Evolutionary silencing of the human elastase I gene (ELA1)
Evolutionary silencing of the human elastase I gene ( ELA1 )Scott D. Rose and Raymond J. MacDonald*
Department of Molecular Biology and Oncology and the Molecular Immunology Center, the University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75235-9140, USA
Received December 11, 1996;Revised and Accepted March 25, 1997
The human pancreatic elastase I gene is transcriptionally silent, despite the apparent integrity of the structural gene. The transcriptional regulatory sequences necessary and sufficient for transcription of the active rat homologue are localized within 205 base pairs (bp) of the transcriptional start and comprise a pancreas-specific transcriptional enhancer of 134 bp immediately upstream of a 71 bp non-specific promoter. The human gene has 58 nucleotide differences within this region, 13 of which are in the three functional elements (A, B and C) that constitute the enhancer. Through cell transfection analyses with a pancreatic acinar tumor cell line, we show that the nucleotide differences in the human 5' flanking gene sequences have inactivated both the enhancer and the promoter. The changes in the three elements of the human enhancer alone are sufficient to inactivate the enhancer; conversely, restoring these to the rat configuration partially restores the activity of the human enhancer. The two mutations in the A element and the four mutations in the B element abolish the binding of the transcription factors previously shown to mediate the activity of these elements. Replacing the active 71 bp rat promoter with the human promoter also prevents expression. Therefore, the evolutionary silencing of the human elastase I gene appears due to mutations that inactivate crucial enhancer and promoter elements.
The evolution of genomes is a continual process of the formation, duplication, modification, and inactivation of genes. Whereas the inactivation of a single copy, essential gene would have a dramatic phenotypic effect, the inactivation of a non-essential gene may have little or no obvious phenotypic effect. The inactivation of a member of a gene family, for example, may have little effect if the function of that gene is redundant with that of other family members.
Six pancreatic elastase-like enzymes are commonly expressed to high levels selectively in the mammalian exocrine pancreas (1 -5 ). Of these six, elastase I is the major protease with elastin-degrading activity. Not all mammals express all six pancreatic elastases, however. For example, human pancreas is devoid of the archetypal elastase I enzyme (6 ,7 ).
The human elastase I gene (ELA1) has been cloned (6 ), sequenced (8 ), and positioned on chromosome 12q13 (9 ). The nucleotide sequences of the exons of the human ELA1 are 88% and 89% identical to those of the porcine and rat elastase I genes, respectively. The exon sequences appear to be intact and encode a protein with an amino acid sequence 89% identical to that of the rat and pig enzymes. The human coding region is devoid of stop codons or other changes that would prevent the synthesis of a functional enzyme. Moreover, the exon/intron boundaries conform to the consensus sequences required for RNA splicing. However, the mRNA for ELA1 is not detectable in human pancreatic RNA preparations either by hybridization analyses (6 ,8 ) or by sensitive RT-PCR (8 ). Therefore, the defect in ELA1 expression appears to be either transcriptional or in a step in RNA processing other than splicing that prevents the accumulation of mature mRNA.
In contrast to the exonic sequences, the 5' and 3' flanking sequences of the human ELA1 gene are less well conserved when compared to those of the active rat gene. The proximal 205 bp of flanking DNA containing the transcriptional control elements of the rat gene differ by 28% from the corresponding human region; further upstream between -206 and -500 the sequences are not significantly related (68% different) (6 ). Therefore, the accumulation of nucleotide changes in transcriptional control elements may have led to the transcriptional inactivation of the human gene.
We sought to identify the defects in the human gene that prevent its expression. For the homologous, but active, rat ELA1 gene the regulatory sequences necessary and sufficient for pancreatic transcription reside within 205 bp immediately upstream of the transcriptional start site (10 ). This regulatory region comprises a transcriptional enhancer between -205 and -72, which contains all the regulatory information necessary for organ-specific transcription in transgenic mice (11 ), and a minimal promoter between -71 and +8, which responds to the enhancer but does not contribute to organ-specificity (11 ,12 ). The enhancer contains three functional elements (designated A, B and C) necessary for the correct cell- and organ-specific expression of a reporter transgene in mice (13 ). The A and B elements bind pancreas-specific transcription factors that mediate the cell-specific activity of the enhancer (14 -16 ); the C element augments the strength of the enhancer activity in combination with the A and B elements (17 ). Mutations within any one of the three elements inactivate the rat elastase I enhancer when tested by transient or stable transfection assays in mouse 266-6 or rat AR4-2J pancreatic acinar cell lines (13 ,18 ); mutation of any two is required to inactivate the enhancer in transgenic mice (13 ). Therefore, the enhancer is required for transcription of the rat elastase I gene, and the A, B and C elements are crucial for the activity of the enhancer.
We tested whether the nucleotide differences between the human and rat enhancer/promoter region prevent transcription of the human gene. Both the enhancer and promoter regions are defective when tested by transfection into cultured pancreatic acinar tumor cells. Nucleotide differences between the human and rat A, B and C enhancer elements alone are sufficient to abolish transcriptional activity of the enhancer. The differences in the A and B elements prevent the binding of the transcription factors that mediate the activity of these two elements. Therefore, it appears that the silence of the human EI gene is due to the mutation of binding sites for organ-specific transcription factors.
The 5' proximal flanking sequence of the rat ELA1 gene from -205 to +8 spans the enhancer and promoter region both necessary and sufficient for transcription in cultured pancreatic acinar cells and in the pancreas of transgenic mice (11 ,18 ). Fifty-eight out of the 213 nucleotide positions of this region differ between the active rat and the inactive human genes (Fig. 1 ). Twenty-four differences are within the proximal 71 bp promoter, 13 are in the A, B and C elements of the enhancer, and the remaining 21 are in the enhancer region outside the three elements.
Kawashima et al. (8 ) proposed several possible causes for the transcriptional silence of the human ELA1 gene, including the presence of an upstream silencer, loss of elastase-gene specific transcription factors in human acinar cells, and repressive chromosomal position effects, as well as deleterious effects of point mutations in the human enhancer or promoter regions. In this report we demonstrated that mutations in the enhancer and promoter regions are sufficient to prevent the transcription of the human ELA1 gene. Mutations in two crucial elements of the human enhancer prevent the binding of the transcription factors previously shown to mediate the activity of these elements.
The A element of the human and rat ELA1 genes is recognizably conserved in the 5' regulatory regions of eight murine and human genes encoding pancreatic digestive enzymes (Table 1 ). The A element (consensus: CACCTGtNggTTTCCCa) is an important component for the action of pancreas-specific enhancers in cultured cells (14 ,18 ,23 ,24 ) and transgenic animals (13 ). The first five A elements listed in Table 1 have been shown to be critical components of enhancers through mutational analyses and nuclear protein binding experiments. In addition, a sequence closely related to the consensus is present at similar positions upstream of the genes for mouse and rat pancreatic trypsins (14 ,25 ). The A element appears to be the principal, and perhaps sole, mediator of acinar cell-specificity of the pancreatic transcriptional enhancers (14 ,15 ,24 ).
The A element encompasses two essential motifs, an E box (CACCTG) and box A (TTTCCC) (21 ,24 ). The E and A boxes contact different proteins of a heteromeric complex termed PTF1 (21 ). In most of the identified A elements, the two motifs are separated by one DNA helical turn; the three amylase genes have a second box A one turn further away (Table 1 ). The function of the redundant amylase A boxes is unclear, but in cell-free binding assays PTF1 prefers the distal box A (14 ). Box A binds a 64 kDa protein subunit of PTF1 that otherwise has not been identified. The E box binds a 48 kDa subunit that is a member of the helix-loop-helix family of transcription factors (22 ). The 48 kDa protein is pancreas-specific and appears to determine the acinar cell-specificity of the PTF1 complex. Mutation of either the E or A boxes prevents PTF1 binding and inactivates the A element. Therefore, the E and A boxes of the A element cooperatively bind the pancreas-specific PTF1 factor complex, are conserved among pancreas-specific genes and are crucial for enhancer activity.
The single base pair difference in the human E box motif prevents the binding of PTF1, presumably by making the sequence incompatible with binding the 48 kDa subunit. None of the proven or prospective PTF1 binding sites shown in Table 1 contain this nucleotide difference in the E box motif. It is likely that the inability of PTF1 to bind the human E box contributes to the inactivation of the human enhancer.
The B element of the ELA1 enhancer is not nearly as well defined. In transfected acinar tumor cells, a 13 bp core (CAGATAAATGAGT) is required for the B element to be active in the context of the enhancer (20 ). Mutations within this core affect the activity of the element in transfected acinar tumor cells and the binding of a pancreas-specific protein complex (the C complex) to the element in parallel. This coincidence suggests that the C complex mediates the activity of the B element in acinar cells (20 ). The C complex is a heterodimer of the homeodomain factors PDX1 and a member of the PBX factor family (G.Swift, Y.Liu, S.D.Rose and R.J.MacDonald, unpublished observations). PDX1 was identified as a pancreas-specific transcription factor that binds sites in the insulin and somatostatin promoters (26 -28 ). It is a member of the subclass of homeodomain factors that form heterodimers with widely expressed PBX proteins (29 -32 ). Peers et al. (33 ) have shown that PDX1 and PBX1 bind cooperatively to a transcriptional element of the somatostatin gene promoter. The data of Figure 4 show that the nucleotide changes in the human B element disrupt the binding of the PDX1-PBX (the C) complex.
These results show that sequence changes throughout the enhancer/promoter region, and specifically within known functional enhancer elements, are sufficient to render the human ELA1 gene transcriptionally silent. Whereas the exon coding regions of the human ELA1 gene are largely conserved relative to the active rat gene (nearly 90%), the human 5' flanking sequence, which harbors the potential transcriptional control elements, has accumulated a large number of nucleotide changes. The nucleotide sequence of the 200 bp remnant of the enhancer/promoter region for the human gene differs by 25% from that of the rat, and the 300 bp immediately upstream of that has no significant sequence conservation. Moreover, there is no significant conservation in the 3' untranslated and 3' flanking regions further than 40 bp downstream of the translational stop codon. Takiguchi and colleagues (6 ,8 ) showed that ELA1 mRNA was not present in all nine pancreatic RNA samples tested; it is possible, though unlikely, that the human ELA1 gene is active in some individuals. Thus, the human ELA1 gene appears to be a transcriptionally inactive pseudogene that still encodes a potentially active protease.
An explanation for the dissimilar rates of divergence of the protein coding region compared to the 5' and 3' flanking sequences is not readily apparent. The significantly fewer differences in the coding sequences compared to the regulatory sequences and the maintenance of an open reading frame suggest that there was selective pressure to maintain the encoded protein even though it was not expressed in the pancreas. It is possible that there was, or perhaps still remains, an unknown yet important site of expression of elastase I in addition to the pancreas. In this instance an active enzyme may have been maintained at this site while pancreatic expression was lost.
The timing of the evolutionary loss of pancreatic ELA1 expression is not known. It would be informative to determine at what point in phylogeny the ELA1 gene was silenced and whether this silencing can be correlated with the inactivating mutations in the transcriptional enhancer and promoter.
Plasmids for cell transfection were constructed using standard recombinant DNA techniques (34 ) and site-specific mutagenesis (35 ). All transfection plasmids containing ELA1 regulatory sequences used the human growth hormone (hGH) structural gene as the reporter (10 ,36 ). The plasmid re-rp.hGH, which contains rat ELA1 gene enhancer (re) and rat promoter (rp) regions from -205 to +8, was previously designated -205EI.hGH (16 ). The plasmid rp.hGH, which contains the truncated rat ELA1 promoter (rp) region from -71 to +8, has been previously described as E-GH4 (11 ). he-hp.hGH contains the human ELA1 gene enhancer (he) and human promoter (hp) regions from -204 to +8 that correspond to the rat -205 to +8 region. he-rp.hGH contains the human enhancer region (nucleotides -204 to -69 of the human EI gene) fused to the rat promoter region (nucleotides -71 to +8 of the rat EI gene). re-hp.hGH contains the rat enhancer region (nucleotides -205 to -72) fused to the human promoter region (nucleotides -68 to +8). h/re-rp.hGH has the rat enhancer and promoter regions (-205 to +8) with substitutions that correspond to the changes found in the A, B and C elements of the human enhancer. These changes to the rat sequence are in element A at nucleotides -100, -105 and -113, element B at -145, -150, -153 and -160, and element C at -183, -185, -186, -189, -191 and -196; the enhancer region outside the three elements and the promoter region retain the nucleotide sequence of the rat gene. r/he-hp.hGH and r/he-rp.hGH have the human and rat promoter regions, respectively, linked to the human enhancer with changes to the rat sequence in, and limited to, the A, B and C elements. The rat ELA1 gene segment was derived from the genomic clone [lambda]EIb (25 ). The human ELA1 gene sequences were assembled by ligation of synthetic double-stranded oligonucleotides.
The mouse pancreatic acinar tumor cell line 266-6 (ATCC #CRL 2151) was transfected by the calcium phosphate procedure (37 ). The expression of transfected genes was assayed by hGH accumulation in the culture medium (38 ) using a radioimmunoassay (Nichols Institue, San Juan Capistrano, CA). The expression levels were corrected for variations in the efficiency of transfection efficiency by measuring the activity of a co-transfected Rous sarcoma virus (RSV)-mCAT fusion gene construct (18 ) and quantitation of chloramphenicol acetyl-transferase activity (39 ).
Conserved nucleotide sequences of proven and prospective A elements in pancreas-specific genes
Gene
Sequence
Location
E box box A distal box A
rat elastase I
CACCTGtgctTTTCCCt
-113/-97
rat chymotrypsin B
CACCTGtcctTTTCCCa
-207/-191
rat amylase
CAGCTGaaggTTCTTCagaaaCTCTCa
-123/-149
mouse amylase 2.1
CAGCTGaaggTTCTTCagaaaCTCCCa
-127/-153
mouse amylase 2.2
CAGGTGatggacttttagaaaCTCTCa
-180/-154
mouse trypsin d
CACCTGtggtTTTCTCc
-88/-104
rat trypsin I
CACCTGtaggTCTCCCa
-180/-164
rat trypsin II
CACCTGtgggTTTCCCc
-90/-106
human elastase I
CATCTGtgcaTTTCCCt
-113/-97
consensus
CACCTGtNggTTTCCCa
In the consensus, N is either 1 or 11 (1 turn) nucleotides. The two nucleotide differences in the human elastase I are underlined. The locations are relative to the transcriptional start sites of the genes.
Nuclear extracts from rat pancreatic tissue were prepared by the method previously described (15 ,40 ). Nuclear extracts from 266-6 acinar tumor cells were prepared by the method of Dignam et al. (41 ). Electrophoretic mobility shift analyses with 40 femtomoles of 32P-labeled double-stranded oligonucleotides were performed as described by Sawada and Littman (42 ) for the ELA1 A element and by Swift et al. (20 ) for the ELA1 B element. The sequences of the blunt-ended, double-stranded oligonucleotides were (template strand):
rELA1 A, 5'-GTCAcCTGTGcTTTTCCCTGC-3';
hELA1 A, 5'-GTCAtCTGTGaTTTTCCCTGC-3';
rELA1 B, 5'-TAtCAGATAaATgAGTTgACT-3';
and hELA1 B, 5'-TAaCAGATAcATtAGTTtACT-3'.
The bases in lower case identify the nucleotide differences between rat and human. The other sequence variants used in the electrophoretic mobility shift experiments of Figures 3 and 4 were made within the context of these 21 bp sequences.
We thank Dr Galvin Swift for encouragement, help and critical readings of the manuscript and Kimberly Dambach for technical assistance. We also thank Shirley Hall, Lee Railey and Russell Guzzetta of the Macromolecular Analysis Facility for DNA sequence analysis and synthetic oligonucleotides. The Macromolecular Analysis Facility is supported by US Public Health Service grant GM31689. This research was supported by USPHS grant AM27430 from the NIH.
1 MacDonald, R. J., Swift, G. H., Quinto, C., Swain, W., Pictet, R. L., Nikovits, W. and Rutter, W. J. (1982) Primary structure of two distinct rat pancreatic preproelastases determined by sequence analysis of the complete cloned messenger ribonucleic acid sequences. Biochemistry, 21, 1453-1463.
2 Kawashima, I., Tani, T., Shimoda, K. and Takiguchi, Y. (1987) Characterization of pancreatic elastase II cDNAs: two elastase II mRNAs are expressed in human pancreas. DNA, 6, 163-172.MEDLINE Abstract
3 Shen, W.-F., Fletcher, T. S. and Largman, C. (1987) Primary structure of human pancreatic protease E determined by sequence analysis of the cloned mRNA. Biochemistry, 26, 3447-3452.
4 Kang, J., Wiegand, U. and Muller-Hill, B. (1992) Identification of cDNAs encoding two novel rat pancreatic serine proteases. Gene, 110, 181-187.MEDLINE Abstract
5 Tani, T., Ohsumi, J., Mita, K. and Takiguchi, Y. (1988) Identification of a novel class of elastase isozyme, human pancreatic elastase III, by cDNA and genomic gene cloning. J. Biol. Chem., 263, 1231-11239.MEDLINE Abstract
6 Tani, T., Kawashima, I., Furukawa, H., Ohmine, T. and Takiguchi, Y. (1987) Characterization of a silent gene for human pancreatic elastase I: structure of the 5'-flanking region. J. Biochem., 101, 591-599.MEDLINE Abstract
7 Shirasu, Y., Takemura, K., Yoshida, H., Sato, Y., Iijima, H., Shimada, Y., Mikayama, T., Ozawa, T., Ikeda, N., Ishida, A., Tamai, Y., Matsuki, S., Tanaka, J.-I., Ikenaga, H. and Ogawa, M. (1988) Molecular cloning of complementary DNA encoding one of the human pancreatic protease E isozymes. J. Biochem., 104, 259-264.MEDLINE Abstract
8 Kawashima, I., Tani, T., Mita-Honjo, K., Shimoda-Takano, K., Ohmine, T., Furukawa, H. and Takiguchi, Y. (1992) Genomic organization of the human homologue of the rat pancreatic elastase I gene. DNA Seq., 2, 303-312.MEDLINE Abstract
9 Davies, R. L., Yoon, S. J., Weissenbach, J., Ward, D., Krauter, K. and Kucherlapati, R. (1995) Physical mapping of the human ELA1 gene between D12S361 and D12S347 on chromosome 12q13. Genomics, 29, 766-768.
10 Ornitz, D. M., Palmiter, R. D., Hammer, R. E., Brinster, R. L., Swift, G. H. and MacDonald, R. J. (1985) Specific expression of an elastase-human growth hormone fusion gene in pancreatic acinar cells of transgenic mice. Nature, 313, 600-603.
11 Hammer, R. E., Swift, G. H., Ornitz, D. M., Quaife, C. J., Palmiter, R. D., Brinster, R. L. and MacDonald, R. J. (1987) The rat elastase I regulatory element is an enhancer that directs correct cell specificity and developmental onset of expression in transgenic mice. Mol. Cell. Biol., 7, 2956-2967.
12 Ornitz, D. M., Hammer, R. E., Davison, B. L., Brinster, R. L. and Palmiter, R. D. (1987) Promoter and enhancer elements from the rat elastase I gene function independently of each other and of heterologous enhancers. Mol. Cell. Biol., 7, 3466-3472.
13 Swift, G. H., Kruse, F., MacDonald, R. J. and Hammer, R. E. (1989) Differential requirements for cell-specific elastase I enhancer domains in transfected cells and transgenic mice. Genes Dev., 3, 687-696.
14 Cockell, M., Stevenson, B. J., Strubin, M., Hagenbuchle, O. and Wellauer, P. K. (1989) Identification of a cell-specific DNA-binding activity that interacts with a transcriptional activator of genes expressed in the acinar pancreas. Mol. Cell. Biol., 9, 2464-2476.MEDLINE Abstract
15 Rose, S. D., Kruse, F., Swift, G. H., MacDonald, R. J. and Hammer, R. E. (1994) A single element of the elastase I enhancer is sufficient to direct transcription selectively to the pancreas and gut. Mol. Cell. Biol., 14, 2048-2057.
16 Kruse, F., Rose, S. D., Swift, G. H., Hammer, R. E. and MacDonald, R. J. (1993) An endocrine-specific element is an integral component of an exocrine-specific pancreatic enhancer. Genes Dev., 7, 774-786.MEDLINE Abstract
17 Kruse, F., Rose, S. D., Swift, G. H., Hammer, R. E. and MacDonald, R. J. (1995) Cooperation between elements of an organ-specific transcriptional enhancer in animals. Mol. Cell. Biol., 15, 4385-4394.MEDLINE Abstract
18 Kruse, F., Komro, C. T., Michnoff, C. H. and MacDonald, R. J. (1988) The cell-specific elastase I enhancer comprises two domains. Mol. Cell. Biol., 8, 893-902.MEDLINE Abstract
19 Ornitz, D. M., Palmiter, R. D., Messing, A., Hammer, R. E., Pinkert, C. A. and Brinster, R. L. (1985) Elastase I promoter directs expression of human growth hormone and SV40 T antigen genes to pancreatic acinar cells in transgenic mice. Cold Spring Harbor Symp. Quant. Biol., 50, 399-409.
20 Swift, G. H., Rose, S. D. and MacDonald, R. J. (1994) An element of the elastase I enhancer is an overlapping bipartite binding site activated by a heteromeric factor. J. Biol. Chem., 269, 12809-12815.
21 Roux, E., Strubin, M., Hagenbuchle, O. and Wellauer, P. K. (1989) The cell-specific transcription factor PTF1 contains two different subunits that interact with the DNA. Genes Dev., 3, 1613-1624.MEDLINE Abstract
22 Krapp, A., Knofler, M., Frutiger, F., Hughes, G. J., Hagenbuchle, O. and Wellauer, P. K. (1996) The p48 DNA-binding subunit of transcription factor PTF1 is a new exocrine pancreas-specific basic helix-loop-helix protein. EMBO J., 15, 4317-4329.MEDLINE Abstract
23 Boulet, A. M., Erwin, C. R. and Rutter, W. J. (1986) Cell-specific enhancers in the rat exocrine pancreas. Proc. Natl. Acad. Sci. USA, 83, 3599-3603.
24 Meister, A., Weinrich, S. L., Nelson, C. and Rutter, W. J. (1989) The chymotrypsin enhancer core. Specific factor binding and biological activity. J. Biol. Chem., 264, 20744-20751.MEDLINE Abstract
25 Swift, G. H., Craik, C. S., Stary, S. J., Quinto, C., Lahaie, R. G., Rutter, W. J. and MacDonald, R. J. (1984) Structure of the two related elastase genes express in the rat pancreas. J. Biol. Chem., 259, 14271-14278.
26 Ohlsson, H., Karlsson, K. and Edlund, T. (1993) IPF1, a homeodomain-containing transactivator of the insulin gene. EMBO J., 12, 4251-4259.MEDLINE Abstract
27 Leonard, J., Peers, B., Johnson, T., Ferreri, K., Lee, S. and Montminy, M. R. (1993) Characterization of somatostatin transactivating factor-1, a novel homeobox factor that stimulates somatostatin expression in pancreatic islet cells. Mol. Endocrinol., 7, 1275-1283.MEDLINE Abstract
28 Miller, C. P., McGehee, R. E. and Habener, J. F. (1994) IDX-1: a new homeodomain transcription factor expressed in rat pancreatic islets and duodenum that transactivates the somatostatin gene. EMBO J., 13, 1145-1156.
29 Chang, C.-P., Shen, W.-F., Rozenfeld, S., Lawrence, H. J., Largman, C. and Cleary, M. L. (1995) Pbx proteins display hexapeptide-dependent cooperative DNA binding with a subset of Hox proteins. Genes Dev., 9, 663-674.
30 Phelan, M. L., Rambaldi, I. and Feataherstone, M. S. (1995) Cooperative interactions between HOX and PBX proteins mediated by a conserved peptide motif. Mol. Cell. Biol., 15, 3989-3997.
31 Neuteboom, S. T. C., Peltenburg, L. T. C., van Duk, M. A. and Murre, C. (1995) The hexapeptide LFPWMR in Hoxb-8 is required for cooperative DNA binding with Pbx1 and Pbx2 proteins. Proc. Natl. Acad. Sci. USA, 92, 9166-9170.
32 Monica, K., Galili, N., Nourse, J., Saltman, D. and Cleary, M. L. (1991) PBX2 and PBX3, new homeobox genes with extensive homology to the human proto-oncogene PBX1. Mol. Cell. Biol., 11, 6149-6157.MEDLINE Abstract
33 Peers, B., Sharma, S., Johnson, T., Kamps, M. and Montminy, M. (1995) The pancreatic islet factor STF-1 binds cooperatively with Pbx to a regulatory element in the somatostatin promoter: Importance of the FPWMK motif and of the homeodomain. Mol. Cell. Biol., 15, 7091-7097.MEDLINE Abstract
34 Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
35 Kunkel, T. A., Roberts, J. D. and Zakour, R. A. (1987) Methods Enzymol., 154, 367-382.
36 Seeburg, P. H. (1982) The human growth hormone gene family: nucleotide sequences show recent divergence and predict a new polypeptide hormone. DNA, 1, 239-249.
37 Graham, F. L. and van der Eb, A. J. (1973) A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology, 52, 456-467.
38 Seldon, R. F., Burke, H. K., Rowe, M. E., Goodman, H. M. and Moore, D. D. (1986) Human growth hormone as a reporter gene in regulation studies employing transient gene expression. Mol. Cell. Biol., 6, 3173-3179.
39 Nielsen, D. A., Chang, T. C. and Shapiro, D. J. (1989) A highly sensitive, mixed-phase assay for chloramphenicol acetyltransferse activity in transfected cells. Anal. Biochem., 179, 19-23.
40 Gorski, K., Carneiro, M. and Schibler, U. (1986) Tissue-specific in vitro transcription from the mouse albumin promoter. Cell, 47, 767-776.MEDLINE Abstract
41 Dignam, J. D., Lebovitz, R. M. and Roeder, R. G. (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res., 11, 1475-1489.
42 Sawada, S. and Littman, D. R. (1993) A heterodimer of HEB and an E12-related protein interacts with the CD4 enhancer and regulates its activity in T-cell lines. Mol. Cell. Biol., 13, 5620-5628.MEDLINE Abstract
*To whom correspondence should be addressed. Tel: +1 214 648 1923; Fax: +1 214 648 1915; Email: ray@hamon.swmed.edu
-->
This page is maintained by OUP admin. Last updated Mon May 12 18:10:04 BST 1997. Part of the OUP Journals World Wide Web service.
Copyright
Oxford University Press, 1996
