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PubMed2001

Characterization of the human alpha-synuclein gene: Genomic structure, transcription start site, promoter region and polymorphisms.

The human NACP/alpha-synuclein gene has been cloned. This gene consists of 6 exons ranging in size from 42 to 1110 bp. The translation start codon ATG is encoded by exon 2 and the stop codon TAA is encoded by exon 6. The non-Abeta component of Alzheimer's disease amyloid (NAC) is encoded by exon 4. The two previously reported minor isoforms of NACP/alpha-synuclein, NACP112 [29] and NACP126 [6], are alternatively spliced products, in which exon 5 and exon 3 are spliced out, respectively. Exon 1 was found to have different splicing sites, producing different 5'-untranslated sequences in the cDNAs. A previously reported dinucleotide repeat polymorphic marker has been mapped to 8kb upstream of the transcription start site. A highly TC-rich sequence in intron 4 was found to be polymorphic by length and four alleles, A0, A1, A2 and B have been identified in the Caucasian population. Genotyping this polymorphism among pure Alzheimer's, Lewy body variant and Parkinson's subjects and aged normal control subjects did not reveal any significant differences.

Journal Article↗

PubMed1992

A putative two-component regulatory system involved in secondary metabolism in Streptomyces spp.

A DNA fragment stimulating actinorhodin, undecylprodigiosin, and A-factor production in Streptomyces lividans 66 was cloned from Streptomyces coelicolor A3(2). Nucleotide sequencing revealed the presence of an open reading frame of 225 codons, named afsQ1, that showed great similarity in amino acid sequence to the response regulators of typical prokaryotic two-component regulatory systems responsible for adaptive responses. The termination codon, TGA, of afsQ1 overlapped the initiation codon, GTG, of a second open reading frame, afsQ2, of 535 codons. The afsQ2 gene product showed homology with the sensory histidine protein kinases of two-component systems. In agreement with the assumption that the AfsQ1 and AfsQ2 proteins comprise an aspartate-histidine phosphotransfer system, an amino acid replacement from Asp to Glu at residue 52 of AfsQ1, generated by site-directed mutagenesis, resulted in loss of the protein's ability to stimulate antibiotic production in S. lividans. Primer extension experiments indicated that transcription of the afsQ1 and afsQ2 genes initiates at the translational start codon (GTG) of the afsQ1 gene. The afsQ1 and afsQ2 genes were physically mapped at a chromosomal position near the actinorhodin biosynthetic gene cluster (act) by hybridization to Southern blots of restriction fragments separated by pulsed-field gel electrophoresis. Disruption of either afsQ1 or afsQ2 on the S. coelicolor chromosome by use of phage phi C31KC515 led to no detectable change in secondary metabolite formation or morphogenesis. The afsQ1 gene on pIJ922 suppressed the S. coelicolor absA mutation and caused actinorhodin production but did not suppress the absB mutation. Southern blot hybridization showed that sequences homologous to afsQ1 and afsQ2 are present in almost all of the actinomycetes examined.

4-Butyrolactone↗

PubMed2003

Upstream short sequence repeats regulate expression of the alpha C protein of group B Streptococcus.

Group B streptococci (GBS) express a family of repeat-containing surface proteins, the prototype of which is the alpha C protein expressed in type Ia/C strain A909. We have isolated a series of mutant GBS strains by mouse-passage of A909 that do not produce normal levels of the alpha C protein. Polymerase chain reaction amplification and sequencing of the gene encoding the alpha C protein, bca, from four mutant strains revealed the presence of a full-length gene in each strain. However, Northern and RT-PCR analysis revealed greatly reduced levels of RNA encoding the alpha C protein. Sequence analysis of the mutant genes found the coding region unchanged from the wild-type gene in each case, but variation was observed in a specific locus located 110 bp upstream of the start codon. The presence of a 5-nucleotide repeat, AGATT, and a string of adenine residues mark this locus. Both deletion and expansion of the AGATT motif were associated with the complete null phenotype. Deletions in the string of adenine residues were associated with both a decreased-production phenotype and a complete null phenotype. Cloning of this upstream region into a green-fluorescent protein (GFP) reporter system in GBS demonstrated promoter activity that was completely abolished by changes in the pentanucleotide repeat or adenine string. Primer extension studies of the wild-type strain revealed one dominant and two minor transcription start sites. Primer extension studies of the null and low-expression mutant strains revealed that the dominant transcript is completely absent in each mutant. The short sequence repeat locus is located at position - 55 to - 78 relative to the start site of the dominant transcript. We have demonstrated in vitro phase variation in expression of the alpha C protein associated with variation at the pentanucleotide repeat locus. We conclude that this short sequence repeat motif is located upstream of the dominant promoter for the alpha C protein and represents a regulatory site for alpha C protein expression. This is the first evidence of transcriptional regulation by short-sequence repeats in a Gram-positive organism.

Animals↗

PubMed2000

Bernard-Soulier syndrome in a patient doubly heterozygous for two frameshift mutations in the glycoprotein ib alpha gene.

We report here the genetic basis of Bernard-Soulier syndrome in a compound heterozygote for two mutant glycoprotein (GP) Ib alpha alleles. One allele contained a novel four base-pair deletion (TGAG) that eliminated the last base of the codon for Ser39 (AGT) and the entire codon for Glu40 (GAG), causing a reading frame shift that yielded a stretch of 51 amino acids before a premature stop codon. The other allele also contained a frame-shift mutation, caused by deletion of the last two bases of the codon for Tyr492 (TAT). This allele produced a truncated glycoprotein Ib alpha that, although not expressed on the surface of the patient's platelets, was detectable in the plasma. The second allele has been identified previously by our group and other investigators as the cause of Bernard-Soulier syndrome in patients of northern European ancestry. This allele carried a haplotype identical to those of the previously reported cases, with the following polymorphic markers: two tandem repeats in the VNTR region, C at nucleotide -5 from the ATG start codon and a substitution of G for A in the third base for codon Arg342. These findings suggest that this particular Bernard-Soulier mutation occurred once on the background of a rare haplotype and has spread throughout the northern European population.

Bernard-Soulier Syndrome↗

PubMed1997

The Rhodobacter capsulatus hupSLC promoter: identification of cis-regulatory elements and of trans-activating factors involved in H2 activation of hupSLC transcription.

The [NiFe]hydrogenase of the photosynthetic bacterium Rhodobacter capsulatus is encoded by the structural hupSLC operon, the expression of which is induced by H2. H2 activation was no longer observable in chromosomal hupR mutants, an indication that HupR is implicated directly in the activation by H2 of hupS gene expression. The transcriptional start site of the hupS promoter, determined by primer extension mapping, was located 55 nucleotides upstream from the translational start codon of the hupS gene. Regulatory sequences were identified by serial 5' deletions of the 300bp hupS promoter-regulatory region (phupS) and phupS-lacZ translational fusions. Cis-regulatory sequences capable of interacting with two transcription factors, IHF and HupR, a response regulator of the NtrC subfamily, were studied by electrophoretic mobility shift assays (EMSAs). The R. capsulatus IHF and HupR proteins were overexpressed in Escherichia coli and purified by affinity chromatography. IHF binds to a site, 5'-TCACACACCATTG, centred at -87 nt from the transcription start site. The HupR protein binds to one site within the -162 to -152 nt region, which contains the palindromic sequence 5'-TTG-R5-CAA. By the use of 5' deletions and site-directed mutagenesis of the -162/-152 region, this palindrome was shown to be required for in vivo hupS transcriptional activation by H2.

Artificial Gene Fusion↗

PubMed1991

A quantitative model for translational control of the GCN4 gene of Saccharomyces cerevisiae.

Expression of the GCN4 gene of Saccharomyces cerevisiae is regulated at the translational level by short open reading frames (uORFs) present in the leader sequence of its mRNA. Under conditions of amino acid sufficiency, these sequences restrict the flow of initiating ribosomes to the GCN4 AUG start codon. Mutational analysis of GCN4 has led to a model in which ribosomes must translate the 5'-proximal uORF1 and reassemble an initiation complex in order to translate GCN4. This reassembly process is thought to be rapid when amino acids are abundant, such that reinitiation occurs at uORF2, uORF3, or uORF4. Reinitiation at these sites prevents translation of GCN4, presumably because ribosomes dissociate from the mRNA following termination at uORFs 2 to 4. Because of reduced initiation factor activity under starvation conditions, a substantial fraction of ribosomal subunits scanning downstream from uORF1 are not ready to reinitiate when they reach uORFs 2 to 4, but become competent to do so while scanning the additional sequences between uORF4 and GCN4. Examination of the effects of point mutations in the ATG codons of the different uORFs suggests a quantitative model for this control mechanism that describes the probability of reinitiation as a function of the distance scanned downstream from uORF1. This model accounts for the phenotypes of a number of deletion and insertion mutations that alter the intercistronic spacing between the uORFs and GCN4. The correspondence between observed and predicted results implies that the differential rates of reinitiation at GCN4 versus uORFs 2 to 4 are determined largely by the different scanning times required to reach each of these start sites following translation of uORF1. In addition, it supports the notion that an increased scanning-time requirement for reinitiation in amino acid-starved cells forms the basis for translational derepression of GCN4 expression.

Amino Acids↗

PubMed1990

High genomic variability in the pre-C region of hepatitis B virus in anti-HBe, HBV DNA-positive chronic hepatitis.

Some chronic HBV carriers have circulating HBV DNA despite the presence of anti-HBeAg antibodies. This observation has recently been related to the presence, in the HBV pre-C region, of a translational stop codon that prevents HBeAg synthesis. In the present study, we analyzed, at the nucleotide level, the pre-C/C region of HBV isolated from the sera of 11 anti-HBe, HBV DNA-positive chronic carriers. Nucleotide sequence analysis of 25 independent clones revealed that the pre-C sequence is highly variable, even among clones derived from the same serum sample. Moreover, our data show that HBeAg synthesis can also be prevented by as yet undescribed T-C substitution at position 1815 that eliminates the start codon of the pre-C transcript. These results suggest that the HBV genome contains segments of high variability that have probably been selected during evolution to favor the segregation of functionally advantaged mutants capable of avoiding host immunity.

Base Sequence↗

PubMed1988

Nucleotide sequence of the phosphinothricin N-acetyltransferase gene from Streptomyces viridochromogenes Tü494 and its expression in Nicotiana tabacum.

The phosphinothricin (Pt) N-acetyltransferase gene (pat) of Streptomyces viridochromogenes Tü494 is located on a 0.8-kb BglII fragment [Strauch et al., Gene 63 (1988) 65-74]. By sequencing a 1.3-kb BglII-SstII fragment, an open reading frame representing the pat gene was found. It encodes a polypeptide of 183 amino acids with an Mr of 20,621. The base composition of the pat gene is typical for Streptomyces [70.1 mol% (G + C) in total and 93.5 mol% (G + C) in the third position]. Translation of pat is initiated by a GTG codon which was identified by frameshift mutations in Escherichia coli as well as in Streptomyces lividans. Significant homology of the pat gene was found to the bialaphos-resistance gene (bar) of Streptomyces hygroscopicus [Thompson et al., EMBO J. 9 (1987) 2519-2523]. However, variations were detected in the 5'-noncoding region of the two resistance genes which may reflect differences in regulation. Since Pt is a potent herbicide, the pat gene was modified and introduced into Nicotiana tabacum by Agrobacterium-mediated leaf-disc transformation. The GTG start codon of pat was replaced by ATG. Subsequently the modified pat-coding region was fused to the 35S promoter of the cauliflower mosaic virus. Transgenic plants could directly be selected on Pt-containing medium.

Acetyltransferases↗

PubMed1983

The structure of the vimentin gene.

The structure of the chromosomal gene encoding the intermediate filament protein vimentin is described. This gene, which is present as a single copy in the hamster genome, comprises about 10 kb of DNA and contains more than 80% of intron sequences. S1 mapping and sequence analysis reveal nine exons with a total length of 1848 nucleotides. For the complete primary structure of hamster vimentin, 464 amino acids are predicted, giving a molecular weight of 53,500 daltons. The intron positions are at codons 186, 206/207, 238/239, 292/293, 334/335, 408, 423, and 451/452. The overall homology with chicken desmin is 60% and is even higher in the central (alpha-helical) regions of both molecules. Cross-hybridization at the DNA level, however, is low. Comparison of the amino acid sequence of vimentin with prekeratin sequences shows that there is lesser homology of primary structure, but both the position and size of alpha-helical regions are strongly conserved. At the 5' end of the gene there is a consensus promoter sequence. The first AUG start codon is found 132 nucleotides downstream of the estimated cap site. The 3' nontranslated sequence shows homologies with the chicken vimentin gene. An interesting feature of the vimentin gene is a stretch of 44 nucleotides of alternating dC and dA within intron 2 that may form left-handed Z-DNA.

Amino Acid Sequence↗

PubMed2003

A novel hormone-sensitive lipase isoform expressed in pancreatic beta-cells.

Hormone-sensitive lipase (HSL) is a key enzyme in fatty acid mobilization in many cell types. Two isoforms of HSL are known to date, namely HSL(adi) (84 kDa in rat) and HSL(tes) (130 kDa in rat). These are encoded by the same gene, with exons 1-9 encoding the parts that are common to both and an additional 5'-exon encoding the additional amino acids in HSL(tes). HSL of various tissues, among these the islet of Langerhans, is larger than HSL(adi), but not as large as HSL(tes), indicating that there may be other 5'-coding exons. Here we describe the molecular basis for a novel 89-kDa HSL isoform that is expressed in beta-cells, adipocytes, adrenal glands, and ovaries in the rat and that is encoded by exons 1-9 and exon A, which is spliced to exon 1 and thereby introducing an upstream start codon. The additional 5'-base pairs encode a 43-amino acid peptide, which is highly positively charged. Conglomerates of HSL molecules are in close association with the secretory granules of the beta-cell, as determined by immunoelectron microscopy with antibodies targeting two separate regions of HSL. We have also determined that the human genomic sequence upstream of exon A has promoter activity in INS-1 cells as well as glucose sensing capability, mediating an increase in expression at high glucose concentration. The minimal promoter is present within 170 bp from the transcriptional start site and maximal glucose responsiveness is conferred by sequence within 850 bp from the transcriptional start site.

Adipocytes↗

PubMed1989

Sequence analysis of the 47-kilodalton major integral membrane immunogen of Treponema pallidum.

The complete primary amino acid sequence for the 47-kilodalton (kDa) major integral membrane immunogen of Treponema pallidum subsp. pallidum was obtained by using a combined strategy of DNA sequencing (of the cloned gene in Escherichia coli) and N-terminal amino acid sequencing of the native (T. pallidum subsp. pallidum-derived) antigen. An open reading frame believed to encode the 47-kDa antigen comprised 367 amino acid codons, which gave rise to a calculated molecular weight for the corresponding antigen of 40,701. Of the 367 amino acids, 113 (31%) were sequenced by N-terminal amino acid sequencing of trypsin and hydroxylamine cleavage fragments of the native molecule isolated from T. pallidum subsp. pallidum; amino acid sequence data had a 100% correlation with that of the amino acid sequence predicted from DNA sequencing of the cloned gene in E. coli. Although no consensus sequences for the initiation of transcription or translation were readily identifiable immediately 5' to the putative methionine start codon, a 63-base-pair PstI fragment located 159 nucleotides upstream was required for expression of the 47-kDa antigen in E. coli. The 47-kDa antigen sequence did not reveal a typical leader sequence. The overall G+C content for the DNA corresponding to the structural gene was 53%. Hydrophilicity analysis identified at least one major hydrophilic domain of the protein near the N terminus of the molecule which potentially represents an immunodominant epitope. No repetitive primary sequence epitopes were found. The combined data provide the molecular basis for further structural and functional studies regarding the role of the antigen in the immunopathogenesis of treponemal disease.

Amino Acid Sequence↗

PubMed1997

Two Sp sites are important cis elements regulating the upstream promoter region of the gene for rat type I hexokinase.

Multiple transcriptional start sites have been identified for the gene encoding the rat Type I isozyme of hexokinase (White, J.A., Liu, W., and Wilson, J. E., Arch. Biochem. Biophys. 335, 161-172, 1996); these are clustered at positions approximately -460, -300, and -100 relative to the translational start codon (ATG, with A being +1). PC12 cells and H9c2 cells were transfected with luciferase reporter constructs containing genomic sequence between positions -3366 and -171. Marked (85%) decrease in promoter activity was associated with deletion of sequence between -742 and -516. In DNase I footprinting experiments, two regions, called P1 (-552 to -529) and P2 (-480 to -458) boxes, were protected by proteins present in nuclear extracts from PC12 cells. Mutation or deletion of the P2 box had no effect on promoter activity; protection in this region, which includes the most upstream cluster of transcriptional start sites, is attributed to binding of RNA polymerase II or associated factors. In contrast, mutations or deletions in the P1 box had markedly detrimental effects on promoter activity and on binding of proteins in PC12 cell nuclear extracts. Maintenance of a consensus Sp1 binding site centrally located in the P1 box was critical for both promoter activity and binding. A second Sp1 site (-570), just upstream from the P1 box, was also shown to be functionally important but no protection of this region was detected in footprinting experiments, presumably reflecting lower affinity at this site under the conditions used. Supershift experiments demonstrated the involvement of Sp1, Sp3, and Sp4 in formation of complexes with the P1 box region and implicate these transcription factors in regulating promoter activity associated with this region. Another series of reporter constructs, including sequence between -171 and -1, permitted detection of an additional promoter activity downstream from -364. While not yet extensively characterized, it is already evident that the cis elements influencing the downstream promoter activity are distinct from the Sp factors determined to be important in expression from the upstream promoter region.

Animals↗

PubMed1980

Polyoma virus DNA: complete nucleotide sequence of the gene which codes for polyoma virus capsid protein VP1 and overlaps the VP2/VP3 genes.

The nucleotide sequence of part of the late region of the polyoma virus genome was determined. It contains coding information for the major capsid protein VP1 and the C-terminal region of the minor proteins VP2 and VP3. In the sequence with the same polarity as late mRNA's, all coding frames are blocked by termination codons in a region around 48 units on the physical map. This is the region where the N-terminus of VP1 and the C-termini of VP2 and VP3 have been located (T. Hunter and W. Gibson, J. Virol. 28:240-253, 1978; S. G. Siddell and A. E. Smith, J. Virol. 27:427-431, 1978; Smith et al., Cell 9:481-487, 1976). There are two long uninterrupted coding frames in the late region of polyoma virus DNA. One lies at the 5' end of the sequence and contains potential coding sequences for VP2 and VP3. The other contains 383 consecutive sense codons starting with the ATG at nucleotide position 1,218, extends from 47.5 to 25.8 units counterclockwise on the physical map, and is located where the VP1 gene has been mapped. The VP1 gene overlaps the genes for proteins VP2/VP3 by 32 nucleotides and uses a different coding frame. From the DNA sequence, the amino acid sequence of VP1 was predicted. The proposed VP1 sequence is in good agreement with other data, namely, with the partial N-terminal amino acid sequence and the total amino acid composition. The VP1 coding frame terminates with a TAA codon at 25.8 map units. This is followed by an AATAAA sequence, which may act as a processing signal for the viral late mRNA's. When both nucleotide and amino acid sequences are compared with their counterparts in the related simian virus 40, extensive homologies are found over the entire region of the two viral genomes. Maximum homology appears to occur in those regions which code for the C-termini of the VP1 proteins. The overlap region of VP1 with VP2/VP3 of polyoma virus is shorter by 90 nucleotides than is that of simian virus 40 and shows very limited homology with the simian virus 40 sequence. This leads to the suggestion that the overlap segments of both viruses have been freed from stringency imposed on drifting during evolution and that proteins VP2 and VP3 of polyoma virus may have been truncated by the appearance of a termination codon within the sequence.

Amino Acid Sequence↗

PubMed1986

nprR1 and nprR2 regulatory regions for neutral protease expression in Bacillus subtilis.

The gene coding for the Bacillus subtilis extracellular neutral protease was isolated from strain BGSC 1A341, an overproducer carrying the nprR2 region, and from strain 168, a normal producer with the nprR1 sequence. The sequence of about 600 nucleotides upstream from the start codon of the protease gene was determined for both strains. The two regions are highly homologous except for a stretch of 66 base pairs close to the promoter region, which is absent in the BGSC 1A341 gene. Northern blot analysis of the in vivo RNAs indicated that the different levels of enzyme secreted by the two strains were due to different amounts of transcripts that accumulated in the cells. Furthermore, at the end of exponential growth, the amount of transcript increased dramatically in the overproducer strain but remained approximately constant in the normal producer strain. The start point(s) for transcription, however, as determined by S1 nuclease mapping of the in vivo transcripts, appeared to be the same for both genes.

Bacillus subtilis↗

PubMed1996

Two beginnings for a single purpose: the dual-start holins in the regulation of phage lysis.

For most large phages of both Gram-positive and Gram-negative bacteria, there appears to be a single pathway for achieving disruption of the host envelope, requiring at least two phage-encoded lysis functions (a holin and an endolysin). The holin is a small membrane protein which causes a non-specific lesion in the cytoplasmic membrane, which allows the endolysin to gain access to its substrate, the peptidoglycan. The scheduling of host lysis is effected by regulatory mechanisms which govern the synthesis and activity of the holin protein accumulating in the membrane. Accordingly, aspects of expression and function of holin genes are considered here, focusing mainly on the lambdoid S genes. This group of genes, of which lambda S is the prototype, are characterized by a dual-start motif consisting of two Met start codons separated by one or two codons, at least one of which specifies Arg or Lys. Two protein products are elaborated, differing only by two or three N-terminal residues but apparently possessing opposing functions: the shorter polypeptide is the active holin, or lysiseffector, whereas the longer polypeptide apparently acts as an inhibitor of holin function. Models will be considered which may account for the ability of the holin to form a 'hole' in the cytoplasmic membrane at a programmed time, as well as for the inhibitory properties of the longer product. Finally, we discuss recent results suggesting that the dual-start motif can be viewed as a level of regulation superimposed on a timing function intrinsic to the canonical holin structure.

Amino Acid Sequence↗

PubMed1993

Escherichia coli rpiA gene encoding ribose phosphate isomerase A.

The rpiA gene encoding ribose phosphate isomerase A was cloned from phage 1A2(471) of the Kohara gene library. Subcloning, restriction, and complementation analyses revealed an 1,800-bp SspI-generated DNA fragment that contained the entire control and coding sequences. This DNA fragment was sequenced and shown to harbor an open reading frame of 219 codons, sufficient to encode a polypeptide with an M(r) of 22,845. The synthesis of the rpiA-encoded polypeptide was detected by analysis of minicells, which established the subunit M(r) as 27,000. The assignment of the correct reading frame was confirmed by amino-terminal analysis of partially purified ribose phosphate isomerase A. Our data indicate that the enzyme is composed of two identical subunits. The 5' end of the rpiA-specified transcript was analyzed by primer extension, which revealed a well-conserved -10 region 34 bp upstream of the presumed translation start codon. Analysis of the 3' end of the transcript by S1 nuclease mapping showed that transcription termination occurred within an adenylate-rich sequence following a guanylate-cytidylate-rich stem-loop structure resembling a rho factor-independent transcription terminator. Host strains harboring the rpiA gene in a multicopy plasmid contained up to 42-fold as much ribose phosphate isomerase A activity as the haploid strain.

Aldose-Ketose Isomerases↗

PubMed2005

Tissue-specific and glucose-responsive expression of the pancreatic derived factor (PANDER) promoter.

Pancreatic derived factor (PANDER) is a recently identified cytokine-like protein that is dominantly expressed in the islets of Langerhans of the pancreas. To investigate the mechanism of tissue-specific regulation of PANDER, we identified and characterized the promoter region. The transcriptional start site was identified 520 bp upstream of the translational start codon by 5'-RLM-RACE. Computer algorithms identified several islet-associated and glucose-responsive binding motifs that included A and E boxes, hepatocyte nuclear factors 1 and 4, Oct-1, and signal transducer and activator of transcription 3, and 5. Reporter gene analysis revealed cell type-specific PANDER promoter expression in islet and liver-derived cell lines. Levels of PANDER mRNA were directly concordant to the observed cell type-specific PANDER promoter gene expression. The minimal element was mapped to the 5'-UTR and located between +200 and +491 relative to the transcriptional start site and imparted maximal gene expression. In addition, several putative glucose-responsive binding sites were further functionally characterized to reveal critical regulatory elements of PANDER. The PANDER promoter was demonstrated to be glucose-responsive in a dose-dependent manner in murine insulinoma beta-TC3 cells and primary murine islets, but unresponsive in glucagon-secreting alpha-TC3 cells. Our findings revealed that the 5'-UTR of PANDER contains the minimal element for gene expression and imparts both tissue-specificity and glucose-responsiveness. The regulation of PANDER gene expression mimics that of insulin and suggests a potential biological function of PANDER involved in metabolic homeostasis.

5' Untranslated Regions↗

PubMed2001

Functional analysis of the chimpanzee and human apo(a) promoter sequences: identification of sequence variations responsible for elevated transcriptional activity in chimpanzee.

Lp(a) concentrations vary considerably among individuals and are primarily determined by the apo(a) gene locus. We have previously shown that mean plasma Lp(a) levels in the chimpanzee are significantly higher than those observed in humans (Doucet, C., Huby, T., Chapman, J., and Thillet, J. (1994) J. Lipid Res 35, 263-270). To evaluate the possibility that this difference may result from a high level of expression of chimpanzee apo(a), we cloned and sequenced 1.4 kilobase (kb) of the 5'-flanking region of the gene and compared promoter activity to that of its human counterpart. Sequence analysis revealed 98% homology between chimpanzee and human apo(a) 5' sequences; among the differences observed, two involved polymorphic sites associated with Lp(a) levels in humans. The TTTTA repeat located 1.3 kb 5' of the apo(a) gene, present in a variable number of copies (n = 5-12) in humans, is uniquely present as four copies in the chimpanzee sequence. The second position concerns the +93 C>T polymorphism that creates an additional ATG start codon in the human apo(a) gene, thereby impairing translation efficiency. In chimpanzee, this position did not appear polymorphic, and a base difference at position +94 precluded the presence of an additional ATG. In transient transfection assays, the chimpanzee apo(a) promoter exhibited a 5-fold elevation in transcriptional activity as compared with its human counterpart. This marked difference in activity was maintained with either 1.4 kb of 5' sequence or the minimal promoter region -98 to +141 of the human and chimpanzee apo(a) genes. Using point mutational analyses, nucleotides present at positions -3, -2, and +8 (relative to the start site of transcription) were found to be essential for the high transcription efficiency of the chimpanzee apo(a) promoter. High transcriptional activity of the chimpanzee apo(a) gene may therefore represent a key factor in the elevated plasma Lp(a) levels characteristic of this non-human primate.

Animals↗