TATA Consensus Sequence Research Articles

Characterization of cis-acting elements required for autorepression of the equine herpesvirus 1 IE gene

The immediate-early protein (IEP), the major regulatory protein encoded by the IE gene of equine herpesvirus 1 (EHV-1), plays a crucial role as both transcription activator and repressor during a productive lytic infection. To investigate the mechanism by which the EHV-1 IEP inhibits its own promoter, IE promoter-luciferase reporter plasmids containing wild-type and mutant IEP-binding site (IEBS) were constructed and used for luciferase reporter assays. The IEP inhibited transcription from its own promoter in the presence of a consensus IEBS (5′-ATCGT-3′) located near the transcription initiation site but did not inhibit when the consensus sequence was deleted. To determine whether the distance between the TATA box and the IEBS affects transcriptional repression, the IEBS was displaced from the original site by the insertion of synthetic DNA sequences. Luciferase reporter assays revealed that the IEP is able to repress its own promoter when the IEBS is located within 26-bp from the TATA box. We also found that the proper orientation and position of the IEBS were required for the repression by the IEP. Interestingly, the level of repression was significantly reduced when a consensus TATA sequence was deleted from the promoter region, indicating that the IEP efficiently inhibits its own promoter in a TATA box-dependent manner. Taken together, these results suggest that the EHV-1 IEP delicately modulates autoregulation of its gene through the consensus IEBS that is near the transcription initiation site and the TATA box.

Cloning and sequencing of phenol oxidase 1 (pox1) gene from Pleurotus ostreatus

The gene (pox1) encoding a phenol oxidase 1 from Pleurotus ostreatus was sequenced and the corresponding pox1-cDNA was also synthesized, cloned and sequenced. The isolated gene is flanked by an upstream region called the promoter (399 bp) prior to the start codon (ATG). The putative metal-responsive elements (MREs) were determined in the promoter region, where MRE 1, 2 and 3 were located in positions -20, -62 and -389, respectively. Functional TATA consensus sequences were recognized in positions -78 and -245, while CAAT consensus sequence was recognized in position -171. The putative GC boxes consensus sequences were recognized in positions -175 and -344, and xenobiotic-responsive elements (XREs) in positions -100 and -270. The pox1-DNA gene consists of 2656 bp, with the coding sequence being interrupted by 19 introns. The nucleotide sequence of cDNA (pox1-cDNA) was found to contain an ORF of 1590 bp capable of coding for a protein of 529 amino acid residues. The signal peptide was predicted to be 23 amino acids in length using SIGNALP 3.0 program. Northern blot analysis revealed that strong transcriptional induction was observed in the copper-supplemented cultures for pox1 gene. Key words: Pleurotus, cDNA, pox1, gene promoter, putative sequences, northern blot analysis, copper.

Cloning and sequencing of phenol oxidase 1 (pox1) gene from Pleurotus ostreatus

The gene (pox1) encoding a phenol oxidase 1 from Pleurotus ostreatus was sequenced and the corresponding pox1-cDNA was also synthesized, cloned and sequenced. The isolated gene is flanked by an upstream region called the promoter (399 bp) prior to the start codon (ATG). The putative metalresponsive elements (MREs) were determined in the promoter region, where MRE 1, 2 and 3 were located in positions -20, -62 and -389, respectively. Functional TATA consensus sequences were recognized in positions -78 and -245, while CAAT consensus sequence was recognized in position -171. The putative GC boxes consensus sequences were recognized in positions -175 and -344, and xenobiotic-responsive elements (XREs) in positions -100 and -270. The pox1-DNA gene consists of 2656 bp, with the coding sequence being interrupted by 19 introns. The nucleotide sequence of cDNA (pox1- cDNA) was found to contain an ORF of 1590 bp capable of coding for a protein of 529 amino acid residues. The signal peptide was predicted to be 23 amino acids in length using SIGNALP 3.0 program. Northern blot analysis revealed that strong transcriptional induction was observed in the coppersupplemented cultures for pox1 gene. Key words : Pleurotus, cDNA, pox1, gene promoter, putative sequences, northern blot analysis, copper.

Molecular characterization of the phenol oxidase (pox2) gene from the ligninolytic fungusPleurotus ostreatus

The gene (pox2) encoding a phenol oxidase from Pleurotus ostreatus, a lignin-degrading basidiomycete, was sequenced and the corresponding pox2-cDNA was also synthesized, cloned and sequenced. The isolated gene consisted of 2674 bp, with the coding sequence interrupted by 19 introns and flanked by an upstream region in which the putative metal-responsive elements (MREs) were determined in the promoter region (849 bp), where MRE 1, 2, 3 and 4 were located in positions -20, -60, -236 and -297. A functional TATA consensus sequence was recognized in position -85, while CAAT and its inversion consensus sequences were recognized in positions -284, -554, -689 and -752. The putative GC box consensus sequences were recognized in positions -181 and -460, and xenobiotic-responsive elements in positions -107, -277 and -390. The isolation of a second cDNA (pox2-cDNA), the nucleotide sequence of pox2, was found to contain an ORF of 1665 bp capable of coding for a protein of 533 amino acid residues. Northern blot analysis revealed that strong transcriptional induction was observed in the copper-supplemented cultures for the pox2 gene.

Polymorphisms in the 5′ flanking region of the insulin-like growth factor I gene are associated with growth traits in largemouth bass Micropterus salmoides

Insulin-like growth factor I (IGF-1) plays an important role in fish growth and development. As the promoter of IGF-I regulates the expression of IGF-I, polymorphisms in the promoter region could be functionally important to differences in IGF-I expression and function. In this study, the 5′ flanking region (1,770 bp) of the largemouth bass IGF-I gene was cloned, sequenced and characterized. Several putative liver-enriched transcriptional factor binding sites, including HNF-1α, HNF-3β and C/EBP, were found to have multiple copies in the promoter sequence of the largemouth bass IGF-I gene. It did not contain consensus TATA, CCAAT-like or GAGA sequences. Sequencing of 5′ flanking sequences from individual fish revealed the presence of a deletion (GTTT) and two single-nucleotide substitutions, causing two different haplotypes: ATTTTTGTTTTT(A) and ATAATT—TT(B). The haplotypes were tested for their association with growth traits in a population of 91 largemouth bass individuals using polymerase chain reaction. The association analysis was carried out using a general linear model. Results showed the haplotypes had significant effects on body weight and body width (P < 0.05). The individuals with the AA genotype had significantly higher body weight and body width than those with the AB and BB genotypes (P < 0.05). Moreover, the effect of the genotypes on IGF-I mRNA expression in the liver was evaluated through the use of a semiquantitative RT-PCR method. The results showed that the IGF-I mRNA expression levels were affected by the promoter polymorphisms (P < 0.01), and that levels of IGF-I mRNA of the AA genotype in liver were greater than those of both the AB and the BB genotypes (P < 0.05 and P < 0.01). Our results implied that the IGF-I gene could be a potential quantitative trait nucleotide that affects growth traits in largemouth bass.

Minimal components of the RNA polymerase II transcription apparatus determine the consensus TATA box

In Saccharomyces cerevisiae, multiple approaches have arrived at a consensus TATA box sequence of TATA(T/A)A(A/T)(A/G). TATA-binding protein (TBP) affinity alone does not determine TATA box function. To discover how a minimal set of factors required for basal and activated transcription contributed to the sequence requirements for a functional TATA box, we performed transcription reactions using highly purified proteins and CYC1 promoter TATA box mutants. The TATA box consensus sequence is a good predictor of promoter activity. However, several nonconsensus sequences are almost fully functional, indicating that mechanistic requirements are not the only selective pressure on the TATA box. We also found that the effect of a mutation at a certain position is often dependent on other bases within a particular TATA box. Although activators and coactivators strongly influence TBP recruitment and stability at promoters, neither Mediator, the activator Gal4-V16, nor TFIID specifically compensate for the low transcription levels of the weak TATA boxes. The addition of Mediator to purified transcription reactions did, however, increase the functional selectivity for certain consensus TATA sequences. Transcription in whole-cell extracts or in vivo with these TATA box mutants indicated that factors, other than those in our purified system, may help initiate transcription from weak TATA boxes.

Gene structure and promoter functional analysis of the marmoset type II GnRH receptor

The marmoset type II GnRH receptor (GnRH-R) gene has the same structure and genomic organisation as the human and other type II GnRH-R genes. The gene consists of three exons and two introns and overlaps in the antisense orientation on its 5' end with peroxisomal membrane protein 11beta and on its 3' end with the RNA-binding motif protein 8A. However, these genes occur only at one locus in the marmoset genome, while in the human at two loci. Employing 5' rapid amplification of cDNA ends demonstrated that the marmoset type II GnRH-R gene has two transcriptional start sites at -341 and -567 nucleotides relative to the translational start codon and both start sites lack TATA and CAAT consensus sequences. A luciferase reporter construct with a 2.3 kb 5' flanking region of the type II GnRH-R gene was active in a wide variety of cell lines tested, consistent with the wide tissue expression of the receptor. Progressive 5' and 3' deletions were employed to identify sequences required for basal expression of the type II GnRH-R gene. This analysis identified negative regulatory elements in the regions -2342/-1995, -1679/-1084 and -458/-1 and positive regulatory elements in the regions -1995/-1679, -1084/-458 and -458/-1 relative to the translational start site. The strongest of the positive regions located between -766/-665 has enhancer activity when cloned in front of a heterologous minimal promoter and is critical for basal expression of the type II GnRH-R.

Real-time monitoring of functional interactions between upstream and core promoter sequences in living cells of sea urchin embryos

There are some functional compatibilities between upstream and core promoter sequences for transcriptional activation in yeast, Drosophila and mammalian cells. Here we examined whether similar compatibilities exist in sea urchin embryos, and if so, whether they are dynamically regulated during early development. Two reporter plasmids, each containing a test promoter conjugated to either CFP or YFP, were concurrently introduced into embryos, and their expression patterns were studied by fluorescence microscopy. The upstream sequence of the Hemicentrotus pulcherrimus (Hp) OtxE promoter drives the expression of its own core promoter and that of Strongylocentrotus purpuratus (Sp) Spec2a in different embryonic regions, especially at the late gastrula stage. Interestingly, when the four putative transcription factor binding sites of this upstream sequence were individually mutated, the resulting sequences directed different spatiotemporal expression from the same set of two core promoters, indicating that combinations of upstream factors may determine core promoter usage in sea urchin embryos. In addition, the insertion or deletion of consensus or nonconsensus TATA sequences changed the expression profile significantly, irrespective of whether the upstream sequence was intact or mutated. Thus, the TATA sequence may serve as a primary determinant for core promoter selection in these cells.

Rate of promoter class turn-over in yeast evolution

BackgroundPhylogenetic conservation at the DNA level is routinely used as evidence of molecular function, under the assumption that locations and sequences of functional DNA segments remain invariant in evolution. In particular, short DNA segments participating in initiation and regulation of transcription are often conserved between related species. However, transcription of a gene can evolve, and this evolution may involve changes of even such conservative DNA segments. Genes of yeast Saccharomyces have promoters of two classes, class 1 (TATA-containing) and class 2 (non-TATA-containing).ResultsComparison of upstream non-coding regions of orthologous genes from the five species of Saccharomyces sensu stricto group shows that among 212 genes which very likely have class 1 promoters in S. cerevisiae, 17 probably have class 2 promoters in one or more other species. Conversely, among 322 genes which very likely have class 2 promoters in S. cerevisiae, 44 probably have class 1 promoters in one or more other species. Also, for at least 2 genes from the set of 212 S. cerevisiae genes with class 1 promoters, the locations of the TATA consensus sequences are substantially different between the species.ConclusionOur results indicate that, in the course of yeast evolution, a promoter switches its class with the probability at least ~0.1 per time required for the accumulation of one nucleotide substitution at a non-coding site. Thus, key sequences involved in initiation of transcription evolve with substantial rates in yeast.

Efficient Binding of NC2·TATA-binding Protein to DNA in the Absence of TATA

Negative cofactor 2 (NC2) forms a stable complex with TATA-binding protein (TBP) on promoters. This prevents the assembly of transcription factor (TF) IIA and TFIIB and leads to repression of RNA polymerase II transcription. Here we have revisited the interactions of NC2.TBP with DNA. We show that NC2.TBP complexes exhibit a significantly reduced preference for TATA box sequences compared with TBP and TBP.TFIIA complexes. In chromatin immunoprecipitations, NC2 is found on a variety of human TATA-containing and TATA-less promoters. Substantial amounts of NC2 are present in a complex with TBP in bulk chromatin. A complex of NC2.TBP displays a K(D) for DNA of approximately 2 x 10(-9) m for a 35-bp major late promoter oligonucleotide. While preferentially recognizing promoter-bound TBP, NC2 also accelerates TBP binding to promoters and stabilizes TBP.DNA complexes. Our data suggest that NC2 controls TBP binding and maintenance on DNA that is largely independent of a canonical TATA sequence.

Differential expression and interaction of transcription co-activator MBF1 with TATA-binding protein (TBP) in the apicomplexan Cryptosporidium parvum.

All gene-specific transcriptional activators initiate gene transcriptions by binding to promoter sequences and recruiting general transcription factors including TATA-binding protein (TBP) to upstream of targeted genes. Some of them require multiprotein bridging factors (MBFs); for example, the type 1 MBF (MBF1) which interconnects the gene activator with TBP. In this study, the properties of a previously cloned type 1 multiprotein bridging factor (CpMBF1) and a newly identified TBP (CpTBP1) from the apicomplexan Cryptosporidium parvum were investigated. Genes encoding both proteins were differentially expressed as determined by semi-quantitative RT-PCRs during the parasite life cycle, but in different patterns. The highest level of expression of CpMBF1 was in the well-developed intracellular parasites, whereas that of CpTBP1 was found in intact oocysts and late intracellular stages, possibly correlated with the formation of oocysts. Both CpMBF1 and CpTBP1 were expressed as maltose-binding protein fusion proteins. The function of CpTBP1 was confirmed by its ability to bind a biotinylated DNA oligonucleotide containing TATA consensus sequence. The interaction between CpMBF1 and CpTBP1 was also observed by an electrophoretic mobility shift assay. Since little is known about the regulation and control of gene activity in C. parvum, this study may point to a new direction for the study of gene activation associated with the development of the complex life cycle of this parasite.

Genomic organization, alternative splicing, and promoter analysis of human dynamin-like protein gene

The human dynamin-like protein, HdynIV, has recently been cloned and shown to be involved in the formation and trafficking of coated vesicles. In particular, one of the HdynIV variant overexpressions has been suggested to contribute to the pathogenesis of brain tumors. In this paper, we report on the genomic organization of the human HdynIV gene. The gene was found to correspond to 20 exons of genomic sequence on human chromosome 12, distributed over 64 kb of genomic DNA. The two exons, numbers 15 and 16, are subjected to differential splicing, generating four different transcripts of a perfect match to our recent report on the four different spliced HdynIV variants [DNA Cell Biol. 19 (2000) 189]. We have also characterized the 5 ′ regulatory region of the HdynIV gene in order to understand the molecular mechanisms regulating its expression. The transcriptional initiation site was identified by 5 ′-RACE. The 5 ′-flanking sequence of the HdynIV gene contains three GC boxes that concatenate Ap2- and Sp1-binding motifs, but that does not contain either the TATA or CAAT consensus sequence. A region between −140 and +92 contributed to high promoter activity. Deletion analysis demonstrated that the minimal promoter activity required the region of −110 to −100. Electrophoretic mobility shift assay demonstrated that a putative transcriptional factor bound to the region of −119 to −90. Site-directed mutagenesis analysis of this region revealed that nucleotides at −108 to −100 were essential for transactivation mediated by this transcriptional factor. In conclusion, we have characterized the minimal HdynIV promoter and shown that CTCCCAGCA (−108 to −100) sequence may act as a novel transcriptional element for regulating HdynIV gene expression.

Characterization of the rat cytoplasmic C 1-tetrahydrofolate synthase gene and analysis of its expression in liver regeneration and fetal development

The eukaryotic trifunctional enzyme, C 1-tetrahydrofolate (THF) synthase, interconverts folic acid derivatives between various oxidation states and is critical for normal cellular function, growth, and differentiation. Using a rat C 1-THF synthase cDNA and synthetic oligonucleotides, the rat C 1-THF synthase gene was isolated and characterized. The gene consists of 28 exons and spans 67.5 kbp. Primer extension, RNase protection, and rapid amplification of cDNA ends (RACE) experiments indicate the presence of multiple transcription start points (tsp) within a 250-bp window located between 50 and 300 bp upstream from the start codon. The 5′ flanking region is devoid of a TATA consensus sequence motif, but putative regulatory elements, including NF-κβ, HNF-4α1, RARα1, C/EBP, and PPAR are present in the promoter region. The 5′ flanking region also contains two sets of tetranucleotide repeats and two short interspersed nuclear elements (SINES). The initial 2500 bp of 5′ flanking sequences of the rat and mouse cytoplasmic C 1-THF synthase genes share 70% identity. However, comparison with the human gene from the Human Genome Data Bank revealed no significant homology in the 5′ flanking region. The gene structure characterization led to the identification of a pseudogene that is 94% identical to the C 1-THF synthase gene and probably diverged 10–12 million years ago. In addition, the gene expression patterns of C 1-THF synthase were investigated during liver regeneration and liver and kidney organogenesis, two highly regulated events. In both processes, C 1-THF synthase expression correlated with increased nucleotide metabolism. This pattern suggests that the gene is regulated in response to changes in the demand for folate-dependent one-carbon units.

Native Human TATA-binding Protein Simultaneously Binds and Bends Promoter DNA without a Slow Isomerization Step or TFIIB Requirement

The association of TATA-binding protein (TBP) with promoter DNA is central to the initiation and regulation of eukaryotic protein synthesis. Our laboratory has previously conducted detailed investigations of this interaction using yeast TBP and seven consensus and variant TATA sequences. We have now investigated this key interaction using human TBP and the TATA sequence from the adenovirus major late promoter (AdMLP). Recombinant native human protein was used together with fluorescently labeled DNA, allowing real time data acquisition in solution. We find that the wild-type hTBP-DNAAdMLP reaction is characterized by high affinity (Kd < or = 5 nm), simultaneous binding and DNA bending, and rapid formation of a stable human TBP-DNA complex having DNA bent approximately 100 degrees. These data allow, for the first time, a direct comparison of the reactions of the full-length, native human and yeast TBPs with a consensus promoter, studied under identical conditions. The general reaction characteristics are similar for the human and yeast proteins, although the details differ and the hTBPwt-induced bend is more severe. This directly measured hTBPwt-DNAAdMLP interaction differs fundamentally from a recently published hTBPwt-DNAAdMLP model characterized by low affinity (microM) binding and an unstable complex requiring either a 30-min isomerization or TFIIB to achieve DNA bending. Possible sources of these significant differences are discussed.

Organization of the human PTK7 gene encoding a receptor protein tyrosine kinase-like molecule and alternative splicing of its mRNA

Protein tyrosine kinase-7 (PTK7) is a receptor protein tyrosine kinase (RPTK)-like molecule that contains a catalytically inactive tyrosine kinase domain. We report here the genomic structure of the human PTK7 gene by screening a BAC library and DNA sequencing. The PTK7 gene is organized into 20 exons. All of the splicing junctions followed the conserved GT/AG rule. The exon–intron structure of the PTK7 gene in the region that encodes the catalytic domain was distinct from those of other RPTKs with strong homology. The 5′-flanking sequence of the PTK7 gene contains two GC boxes that concatenate Sp1 binding motifs, but does not contain either the TATA or CAAT consensus sequence. Using a luciferase reporter assay, it was demonstrated that the 883-bp 5′-flanking sequence is functional as a promoter of the PTK7 gene. We identified four new splicing variants in testis that could be derived from alternative splicing of exons 8–10, 10, a part of 12–13, and 16. The expression patterns of the splicing variants in the hepatoma and colon cancer cells were different from those of the testis. Our findings suggest that PTK7 is evolutionarily distinct from other RPTKs, and that the alternative splicing of PTK7 mRNA may contribute to its diverse function in cell signaling.

Characterization of the major laccase isoenzyme from Trametes pubescens and regulation of its synthesis by metal ions.

The major laccase isoenzyme LAP2 secreted by the white-rot basidiomycete Trametes pubescens in response to high copper concentrations was purified to apparent electrophoretic homogeneity using anion-exchange chromatography and gel filtration. The monomeric protein has a molecular mass of 65 kDa, of which 18% is glycosylation, and a pI value of 2.6. The pH optima of the laccase depend on the substrates oxidized and show bell-shaped pH activity profiles with an optimum of 3-4.5 for phenolic substrates such as 2,6-dimethoxyphenol or syringaldazine, while the non-phenolic substrates ABTS [2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)] and ferrocyanide show a monotonic pH profile with a rate increasing with decreasing pH. The catalytic efficiencies k(cat)/K(m) determined for some of its substrates were 48 x 10(6), 47 x 10(6), 20 x 10(6) and 7 x 10(6) M(-1) s(-1) for ABTS, syringaldazine, ferrocyanide and oxygen, respectively. Furthermore, the gene lap2 encoding the purified laccase was cloned and its nucleotide sequence determined. The gene consists of 1997 bp, with the coding sequence interrupted by eight introns and flanked by an upstream region in which putative CAAT, TATA, MRE and CreA consensus sequences were identified. Based on Northern analysis containing total RNA from both induced and uninduced cultures, expression of lap2 is highly induced by copper, which is also corroborated by an increase in laccase activity in response to copper. A stimulating effect of various other heavy metal ions on laccase synthesis was also observed. In addition to induction, a second regulatory mechanism seems to be repression of lap2 transcription by glucose.

A constitutive gene expression system derived from the tCUP cryptic promoter elements.

A limited number of constitutive promoters have been used to direct transgene expression in plants and they are often derived from non-plant sources. Here, we describe novel gene-regulatory elements which are associated with a cryptic constitutive promoter from tobacco, tCUP, and modifications that were made to create a strong gene-expression system that is effective across all living cell types from a wide range of plant species, including several important crops ( Arabidopsis, canola, flax, alfalfa, tobacco). The tCUP 5' untranslated region was mutated to eliminate translational interference by upstream ATGs, and the influence of the Kozak consensus sequence on the levels of a beta-glucuronidase (GUS) reporter gene activity was demonstrated. These modifications resulted in expression that was greatly enhanced in all organs. A TATA consensus sequence was added to the core promoter to complement an existing Initiator (Inr) sequence. Although this addition was known to elevate core promoter activity by 3-fold the additive effect on the overall gene-expression system was marginal in all of the transgenic plants tested. Two transcriptional enhancers were identified and the region containing them were oligomerized, yielding a significant increase in marker gene-expression in some but not all plant species. In general, the enhanced tCUP gene-expression system generated levels of GUS activity which exceeded that of the 35S promoter in most plant species and the elevation in activity occurred uniformly among the various plant organs. The potential benefit of cryptic elements for the construction of gene-expression systems for crop species is discussed

Enhancers and core promoter elements are essential for the activity of a cryptic gene activation sequence from tobacco, tCUP.

Cryptic gene regulatory elements are sequences that are inactive at their native locations in the genome but have the ability to become functional when positioned adjacent to genes. We have recently isolated such a cryptic sequence from tobacco, tCUP, that can act as a promoter. A 135-bp fragment spanning extending from position -197 to -62, relative to the transcription start site, was found to promote GUS expression in all of the major organs of transgenic Arabidopsis plants. Furthermore, this 135-bp fragment complemented the -46 minimal promoter of CaMV 35S and conferred constitutive expression on transgenic Arabidopsis plants. An electrophoretic mobility-shift assay showed that nuclear proteins prepared from tobacco leaves interact with the 135-bp fragment. tCUP has a core promoter that lacks the TATA consensus sequence but addition of a TATA-box sequence increased the core promoter activity by three-fold. The sequence surrounding the transcription start site of tCUP has sequence similarity with the initiator element (Inr), and deletion of this sequence significantly reduced promoter activity, suggesting that an essential Inr element may exist in the tCUP core promoter. Fusion of the GCC-box enhancer element from pathogenesis-related genes to the core promoter elevated tCUP core promoter activity. Our study indicates that cryptic promoters are similar in composition and organization to promoters associated with expressed genes and that their promoter elements can be combined to create composite promoters that are fully functional. This data provides direct evidence that the expression pattern of plant genes can be influenced by cryptic gene regulatory elements when they are brought into juxtaposition with genes through DNA rearrangements.

TATA box occupancy in the SV40 transcription elongation complex.

In order to gain insight into requirements for template activation and commitment in mammalian transcription, TATA site occupancy was measured in native SV40 viral transcription complexes that were in the process of transcription elongation at the time of cell lysis. This was accomplished by quantifying resistance to restriction enzyme digestion of transcription complexes in nuclear lysate. The rate of cleavage at the TATA site of the late gene in the native complex was slower than that of a bare DNA control, both for wild-type virus and for a virus containing a TATA consensus sequence. These results suggest that the TATA site in the transcription elongation complex in vivo is occupied with transcription factor TBP/TFIID. When considered in light of previous work, these findings support a model in which transcription activation involves reinitiation from a promoter that contains both activator and TFIID bound in a stable complex.

Identification and Characterization of Asporin: A NOVEL MEMBER OF THE LEUCINE-RICH REPEAT PROTEIN FAMILY CLOSELY RELATED TO DECORIN AND BIGLYCAN

Asporin, a novel member of the leucine-rich repeat family of proteins, was partially purified from human articular cartilage and meniscus. Cloning of human and mouse asporin cDNAs revealed that the protein is closely related to decorin and biglycan. It contains a putative propeptide, 4 amino-terminal cysteines, 10 leucine-rich repeats, and 2 C-terminal cysteines. In contrast to decorin and biglycan, asporin is not a proteoglycan. Instead, asporin contains a unique stretch of aspartic acid residues in its amino-terminal region. A polymorphism was identified in that the number of consecutive aspartate residues varied from 11 to 15. The 8 exons of the human asporin gene span 26 kilobases on chromosome 9q31.1-32, and the putative promoter region lacks TATA consensus sequences. The asporin mRNA is expressed in a variety of human tissues with higher levels in osteoarthritic articular cartilage, aorta, uterus, heart, and liver. The deduced amino acid sequence of asporin was confirmed by mass spectrometry of the isolated protein resulting in 84% sequence coverage. The protein contains an N-glycosylation site at Asn(281) with a heterogeneous oligosaccharide structure and a potential O-glycosylation site at Ser(54). The name asporin reflects the aspartate-rich amino terminus and the overall similarity to decorin.