example is IVS2 of human [alpha]-globin, which contains a G3X0-4G3 motif ISE, in which only the first and third Gs of the G3 repeats are important for its function (8). Copies of this ISE also act additively to regulate alternative splicing of [alpha]-globin transcripts. This ISE regulates splicing through an intron definition pathway, characterized by intron retention, rather than exon skipping, when the 5[prime]SS of the intron is mutated (15).

Cogan et al. reported previously that some mutations located 28-45 nucleotides (nt) into IVS3 (92 nt long) of the human GH-1 gene cause skipping of exon 3 (E3), resulting in an autosomal dominant form of isolated growth hormone deficiency (IGHD-II) (16-19). Since these IGHD-II mutations are buried in IVS3 and do not involve consensus sequences at its boundaries or its putative branch site, we hypothesized that they perturb an ISE which regulates IVS3 splicing. We localized this ISE within IVS3 by mutating the GH-1 expression vector pXGH5, transfecting mutant constructs into GH3 cells and determining their splicing patterns by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). To do this we analyzed the effects of point mutants (nucleotides +25-36) and deletions of IVS3, as well as addition of a second ISE, on alternative splicing. Our results show that the GH-1 IVS3 ISE is located 26 nt into this small intron and has a G3X3G3 sequence. Mutations of the second or third G, in either G3 of the sequence, increased E3 skipping by ~20 and 49% above normal levels, respectively; mutations of the first G increased skipping only slightly when occurring in the second but not the first G3. Changing the length of the X3 spacer from 1 to 4 nt did not affect E3 skipping, but when 0 nt were present, E3 skipping increased from 6% in normal to 14%. Also, G3X3G3 copy numbers of 0, 1, 2 or a partial G3X3G3 construct containing only one G3 resulted in totals of 60, 6, 3 and 57% E3 skipped splice products, respectively. We also found that the effect of the ISE can be modified by an adjacent AC element, which may be analogous to ACEs (5). Finally, constructs containing mutations reported to cause IGHD-II showed dramatic increases in E3 skipping. Our results show that an ISE regulating splicing of GH-1 transcripts (i) resides between nucleotides 26-34 of IVS3; (ii) has a G2X1-4G3 motif; (iii) requires both runs of Gs for its function; (iv) can be modulated by an AC element and (v) unlike the only other human ISE reported, does not work in an additive fashion. Our findings demonstrate a new mechanism (ISE mutations that perturb splicing) that can cause inherited human endocrine disorders and suggest that (i) homologous ISEs may regulate splicing of transcripts of other genes and (ii) mutations of these ISEs may cause other genetic disorders, including endocrinopathies.

RESULTS

Location of the GH-1 IVS3 ISE

Figure 1A shows the alternative GH-1 splice products obtained by quantitative RT-PCR analysis of mRNA from GH3 cells transfected with GH expression vector constructs. Typical data collected from the phosphorimager are shown in Figure 1B. The primers used for amplification were specific to the human GH-1 gene and do not amplify endogenous rat GH transcripts in the untransfected control samples (Fig. 1B, lanes 1 and 2). Transcripts from GH3 cells transfected with the wild-type GH-1 expression vector contain the same proportion of GH mRNA isoforms as those from normal human pituitary (~6% E3 skipping; Fig. 1B, lanes 3, 4 and 15, and Fig. 2B, lanes 1 and 2). As reported previously, mutation of the 5[prime]SS (+1G->A) of IVS3 of GH-1 results in 100% E3 skipping (Fig. 1B, lanes 5 and 6, and Fig. 2B, lane 3) (17). The GH-1 IVS3 ISE was localized by examining the effects of a series of contiguous 5 bp IVS3 deletions on the distribution of cDNA isoforms (Figs 1B and 2). To do this, we determined the amount of E3 skipped splice product relative to the full-length splice product in each sample. The [Delta]23-27, [Delta]28-32 and [Delta]33-37 mutants all significantly increased E3 skipping (53%, P < 0.01; 14%, P = 0.02; 17%, P < 0.01; Fig. 1B, lanes 9 and 10, 11 and 12, and 13 and 14, and Fig. 2B, lanes 5-7, respectively). In contrast, the [Delta]18-22 and [Delta]38-42 mutants did not increase E3 skipping significantly (3%, P > 0.7; 4%, P > 0.4; Fig. 2B, lanes 4 and 8). We restored the IVS3 length by filling the 18 bp deletion with IVS4 sequence (CACTGCTGCCCTCTTTTT) and still observed 60% E3 skipping (data not shown). This suggests that the sequence of the ISE, not the length of the intron, controls E3 skipping.


Figure 1. (A) Diagram of GH-1 pre-mRNA splicing showing alternative splice products and the sizes of their corresponding translation products. (B) Typical results of quantitative RT-PCR analyses of transcripts from normal human pituitary (lane 15) and GH3 cells transfected with either wild-type or mutant GH-1 expression vectors (lanes 1-14).


Figure 2. Effects of small deletions within IVS3 on GH-1 pre-mRNA splicing. (A) Diagram of sequential 5 bp deletions (underlined or large font) are shown below the sequence of IVS3. Additional deletions of groups of Gs are denoted above the sequence with arrows and the addition of a second ISE (Add 1 ISE) 3[prime] to the natural ISE is shown below. (B) Graphic representation of quantitative RT-PCR analyses of effects of IVS3 deletions/additions on the proportions of GH-1 mRNA isoforms. gray, full-length (22 kDa) splice products; diagonal hatching, cryptic (20 kDa) splice products; black, E3 skipped (17.5 kDa) splice products; horizontal hatching, abnormal splice products produced only by the [Delta]25-36 mutant. Column heights reflect corresponding mean percentages that each splice product comprises of the total splice products and the number of individual transfections and standard deviations are shown above the graph. Pituitary data were derived from multiple analyses of one pituitary sample.

Importance of runs of Gs within the GH-1 IVS3 ISE

The contribution of individual runs of Gs to regulation of E3 splicing was also evaluated. When IVS3 contained no runs of Gs ([Delta]25-36), E3 skipping increased to 60% (P < 0.01; Fig. 2B, lane 11) compared with human pituitary and wild-type transfections (6 and 5%; Fig. 2B, lanes 1 and 2, respectively). In addition, the [Delta]25-36 mutant caused two novel splice products to appear (total of ~13%; Fig. 2B, lane 11). These novel splice products were not observed in transcripts from wild-type or any other mutant transfections, and may arise because the [Delta]25-36 mutation shortens IVS3 to 80 nt, and are currently being sequenced. We suspect that these splice products may be identical to unusual splice products observed previously in the [Delta]28-45 mutant (J.D. Cogan, personal communication). In the case of the [Delta]28-45 mutant, a cryptic 3[prime]SS located at +98 of E3 was activated, causing the last 23 nt of E3, IVS3 and E4 to be treated as one exon. Single runs of Gs (mutants [Delta]25-29 and [Delta]31-36; Fig. 2B, lanes 9 and 10) increased E3 skipping (55 and 59%, respectively, both P < 0.01) to amounts seen in constructs having no runs of Gs (60%, mutant [Delta]25-36; Fig. 2B, lane 11). When an additional G3X3G3 ISE was added (Fig. 2A), no significant decrease in E3 skipping from wild-type levels was observed (3%, P > 0.1; Fig. 2B, lane 12).

Importance of individual nucleotides within the GH-1 IVS3 ISE

To determine the importance of individual nucleotides within the ISE, a series of point mutants were constructed and expressed in GH3 cells (Fig. 3A). Mutation of the second and third Gs in each G3 (G27A, G28A, G33A and G34A mutants) all increased E3 skipping significantly (16, 49, 23 and 53%, all P < 0.01; Fig. 3B, lanes 6, 7, 12 and 13, respectively). On the other hand, mutations of the first Gs (G26A and G32A) in both G3s caused only 3.7 and 8.9% E3 skipping, respectively (Fig. 3B, lanes 5 and 11).


Figure 3. Graphic representation of effects of IVS3 point mutations on GH-1 pre-mRNA splicing. (A) Individual point mutations and the double mutation are denoted by arrows above and below the sequence, respectively. (B) Graphic representation of quantitative RT-PCR analyses of effects of IVS3 mutations on GH-1 pre-mRNA splicing. gray, full-length (22 kDa) splice products; diagonal hatching, cryptic (20 kDa) splice products; black, E3 skipped (17.5 kDa) splice products. Column heights reflect corresponding mean percentages that each splice product comprises of the total splice products and the number of individual transfections and standard deviations are shown above the graph. Pituitary data were derived from multiple analyses of one pituitary sample. (C) Mutations made to test the effect of adjacent AC element on ISE control of IVS3 splicing. Sequences comprising the putative `AC' element are shown in large, bold text.

The G34A mutation, which significantly disrupted splicing, reduced the second run of Gs to two. To evaluate whether at least three Gs, in the second run of Gs, were required for ISE functionality, we added another G to the 5[prime] end of the second run of Gs of the G34A mutant (Fig. 3A, T31G+G34A). This double mutant, which yields a G3X1G3 motif, restored normal splicing (6% E3 skipping, P = 0.08; Fig. 3B, lane 16).

Interestingly, while the [Delta]33-37 and the G34A mutants both had a G3X2-3G2 sequence, they differed in their effects on splicing, in that they increased E3 skipping by 17 and 53%, respectively. We noted that the [Delta]33-37 mutation brought an AC sequence 2 nt closer to the G3X2-3G2 sequence, suggesting that this might be a modifying element similar to reported ACEs. To test if the AC was modulating the effect of the ISE, we replaced the wild-type ISE (G4ATG5AGACC) with G3TTATG3AGACC (M1) or G3TTATG3ACC (M2), to convert both to G3X4G3 constructs. The M1 mutant had wild-type levels of E3 skipping, while the M2 had no E3 skipping (Fig. 3B, lanes 9 and 10, and Fig. 3C).

DISCUSSION

Alternative splicing of GH-1 IVS3 is regulated by an ISE

Our results show that the GH-1 gene contains an ISE between nucleotides 26-34 of IVS3 (Fig. 3). From the effects of changes in the ISE (Figs 1-3, summarized in Fig. 4), we conclude that the structural motif of the GH-1 ISE is G2X1-4G3. The relative functional importance of the Gs in the runs of Gs is G3 > G2 > G1 (Figs 3 and 4A). Furthermore, our finding that site-specific mutations of the third G of either of the G3s (G28A and G34A) cause ~50% E3 skipping (Fig. 3, lanes 7 and 13) agrees with previous reports that natural mutations of either of these two nucleotides to an A is sufficient to cause IGHD-II (18-20). While constructs containing single G3s have decreased ISE function (Fig. 2, lanes 9 and 10), all those containing two G3 motifs in tandem separated by 1-4 spacer nucleotides (G3X1-4G3) retain normal ISE function (Figs 3 and 4). Addition of a second ISE (CGGGGATGGGGG) does not significantly increase E3 inclusion (3% E3 skipping, P = 0.14; Fig. 2B, lane 12), suggesting that copies of the GH-1 IVS3 ISEs do not act additively to regulate splicing. In addition, the ability of the ISE to regulate splicing of E3 is apparently modulated by a nearby AC element (Fig. 3C). When the AC element is immediately adjacent to the ISE, as opposed to 2 nt 3[prime] to the ISE, E3 skipping is non-detectable versus 9% (Fig. 3B, lanes 17 and 18, respectively). This suggests that the AC element may be similar to ACEs observed in Drosophila doublesex, human calcitonin and chicken cardiac troponin T (5).


Figure 4. (A) Location and consensus sequence (G3X1-3G3) of GH-1 IVS3 ISE. The relative importance of each nucleotide within the runs of Gs is proportional to its size and the X1-4 denotes that the two runs of Gs must be separated by 1-4 nt. The putative `AC' element is shown in italics. (B) Correlation of ISE motif changes with changes in GH-1 pre-mRNA splicing.

The IVS2 ISE of the human [alpha]-globin gene reported by McCullough and Berget contains a G3X0-4G3 motif (8). Because mutations of the [alpha]-globin IVS2 5[prime]SS cause inclusion of this IVS (i.e. E2, IVS2 and E3 are treated as one long exon), they propose that the ISE regulates splicing through an intron definition pattern (Fig. 5A and B). Since, in contrast, 5[prime]SS mutations of GH-1 IVS3 cause skipping of the upstream exon (i.e. IVS2, E3 and IVS3 are treated as one long IVS), we propose that the GH-1 ISE regulates splicing through an exon definition pattern (Fig. 5C and D). Thus, while the [alpha]-globin and GH-1 ISEs have nearly identical motifs, the splicing patterns (intron versus exon definition) of the IVSs in which they reside differ. Taken together, these data suggest that different human ISEs can regulate splicing of small IVSs to give different patterns of splicing. Regarding this, Talerico and Berget found that in small Drosophila IVSs with pyrimidine poor 3[prime]SSs the intron definition pattern of splicing was usually seen, while an exon definition pattern was favored when the 3[prime]SS was pyrimidine rich (14). Interestingly, the difference seen in pyrimidine tract strengths of the 3[prime]SSs of the human [alpha]-globin IVS2 and GH-1 IVS3 ([alpha]-globin has 5 Us + 6 Cs, while GH-1 has 4 Us + 9 Cs) agrees with the intron and exon definition patterns of splicing predicted from Talerico and Berget's findings. This suggests that pyrimidine tract strength may also affect ISE regulation of splice site selection in humans.


Figure 5. Graphic representation of the effects of 5[prime]SS mutations on splicing following either an intron definition (A) or an exon definition (C) pattern of splicing. Effects of ISE mutations on splicing following either an intron definition (B) or an exon definition (D) pattern.

Tandem repeats of G3s are sufficient to comprise a cis splice element

Several observations suggest that, while single G3s alone cannot, groups of G3s can modulate splice site selection. In a search of the Genome Database for cis splicing elements, Engelbrecht et al. determined that the sequence GGG occurs almost four times as often in the first 50 nt of introns compared with random sequences, and that the 6 nt consensus sequence with the highest information content is CCTGGG (21). Second, McCullough and Berget's analysis of the occurrence of triplets in 693 small IVSs supports the importance of G3s (8). They found that 20% of the small IVSs examined contained more than three times the number of G3s expected in a random sequence, when only 2% were expected to have that many G3s. In contrast, they found that only 1% of the IVSs analyzed had a similar number of A3s. Third, our results indicate that two G3s in tandem, separated by 1-3 nt, are required for the IVS3 ISE that regulates splice site selection of GH-1 transcripts.

G3 repeats in GH-1 IVSs

All of the GH-1 introns are short (92-260 bp) with IVS3 being the shortest. The number of G3s in IVS1-4 are six, seven, two and six, respectively. Thus IVS3 is distinct in having only two G3s, both of which are essential components of the ISE that is required for its correct splicing. The lack of additional G3s to compensate for mutation of the ISE in IVS3 may explain why IGHD-II mutations affecting G3s have only been found in IVS3, and not IVS1, -2 or -4. The physiologic importance of the IVS3 ISE is supported by reports of IVS3 mutations that cause IGHD-II because of exon skipping (18-20). The lack of identified mutations in G3s of IVS1, -2 and -4 may be due to the presence of multiple G3s in each of these IVSs and/or that these other IVSs are sufficiently long as to not require an ISE.

ISEs may regulate other endocrine pathways

Many human genetic disorders result from mutations that induce exon skipping. Our findings demonstrate that mutations in an ISE buried in an intron can disrupt splicing, resulting in exon skipping that produces a disease phenotype (IGHD-II). Our findings indicate that the GH-1 IVS3 ISE regulates splicing of IVS3 to give consistent proportions of alternatively spliced mRNAs, and presumably protein isoforms from the transcripts of a single gene. We found that a variety of site-specific GH-1 IVS3 mutations can perturb the function of this ISE, resulting in a shift in the proportions of different mRNA isoforms. Some of the mutations cause sufficient production of truncated protein products to cause IGHD-II. In addition to [alpha]-globin, chicken [beta]-tropomyosin and GH-1, this model may apply to other genes whose splicing may be regulated by ISEs. Finally, qualitative or quantitative changes in the trans-acting factors that interact with ISEs may adversely affect splicing to yield diverse protein isoforms, which have different metabolic functions.

MATERIALS AND METHODS

Site-directed mutagenesis

Some site-directed mutations of IVS3 of GH-1 were made as described previously (18). Others were made by wrap-around PCR mutagenesis of pXGH5, a GH-1 expression vector, using the Boehringer Mannheim Expand High Fidelity PCR System according to the manufacturer's instructions, with the addition of 0, 5 or 10% DMSO. Templates consisted of 600 ng of pXGH5. Mutagenic primer sets were designed with the point mutations contained at the 5[prime] end of one of the two abutting mutagenic primers. For mutations with multiple changes, each primer in the set contained half of the mutation at its 5[prime] end. Amplicons were evaluated on 1% agarose gels, and desalted using Microcon100 (Amicon) spin filters and water. Aliquots of the amplicons sufficient to be seen with a hand-held long-wave UV lamp, after electrophoresis in agarose gels containing ethidium bromide, were treated with T4 polynucleotide kinase (Gibco BRL). The kinased amplicons were then subjected to electrophoresis in a 1% SeaPlaque GTG low melting point agarose gel (FMC) containing 0.5 µg/ml ethidium bromide in 1× TAE buffer at 4°C. The linear amplicons were excised from the gel, melted at 68°C, then cooled to 37°C. Aliquots (6 µl) of the melted gel slices were added to pre-warmed in-gel ligation reactions, using Gibco BRL T4 DNA ligase (30 µl total volume), and incubated for 1 h at 37°C. XL1-Blue Escherichia coli were then transformed with 3-6 µl of these ligation reactions. Resulting colonies were picked and the mutant pXGH5 constructs were confirmed by sequencing.

Cell culture, transfections and RNA purification

Multiple 100 mm2 dishes of GH3 cells (rat sommatotrophs, a gift from Dr Ron Emeson at Vanderbilt University School of Medicine) were transfected with wild-type or mutant pXGH5 constructs, using Lipofectin (Gibco BRL) according to the manufacturer's directions. Forty-eight hours post transfection, total RNA was harvested using a Qiagen RNeasy total RNA isolation kit and treated with DNase1 (Ambion). mRNA was purified with a Qiagen Oligotex mRNA purification kit. Human pituitary total RNA was purified from anonymously donated human pituitaries (sample #338), obtained from the National Hormone and Pituitary Program (NIDDK, NICHD and USDA), using Trizol Reagent (Gibco BRL).

Quantitative RT-PCR analysis of mRNA

GH-1 cDNA was synthesized with the GH-1 specific primer 5[prime]-ACAAGGCTGGTGGGCACTGGAGT-3[prime], using InVitrogen's 1st Strand Synthesis Kit according to the manufacturer's directions. The resulting cDNA was desalted with a Microcon100 (Amicon) and recovered in 60 µl TE. Aliquots (13 µl) were used as templates for 25 µl PCR reactions for cycle curves and quantitative PCR analysis, based on the methods of McCarthy et al. (22). An aliquot of GH-1 specific PCR primers (5[prime]-CGTCTGCACCAGCTGGCCTTT-3[prime] and 5[prime]-CCACAGCTGCCCTCCACAGA-3[prime]) sufficient to generate cycle curves and to perform the subsequent quantitative PCRs was end-labeled with [[gamma]-32P]ATP. The number of cycles (usually 25-27) at which cDNAs of different lengths amplified at the same rate was determined using cDNAs from two independent transfections and the following cycling conditions: 4 min denature (94°C), followed by 20-32 cycles of 1 min denature (94°C) and 2 min annealing/extension (73°C). Amplicon lengths varied from full length (exons 2-5, 22 kDa protein), cryptic (exons 2-5, using the cryptic acceptor in E3, 20 kDa protein) and E3 skipped (E2 + exons 4-5, 17.5 kDa protein) splice products. After the optimal cycle number was determined, 25 µl quantitative PCR reactions were performed using the cDNA templates generated from human pituitary and from the wild-type and mutant transfections using the above cycling conditions. Upon completion, 5 µl of loading dye was added to the reactions, and 20 µl of the PCR products were loaded onto 20 cm 5% non-denaturing polyacrylamide gels (1× TBE). After the samples were subjected to 5 h of electrophoresis at 100 V, the gels were dried and exposed to a phosphorimager screen for 24 h. The relative amounts of full length, cryptic and E3 skipped cDNA amplicons were quantitated with a Molecular Dynamics phosphorimager and ImagQuant software.

Statistical analysis

A Wilcoxon ranked sum test was performed to test the null hypothesis that the amount of full-length and E3 skipped splice products of each mutant group was identical to that of the wild-type group. Due to the large number of tests performed, the more conservative P < 0.02 was considered significant.

ACKNOWLEDGEMENTS

This work was supported in part by NIH grant DK35592 ( J.A.P.). Human pituitaries (sample #338) were provided by the National Hormone and Pituitary Program (NIDDK, NICHD and USDA).

ABBREVIATIONS

3[prime]SS, 3[prime] splice site (acceptor); 5[prime]SS, 5[prime] splice site (donor); ACE, AC-rich enhancer; DMSO, dimethylsulfoxide; E3, exon 3; ESE, exon splice enhancer; GH-1, human growth hormone gene; hGH, human growth hormone; IGHD-II, isolated growth hormone deficiency type II; ISE, intron splice enhancer; IVS, intervening sequence (intron); IVS3, intron 3; PCR, polymerase chain reaction; pXGH5, GH-1 expression vector; RT-PCR, reverse transcriptase-polymerase chain reaction; TAE, Tris-ammonium acetate-EDTA buffer; TBE, Tris-borate-EDTA buffer.

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21. Engelbrecht, J., Knudsen, S. and Brunak, S. (1992) G+C-rich tract in 5[prime] end of human introns. J. Mol. Biol., 227, 108-113. MEDLINE Abstract

22. McCarthy, M.J., Rosenblatt, J.I. and Lloyd, R.S. (1996) A modified quantitative polymerase chain reaction assay for measuring gene-specific repair of UV photoproducts in human cells. Mutat. Res. DNA Repair, 363, 57-66.

*To whom correspondence should be addressed. Tel: +1 615 322 7601; Fax: +1 615 343 9951; Email: mccartem@ctrvax.vanderbilt.edu

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