WO1994029468A1

WO1994029468A1 - Untranslated exon-1 sequences of eukaryotic genes as promoters

Info

Publication number: WO1994029468A1
Application number: PCT/GB1994/001251
Authority: WO
Inventors: Ciao Chang Bleasdale; Catherine Demoliou-Mason; Vijay Vir Kakkar
Original assignee: Thrombosis Research Institute
Priority date: 1993-06-11
Filing date: 1994-06-10
Publication date: 1994-12-22
Also published as: AU6855094A; GB9312070D0

Abstract

Untranslated exon 1 sequences of certain eukaryotic genes, particularly SMHC and sTnI genes, without any 5' or 3' flanking sequences, have strong promoter activity in the expression of reporter genes such as CAT and β-gal in a variety of mammalian host cells, e.g. vascular smooth muscle cells. The invention provides methods of such enhanced gene expression, eukaryotic expression vectors therefor, and use of such promoters in the expression of such genes. The invention has particular applicability to the expression of genes for use in human gene therapy.

Description

UNTRANSLATED EXON-1 SEQUENCES OF EUKARYOTIC GENES AS PROMOTERS

FIELD OF THE INVENTION

This invention relates to a novel function of untranslated TATA-less first exon sequences in eukaryotic genes, the use of these sequences in the expression regulation of eukaryotic genes, and for the construction of novel expression vectors which provide fine controlling of expression levels of specific genes in vitro and in vivo, for use for example in human gene therapy.

BACKGROUND OF THE INVENTION AND PRIOR ART

Background literature references are referred to herein by way of parenthetical numerical citation in the text to the appended bibliography. The disclosures of these references are incorporated herein by reference.

As is well known, all eukaryotic genes contain exons and introns, the multiftinctions of which in gene regulation processes are still the subject of much investigation. A striking finding in recent progress of human genome mapping is the high degree of conservation of introns sequences, which predict the importance of these large regions whose functions are still largely unknown. Another interesting phenomenon is that in the case of many eukaryotic genes, their first exons are highly conserved, actively transcribed, but untranslated. Remarkable progress in the study of untranslated first exons has been made during recent years. For example, the first exons of a number of genes have been shown to play roles in transcription regulation; they are required either for the full activity of their promoters, or for interaction with upstream cis-elements to regulate the expression level of genes. Those exonic control regions have been described for example in the case of the HSV tk gene where a portion of its first exon is required for full activity from the tk promoter (1), and in the case of skeletal troponin I (sTnl) gene, where the first exon is required for full muscle-specific activity of the sTnl promoter. The conclusion here is that the sTnl exon 1 does not contain a transcriptional enhancer, but the interaction between sTnl exon 1 and the distal upstream region is necessary for the expression regulatory mechanism (2). Other exonic control regions previously described include: RNA polymerase III transcribed rRNAs (3,4) and tRNAs (5), heat shock genes (6 - 9), the gastrin gene (10); c-myc is an interesting model, and shows it has at least four different promoters. The majority of transcripts start at the P_[ (-161) and P₂ promoter (+1), in normal Burkitt's lymphoma cells (BL), which account for about 10-

25% and 75-90% of c-myc RNA. In BL cells with the chromosomal breakpoint upstream of the gene, c-myc is preferentially transcribed from the P, promoter, shifting the ratio of P^ usage to >1. Notably the P2 promoter is located in die c-myc exon 1 region and contains a potential TATA box (29 - 23) which can be recognized by both polymerase II & III. The sequences within the P2 promoter affect the elongation and premature termination of transcripts initiated from me 5' upstream pi promoter.

Two further c-myc promoters³ are the P₀ promoter which is located 55-

650 bp upstream of P, and the P₃ promoter which is located in the first intron of the gene. The potential coding capacity of these P,/P₂, P₀ and P₃ RNAs is different. P,/P₂ RNAs contain a single large open reading frame and encode two c-myc proteins of 64 and 67 kd translation of the 67 kd protein is initiated at a CTG codon at the end of exon 1, whereas the 64 kd protein is initiated in exon 2, using an AUG codon. However, P₀ RNA contains three open reading frames, the 5' and middle open reading frames being initiated upstream of the P, promoter. The 3' open reading frame is identical to the P,/P₂ - 64/67 kd proteins. P₃ RNA is initiated at multiple start sites within the first intron and lacks the exon 1 sequences, and is only able to code the 64 kd c-myc protein (11 - 14).

In eukaryotes, transcription is carried out by three different RNA polymerases: RNA polymerase I, II and III, each of which is dedicated to the transcription of different sets of genes. The genes in each class contain characteristic promoters, which usually consist of two types of functional elements: core (basal) promoter elements and modulator (upstream) promoter elements. The core promoter elements are sufficient to determine RNA polymerase specificity and direct low levels of transcription, whereas the modulator elements enhance or reduce the basal levels of transcription. The core promoter elements are first recognized by specific transcription factors, which then recruit the specific RNA polymerase. However recent studies have discovered that one general factor, the TATA box-binding protein (TBP), plays a role in all three polymerase systems, and the mechanisms of initiation complex assembly are strongly conserved. There are thus two fundamental mechanisms of initiation complex assembly: for TATA - containing promoters (polymerase II and III), TBP binding to TATA box directly with its concave DNA-building surface, and its large convex surface to which other proteins i.e. activators, general factors, polymerase can bind; for non - TATA promoters (all three polymerases), another protein binds to the DNA, i.e. upstream binding factor for polymerase I family, a transcription factor like SP1 for polymerase II family, and TFiπC for classical polymerase III promoters. These proteins then recruit TBP directly or indirectly via a TBP-associated factor (TAF), once the TBP is in the initiation complex. It can then facilitate the binding of other proteins (15 - 17).

Transcription initiation is one of the most important ways to regulate gene expression. Although most protein-encoding genes contain a TATA motif, many promoters transcribed by polymerase II, especially those of housekeeping genes, lack a TATA element. These are referred to as TATA-less promoters. In some promoters, a second type of core promoter element called a transcriptional initiator (Inr) has been found to mediate the same functions as the TATA motif. An Inr can be defined as a DNA sequence element that overlaps a transcription start site and is sufficient for determining the start site location in a promoter that lacks a TATA box and also can cooperate witii a TATA box to enhance the promoter activity. Various Inr elements have been described and classified according to sequence homology, for example, the TdT - Inr family, the PBGD - Inr family, the DHFR - Inr family, the ribosomal protein - Inr family, the adeno - associated virus p5 - Inr family etc. Also it has been shown that TFIID is an integral component of the transcription initiation complex from almost all TATA - less promoters studies. It is strongly suggested that recognition of the Irn by universal or multiple Inr binding proteins (ITF) might provide a means by which a transcription competent complex can assemble (18 - 20).

Myosin is one of only three proteins known to convert chemical energy into mechanical work, i.e. to function as a molecular motor. Each myosin molecule is composed of two myosin heavy chains and four myosin light chains. All smooth muscle myosin heavy chain (SMHC) isoforms (SMI, SM2 & SMB) so far have been shown to be transcribed from a single gene by alternative RNA splicing which results in divergences at the carboxyl termini (SMI & SM2), or at the 25/50 kD junction (SMB). The expression of SMHC gene is strictly controlled in a developmental stage - specific and tissue - specific manner. SMI and SM2 so far have been found to be specific to vascular and nonvascular smooth muscle cells. Smooth muscle cell proliferation in experimental arteriosclerosis and atherosclerosis was associated with dedifferentiation of the smooth muscle cells toward the embryonic phenotype on the basis of the SMHC expression. These existing SMHC isoforms are important molecular markers in the studies of the process of atherosclerosis as well as phenotypic modulation of arterial smooth muscle cells (21 - 26).

As a result of the characterization of the 5' upstream promoter region of the rabbit SMHC gene and the role of untranslated SMHC Exon 1 involved in the tissue - specific regulation of SMHC promoter activity, surprisingly we have found that this 78 bp TATA-less SMHC Exon 1 on its own possesses promoter activity, in the absence of a 5' upstream core promoter region, and even in the absence of any 5' upstream or 3' downstream sequences. This Exon 1 promoter sequence does not contain any TATA motif or CAAT motif to provide the second promoter capacity as mentioned above in the c-myc gene. This Exon 1 promoter also does not contain any known initiator sequences, and for its promoter activity does not require any 5' upstream region. (As is well known, the initiator element usually starts in the 5' upstream region and encompasses the transcription start site).

To investigate this new type of Exon 1 promoter, we have synthesized a series of mutant oligonucleotides of the SMHC Exon 1 sequences

Exon 1 (+1-+79), Ex 60 (+1-+61), Ex 40 (+1-+41), and Ex 20 (+1- +21), cloned into four pCAT vectors (pCATbasic, pCATpromoter, pCATenhancer, and pCATcontrol.; Promega) at either promoter or enhancer positions. These vectors were designed specially for testing promoter or enhancer activity and contain no other eukaryotic promoter or enhancer sequences (pCATbasic), or with SV40 promoter but lacking any eukaryotic enhancer (pCATpromoter), or with SV40 enhancer but lacking any eukaryotic promoter (pCATenhancer) or with both SV40 promoter plus enhancer as the positive control (pCATcontrol). Six differentially positioned pCAT - EXON 1 constructions were used to transfect primary rabbit vascular smooth muscle cells (RSMC). Transient expression assays showed that in the absence of the 5' upstrea region, (including the SMHC core promoter, a strong TATA - promoter which can drive CAT gene expression to reach the equivalent level of pCATcontrol) or any other cis-acting regulatory elements located in the 5' upstream or intron 1 regions of the SMHC gene, the whole SMHC Exon 1 sequence (79 bp) alone is sufficient to function as an alternative promoter, to drive the chloramphenicol acetyltransferase (CAT) reporter gene expression in RSMC cells such that the CAT expression level reached 45 - 50% of pCATcontrol, (SV40 promoter plus enhancer). This SMHC exon 1 sequence functions as an alternative promoter, but not enhancer, as the reverse orientation clone of pCATbasic-Exon 1 p could not drive CAT gene expression. The SMHC exon 1 sequence showed no capacity to interact with SV40 promoter (pCATpromoter- Exon 1, Exon 1 -pCATpromoter). Further analysis of the promoter activity region located inside of die SMHC Exon 1 sequence showed d at the alternative promoter activity requires the whole exon 1 region.

The 5' nested deletion mutants of SMHC exon 1 extending from +1 to +21 (Ex20), +41 (Ex40), +61 (Ex60) regions showed no substantial promoter activity. It seems that the 3' end of the SMHC Exon 1 sequence is essential for this alternative promoter activity. Interestingly, a transcription factor binding motifs search using a GCG program package showed there to be a number of putative transcription factor binding sites in the SMHC Exon 1 region, including: two AP 2 sites (activator protein 2)(27) located at +12-+ 19 and +16-+23; three GC- rich/GCF sites (28) located at +11-+17, +61-+67 and +63-+69; one SIF site (c-sis/PDGF induced, activates c-fos gene) (29) at +45-+50; and one E box (bHLH proteins binding concensus site i.e. MyoD family) located at +71 -+76. It may be mat the E box or GC-rich/GCF binding sites at the 3' end region of the SMHC Exon 1 is critical to this exon 1 promoter activity. To answer this question, we have constructed E box and GCF site mutants of SMHC Exon 1, the CACTTG motif of E box having been changed to GGTTTG (EXMUT 1); and the GCGCC central motif of two GC-rich/GCF motifs having been changed to AATTT, (EXMUT 2). Transient expression studies showed diat these single mutations of the 3' end motifs dramatically abolish the Exon 1 promoter activity. This phenomenon distinguishes from all initiators reported so far, and requires die whole length of exon 1 to function as an alternative promoter. This is the first time an untranslated TATA- less first exon has been disclosed to have promoter activity in the absence of 5' upstream core promoter sequences. The essential element for its promoter activity is located at die 3' end of die Exon 1 sequence, but not encompassing the transcription start site as defined for the initiator.

From the view of the multiftinctions of highly conserved untranslated first exons, it seems mese actively transcribed but untranslated sequences are particularly important regions involved in die transcription regulatory mechanisms and pathways which have still to be completely understood. It has been shown that these untranslated first exons can interact with 5 ' upstream TATA - promoter or other regulatory elements to regulate expression levels or tissue-specificity. It has been shown that these untranslated first exons can contain a second TATA- promoter which confers an alternative transcription start site, or can contain partial sequences of initiator which encompass the transcription start site.

As anodier new function of me untranslated first exons of eukaryotic genes, under certain circumstances, for example, in die absence of any 5' upstream core promoter and odier regulatory element sequences as well as any 3' flanking sequences, the untranslated first exons can function as an alternative promoter per se, even without the necessity of the presence of a potential TATA - box. The transcription initiation complex might be assembled eidier via specific exon 1 binding proteins, or via known general transcription factors, for instance TBP, either directly binding, or indirectly binding d rough certain potential tissue- specific and gene-specific transcription factors, as we show here that the MyoD and GC-rich/GCF motifs play a critical role in the SMHC exon 1 promoter activity.

To extend diis discovery of a new function of untranslated first exons to odier eukaryotic genes, we have also synthesized a 61 nt oligonucleotide containing the whole length of chicken skeletal Troponin I exon 1 sequences and cloned d is into pCATbasic vector, to test its promoter activity in mammalian cells. The results show that this untranslated TATA-less first exon of sTnl gene also confers moderate promoter activity in the absence of any 5' upstream sequences including d e sTnl core promoter region and wimout 3' flanking (sTnl intron 1) sequences as well.

SUMMARY OF THE INVENTION As me basis for this invention, we have discovered a new function of untranslated first exons of eukaryotic genes, without any 5' or 3' flanking sequences namely that they function as an alternative promoter per se, under certain circumstances, for instance, in the absence of 5' upstream normal core promoter sequences.

This exon 1 promoter function has been demonstrated by e synthesized 79 nt SMHC Exon 1 and 61 nt sTnl Exon 1 oligonucleotides (both lacking any 5' or 3' flanking sequences) driving CAT reporter gene expression in mammalian cells to a moderate level equivalent to 55

- 60% CAT activity driven by 5' upstream SMHC core promoter or about 45-50% CAT activity driven by SV40 promoter plus enhancer (pCATcontrol).

To dissect d e essential elements in me SMHC exon 1 promoter a series of 3' end deletion mutants of the SMHC Exon 1 sequence have been made and transient expression assays showed that the 3' end 18 nucleotides were critical for this exon 1 promoter activity. Further mutations of the MyoD motif or GC-rich motif which are located in diis 18 nucleotide 3' end region abolished diis exon 1 promoter activity. As is well known, the MyoD motif CANNTG and GC-rich motif GCGCC iemselves could not ftinction as a promoter. Hence in one aspect diese protein binding motifs located inside die first exon can be utilized as important components of transcription initiation complex assembled by me exon 1 promoter.

This strategy can be an alternative way for highly conserved genes, particularly housekeeping genes, to maintain their basal levels of transcription under certain circumstances, for instance, during genomic rearrangements or gene translocations. The normal gene-specific functional 5' promoter regions are truncated or inactivated, then the transcribed first exon becomes the potential initiator which requires the whole genetic information to be carried in the gene-specific Exon 1, to direct d e temperarly expression of these genes.

In one aspect, the present invention provides me use of SMHC exon 1 promoter to construct moderate expression vectors, for use in Melody

Gene Therapy die multiple genes regulation strategy in gene tiierapy, fine controlling of expression levels of concerted genes, and to restore die proper balance genes interactions. The advantages of highly conserved exon 1 promoter are its genetic stability, small size, sequence information always being available, easy manipulation and usually compact in genetic information. The accumulation of exon 1 promoter information provides a new source for die construction of new expression vectors by combining varied motifs, which have been naturally organized inside die first exons to earn some gene-specific character, such as tissue-specific and differentiation - stage specificities.

Furthermore, the invention provides die use of eukaryotic genes first exons sequences in die isolation and characterization of potential transcription factors involved in die initiation and regulation of transcription, as well as in die elucidation of interactions between general and gene-specific transcription factors with eukaryotic polymerase enzymes.

A further aspect of the application of die present invention is as a diagnostic tool in die in vitro testing for the response of a patient to gene therapy targeted to inhibit vascular smooth muscle cell proliferation and phenotype change in adierosclerosis and in intimal hyperplasia. In addition, die SMHC exon 1 promoter can be used as a probe for die identification, isolation, characterization and applications of other genes, particularly of die myosin family including myosin heavy chain genes. These in turn can themselves be used for cell specific expression of phenotype markers and die expression of genes involved in die regulation of cell specific functions, like those of proliferation, migration and phenotype determination.

This invention further provides die use of exon 1 promoter sequences from eukaryotic genes to construct eukaryotic expression vectors for the expression of reporter genes or other marker genes for transient or long term expression studies in mammalian cell cultures or in vivo animal model studies.

This invention also provides me use of these eukaryotic exon 1 mini- promoters for gene therapy studies, which avoid die safety issues from viral vectors used in clinical trials which have had to be addressed up to now.

Further, this invention provides die use of tiiese exon 1 promoter sequences to be modified by subtraction or addition or modification of sequences for the construction of eukaryotic expression vectors to modulate expression levels driven by tiiese new eukaryotic promoters or with cell type specific expression, that can be provided in any suitable form appropriate to the protocol of administration and/or needs of a patient undergoing gene therapy or as an in vitro diagnosis tool for use in gene tiierapy.

BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1 illustrates the sequence of the SMHC Exon 1 (+1 to +79) oligonucleotides synthesized in die 5' to 3' orientation, and the putative transcription factors binding sites.

FIGURE 2 illustrates the constructions of pCAT - SMHC Exon 1 plasmid vectors, in which die SMHC Exon 1 sequence was inserted into different positions related to die CAT reporter gene, to test its function as promoter, enhancer or cis-regulatory element in mammalian cells.

FIGURE 3 illustrates the promoter activity of pCAT - SMHC Exon 1 constructs. RSMC, HSMC and odier cell-types were transfected with tiiese pCAT - SMHC Exon 1 plasmid DNAs, transient expressed CAT activity was measured by liquid scintillation counting (LSC) and the transfection efficiency was normalized by co-transfection of β-galactosidase activity and protein content assay. Relative CAT activity is expressed as % of pCATcontrol.

FIGURE 4 illustrates the constructions of SMHC Exon 1 Promoter mutants, including its 3' nested deletion mutants and the MyoD, GC- rich/GCF motifs mutants. All synthesized mutant oligonucleotides were cloned into pCATbasic vector upstream of the CAT reporter gene to test their promoter activity in vascular smooth muscle cells.

FIGURE 5 illustrates die relative CAT activity driven by the SMHC Exon 1 Promoter mutants of Figure 4 in RSMC and HSMC cells.

FIGURE 6 illustrates die synthesized oligonucleotides sequence of sTnl Exon 1 , the construction of pCATbasic - sTnl EXon plasmid vector and its expression in mammalian cells. DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

In relation to Figures 1 - 6 the following materials and methods were used. Materials: [¹⁴C] chloramphemcol (40 - 60 mCi/mmol) and [α - 35 S] - dATP were purchased from Amersham. Enzymes used in plasmid construction were obtained from Boehringer Mannheim Biochemicals and Promega. Plasmids vectors pCATbasic, pCATpromoter, pCATenhancer, pCATcontrol and pSV-βGal were from Promega. Bacterial chloramphemcol acetyltransferase (CAT) and acetyl coenzyme A were from Pharmacia Inc.

Cell cultures: rabbit smooth muscle cells (RSMC) and rabbit endotiielial cells (RENDO) were obtained from die thoracic aorta of 8 to 10-weeks old rabbits as described (30 - 31). Human aortic or vein smootii muscle cells (HSMC) were prepared by explantation of aortic or vein tissues obtained during cardiovascular surgery as described (32). Human dermal fibroblasts (HDF) were obtained by enzymatic digestion of tissues as described (33). Human Girard heart cells (HGH) were obtained from ICN Biochemicals Ltd. All cells were maintained in DMEM medium supplemented witii 10 - 20% fetal calf serum, 2mM L-glutamine, 0.25 ug/ml fi gizone, 100 u ml penicillin and lOOug/ml streptomycin (all from GIBCO), at 37°C and 10% CO₂. However RENDO cells were maintained in Ml 99 medium supplemented witii

20% fetal calf serum, 2mM L-glutamine, 0.25 ug/ml fungizone, 100u/ml penicillin and lOOu/ml streptomycin, plus 20 ug/ml endotiielial cell growth factor and 80 ug/ml heparin, witiiin gelatin pre-coated flasks at 37°C and 7% CO,. Sequence computering analysis: The SMHC gene and sTnl gene sequences obtained from Genbank database. Totally 162 transcription factors binding sites (34) were searched using the GCG program package (via UNIX system, SEQNET, Daresbury Laboratory, UK). DNA sequence analysis: Sequence analysis of the recombinant plasmids was performed by dideoxy chain termination methods (35) using Sequenase (USB). All constructions have been confirmed by sequencing.

Plasmids construction: Equal amounts of the synthetic oligonucleotides were kinase treated and purified by phenol extraction - ethanol precipitation. dsDNA fragments were obtained by hybridizing the upper strand and lower strand oligonucleotides mixed in hybridization buffer (lOmM Tris-HCL, pH7.5, 150 mM NaCl, lOmM MgCy using single cycle of a PCR instrument (3min/97°C, 15min/65°C, 15min 37°C and 15min/24°C). These dsDNA fragments were cloned into pCAT vectors by appropriate restriction enzyme digestion and blunt-end ligation at die desired insert positions as indicated below. After transformation into DH₅α competent cells, selected recombinants were confirmed by restriction enzyme mapping and DNA sequencing. CsCl gradient method (36) was used to purify the plasmid DNAs for transfection studies.

(1) Exon 1 - pCATbasic: SMHC Exon 1 sequence (+1-+79) was inserted into Sal I site, upstream of die CAT gene, at die promoter position. pCATbasic vector contains pUC19 backbone, poly linker site, CAT gene, SV40 small Tantigen (terminal signal) and ampicillin resistance gene. It lacks any eukaryotic promoter and enhancer sequences. (2) pCATpromoter - Exon i: SMHC Exon 1 sequence (+1-+79) was inserted into Sal I site, downstream of CAT gene and SV40 terminal signal, at the enhancer position. pCATpromoter vector contains SV40 promoter but lacks any eukaryotic enhancer sequence.

(3) Exon 1 - pCATpromoter: SMHC Exon 1 sequence (+1-+79) was inserted into Bgl II site, upstream of SV40 promoter and CAT gene at die cis-regulatory element position.

(4) Exon 1 - pCATenhancer: SMHC Exon 1 sequence (+1-+79) was inserted into Sal I site, upstream of CAT gene, at the promoter position. pCATenhancer vector lacks any eukaryotic promoter sequence, but contains SV40 enhancer which was positioned downstream of CAT gene and SV40 terminal signal.

(5) Exon 1 - pCATcontrol: SMHC Exon 1 sequence (+1 - +79)

was inserted at Bgl II site, upstream of SV40 promoter and CAT gene,

at die cis-regulatory element position. pCATcontrol contains SV40

promoter plus enhancer which can drive a high level of CAT expression

in eukaryotic cells.

(6) pCATcontrol - Exon 1: SMHC Exonl sequence (+1 - +79)

was inserted into Sal I site, downstream of SV40 enhancer, at the

enhancer position.

(7) Ex 20 - pCATbasic: 3' end deletion mutant partial SMHC Exon 1 sequence (+1 - +21) was inserted into Sal I site

of pCATbasic vector, upstream of CAT gene, at die promoter position.

(8) Ex 40 - pCATbasic: 3' end deletion mutant

partial SMHC Exon 1 sequence (+1 - +41) was inserted into Sal I site

of pCATbasic vector, upstream of CAT gene, at the promoter position.

(9) Ex 60 - pCATbasic: 3' end deletion mutant

partial SMHC Exon 1 sequence (+1 - +61) was inserted into Sal I site

of pCATbasic vector, upstream of CAT gene, at the promoter position.

(10) Exonlop - pCATbasic: SMHC Exon 1 sequence (+79 - +1)

was inserted into die Sal I site of pCATbasic vector, but in the reverse

orientation, at the promoter position.

(11) EXMUT1 - pCATbasic: MyoD motif mutant of SMHC

Exon 1 sequence (+1 - +79), its MyoD motif CACTTG (+71 - +76)

was replaced by GGTTTG). Mutated SMHC Exon 1 sequence was

inserted into Sal I site of pCATbasic vector, upstream of CAT gene, at

the promoter position.

(12) EXMUT2 - pCATbasic: GC-rich/CGF motif mutant of SMHC Exon 1 sequence (+1 - +79), Two of the GC-rich/GCF motifs

located at its 3' end GCGCGCC (+60 - +66) & GCGCCCC (+62 -

+69) were mutated by change the central GCGCC motif (+62 - +66)

into AATTT. The mutated SMHC Exon 1 sequence was inserted into

die Sal I site of pCATbasic vector, upstream of the CAT gene, at the

promoter position.

(13) EXMUT2op - pCATbasic: The same insert sequence and

position as mentioned above, but was cloned in die reverse orientation.

(14) sTnl Exonl - pCATbasic: sTnl Exon 1 sequence (+1 -

+ 1) was inserted into the Sal I site of pCATbasic vector, upstream of

the CAT gene, at the promoter position.

Cell transfection: All cells were transfected by electroporation metiiod

(37), using BIO-RAD gene pulser system. 20-40 ug plasmid DNA was

electroporated at 260 V/960 uF (RSMC, HSMC, HGH, HDF) or 230

V/960 uF (RENDO) witii -10⁶ cells suspended in 0.5 ml electroporation

buffer. After 48-60 hr, the transfected cells were harvested for CAT

activity assays. The transfection efficiency was normalized by co-

transfection of β-Gal expression vector (pSV-BGal). CAT enzyme activity: CAT activity was determined as described

(38) with some modifications. Briefly, cell extracts in 0.25 M Tris-HCl

pH8.0 obtained by freeze thaw were heated at 60°C/10min for the

inactivation of endogenous acetylase. Aliquotes of 115 ul cell lysate

were incubated witii 5 ul of ¹⁴C-chloramphenicol (Amersham, 0.025

mCi/ml) and 5 ul of n-butyryl coenzyme A (5ug/ml, Sigma) at 37°C for

60 min. The reaction products were extracted with 300 ul mixed

xylenes (Aldrich), die xylene phase was back-extracted with 0.25 M

Tris-HCl pH8.0 twice to remove all unreacted chloramphenical, and

use for liquid scintillation counting (LSC).

β-Gal enzyme assay: Co-transfected β-Gal activity in Cell lysates

(without heat inactivation) was determined as previously described (39).

Protein content assay: The protein content of cell lysates was

determined using me protein assay kit (BIO-RAD) based on the method

of Bradford (40).

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Claims

1. The nucleotide seuqence from +1 to +79bp (Exon 1) of the rabbit

smooth muscle myosin heavy chain gene or the sequence from +1 to

+61 bp (Exon 1) of the chicken skeletal Troponin I gene.

2. The nucleotide sequence of claim 1 for use as a promoter in die

expression of one or more genes, esepcially reporter genes, in a

mammalian host cell.

3. The nucleotide sequence of claim 1 for use in the construction of

a eukaryotic expression vector comprising a reporter or marker gene.

4. A eukaryotic expression vector comprising the nucleotide

sequence of claim 1, or a portion or mutant thereof.

5. An expression vector according to claim 4, selected from any of

these vectors disclosed herein with reference to the accompanying

drawings.

6. A plasmid comprising the nucleotide sequence of claim 1, or a

portion or mutant thereof.

7. A plasmid according to claim 6, selected from any of those

plasmids disclosed herein with reference to die accompanying drawings.

8. A mammalian host cell transfected witii a gene comprising the

sequence of claim 1, or a portion or mutant thereof, as the promoter

dierefor.

9. A cell according to claim 8, which is selected from rabbit or

human or other vascular smooth muscle cells, human dermal fibroblasts,

human Girard heart cells, rabbit skin fibroblasts, rabbit endothelial cells,

rabbit kidney epitiielial cells or rat skeletal muscle myoblasts.

10. A method of initiating and/or regulating expression levels and/or

tissue specificity of a gene in a mammalian host cell by use of a

promoter therefor which comprises the nucleotide sequence of claim 1

or a portion or mutant thereof.

11. A method according to claim 10 wherein the host cell is a human

cell.

12. A method according to claim 10, wherein the said gene is tiiat

which codes for chloramphemcol acetyl transferase (CAT) or β- galactosidase or the firefly luciferase gene.

13. A method according to claim 10, wherein the host cell is a rabbit

or human or other vascular smooth muscle cell, human dermal

fibroblast, human Girard heart cell, rabbit skin fibroblast, rabbit

endothelial cell, rabbit kidney epithelial cell or rat skeletal muscle

myoblast.

14. Use of the nucleotide sequence of claim 1, or a portion or mutant

thereof, as a promoter in the expression of one or more genes, especially

one or more reporter genes, in a mammalian host cell.

15. Use of the nucleotide sequence of claim 1, or a portion or mutant

thereof, in the construction of a eukaryotic expression vector for the

transient or stable expression of a foreign gene in a mammalian host

cell.

16. Use of the nucleotide sequence of claim 1, or a portion or mutant

thereof, in the construction of a vector comprising one or more marker

genes for vascular SMC phenotype diagnosis.

17. Use of the nucleotide sequence of claim 1, or a portion or mutant thereof, as a gene probe.

18. Use of die nucleotide sequence of claim 1, or a portion or mutant

thereof, in human gene therapy.