Pyrimidine-rich Region Research Articles

Characterization and engineering of the xylose-inducible xylP promoter for use in mold fungal species

Conditional promoters allowing both induction and silencing of gene expression are indispensable for basic and applied research. The xylP promoter (pxylP) from Penicillium chrysogenum was demonstrated to function in various mold species including Aspergillus fumigatus. pxylP allows high induction by xylan or its degradation product xylose with low basal activity in the absence of an inducer. Here we structurally characterized and engineered pxylP in A. fumigatus to optimize its application. Mutational analysis demonstrated the importance of the putative TATA-box and a pyrimidine-rich region in the core promoter, both copies of a largely duplicated 91-bp sequence (91bpDS), as well as putative binding sites for the transcription factor XlnR and a GATA motif within the 91bpDS. In agreement, pxylP activity was found to depend on XlnR, while glucose repression appeared to be indirect. Truncation of the originally used 1643-bp promoter fragment to 725 bp largely preserved the promoter activity and the regulatory pattern. Integration of a third 91bpDS significantly increased promoter activity particularly under low inducer concentrations. Truncation of pxylP to 199 bp demonstrated that the upstream region including the 91bpDSs mediates not only inducer-dependent activation but also repression in the absence of inducer. Remarkably, the 1579-bp pxylP was found to act bi-bidirectionally with a similar regulatory pattern by driving expression of the upstream-located arabinofuranosidase gene. The latter opens the possibility of dual bidirectional use of pxylP. Comparison with a doxycycline-inducible TetOn system revealed a significantly higher dynamic range of pxylP. Taken together, this study identified functional elements of pxylP and opened new methodological opportunities for its application.

Structure of RNA Stem Loop B from the Picornavirus Replication Platform.

The presumptive RNA cloverleaf at the start of the 5'-untranslated region of the picornavirus genome is an essential element in replication. Stem loop B (SLB) of the cloverleaf is a recognition site for the host polyC-binding protein, which initiates a switch from translation to replication. Here we present the solution structure of human rhinovirus isotype 14 SLB using nuclear magnetic resonance spectroscopy. SLB adopts a predominantly A-form helical structure. The stem contains five Watson-Crick base pairs and one wobble base pair and is capped by an eight-nucleotide loop. The wobble base pair introduces perturbations into the helical parameters but does not appear to introduce flexibility. However, the helix major groove appears to be accessible. Flexibility is seen throughout the loop and in the terminal nucleotides. The pyrimidine-rich region of the loop, the apparent recognition site for the polyC-binding protein, is the most disordered region of the structure.

Activation of Src and transformation by an RPTPα splice mutant found in human tumours

Receptor protein tyrosine phosphatase α (RPTPα)-mediated Src activation is required for survival of tested human colon and oestrogen receptor-negative breast cancer cell lines. To explore whether mutated RPTPα participates in human carcinogenesis, we sequenced RPTPα cDNAs from five types of human tumours and found splice mutants in ∼30% of colon, breast, and liver tumours. RPTPα245, a mutant expressed in all three tumour types, was studied further. Although it lacks any catalytic domain, RPTPα245 expression in the tumours correlated with Src tyrosine dephosphorylation, and its expression in rodent fibroblasts activated Src by a novel mechanism. This involved RPTPα245 binding to endogenous RPTPα (eRPTPα), which decreased eRPTPα-Grb2 binding and increased eRPTPα dephosphorylation of Src without increasing non-specific eRPTPα activity. RPTPα245-eRPTPα binding was blocked by Pro210 → Leu/Pro211 → Leu mutation, consistent with the involvement of the structural 'wedge' that contributes to eRPTPα homodimerization. RPTPα245-induced fibroblast transformation was blocked by either Src or eRPTPα RNAi, indicating that this required the dephosphorylation of Src by eRPTPα. The transformed cells were tumourigenic in nude mice, suggesting that RPTPα245-induced activation of Src in the human tumours may have contributed to carcinogenesis.

Comprehensive Analysis of Transcript Start Sites in Ly49 Genes Reveals an Unexpected Relationship with Gene Function and a Lack Of Upstream Promoters

Comprehensive analysis of the transcription start sites of the Ly49 genes of C57BL/6 mice using the oligo-capping 5′-RACE technique revealed that the genes encoding the “missing self” inhibitory receptors, Ly49A, C, G, and I, were transcribed from multiple broad regions in exon 1, in the intron1/exon2 region, and upstream of exon -1b. Ly49E was also transcribed in this manner, and uniquely showed a transcriptional shift from exon1 to exon 2 when NK cells were activated in vitro with IL2. Remarkably, a large proportion of Ly49E transcripts was then initiated from downstream of the translational start codon. By contrast, the genes encoding Ly49B and Q in myeloid cells, the activating Ly49D and H receptors in NK cells, and Ly49F in activated T cells, were predominantly transcribed from a conserved site in a pyrimidine-rich region upstream of exon 1. An ∼200 bp fragment from upstream of the Ly49B start site displayed tissue-specific promoter activity in dendritic cell lines, but the corresponding upstream fragments from all other Ly49 genes lacked detectable tissue-specific promoter activity. In particular, none displayed any significant activity in a newly developed adult NK cell line that expressed multiple Ly49 receptors. Similarly, no promoter activity could be found in fragments upstream of intron1/exon2. Collectively, these findings reveal a previously unrecognized relationship between the pattern of transcription and the expression/function of Ly49 receptors, and indicate that transcription of the Ly49 genes expressed in lymphoid cells is achieved in a manner that does not require classical upstream promoters.

UV-dependent interaction between Cep164 and XPA mediates localization of Cep164 at sites of DNA damage and UV sensitivity

DNA damage response plays a critical role in the maintenance of genomic stability. In response to UV damage, ATR (ataxia telangiectasia mutated and rad3 related kinase)-mediated checkpoint and nucleotide excision repair (NER) machinery are activated. A centrosomal protein, Cep164, has been identified as a chromatin binding mediator protein functioning in ATR-mediated checkpoint activation upon UV damage. However, it was not clear whether there is cross talk between Cep164 and NER machinery. We show that Cep164 knockdown compromises the cell survival upon UV damage. Cep164 localizes to cyclobutane pyrimidine dimmers (CPD), one of the major DNA lesions induced by UV irradiation. Binding of Cep164 to the region of exon 4 to intron 9 within p53 gene, known as a pyrimidine-rich region as well as a photoproduct formation region upon UV damage, increases 2.5 folds upon UV irradiation. Recruitment of Cep164 to CPD sites or photoproduct formation region requires a NER factor, XPA. Furthermore, UV irradiation significantly enhances the interaction between Cep164 and XPA. Cep164 binds to amino acids 4-97 of XPA, a region required for UV resistance. Importantly, co-localization of Cep164 and CPD is aberrant in XPA-deficient fibroblasts complemented with XPAΔ10-88 mutant. In addition, UV-induced CHK1 phosphorylation is impaired in these cells. These findings provide mechanistic insights for the recruitment of Cep164 mediator protein to DNA damage sites and reveal a critical juncture between checkpoint pathways and repair systems.

UNR translation can be driven by an IRES element that is negatively regulated by polypyrimidine tract binding protein

Upstream of N-ras (Unr) has been described as an internal initiation trans-acting factor (ITAF) in the cap-independent translation of some particular viral and cellular mRNAs. Two factors led us to hypothesize that the UNR 5′-untranslated region (5′-UTR) may contain an internal ribosome entry site (IRES). The first was the requirement for persisting Unr expression under conditions that correlate with cap-independent translation. The other was the observation that the primary UNR transcript contains a 447 nt long 5′-UTR including two upstream AUGs that may restrict translation initiation via cap-dependent ribosome scanning. Here we report that the UNR 5′-UTR allows IRES-dependent translation, as revealed by a dicistronic reporter assay. Various controls ruled out the contribution of leaky scanning, cryptic promoter sequences or RNA processing events to the ability of the UNR 5′-UTR to mediate internal initiation of translation. Ultraviolet cross-linking analysis and RNA affinity chromatography revealed the binding of polypyrimidine tract binding protein (PTB) to the UNR IRES, requiring a pyrimidine-rich region (nucleotides 335–355). Whereas overexpression of PTB in several cell lines inhibited UNR IRES activity and UNR protein expression, depletion of endogenous PTB using RNAi increased UNR IRES activity. Moreover, a mutant version of the UNR IRES lacking the PTB binding site was more efficient at directing IRES-mediated translation. In conclusion, our results demonstrate that translation of the ITAF Unr can itself be regulated by an IRES that is downregulated by PTB.

Template requirements for de novo RNA synthesis by hepatitis C virus nonstructural protein 5B polymerase on the viral X RNA.

The hepatitis C virus (HCV)-encoded NS5B protein is an RNA-dependent RNA polymerase which plays a substantial role in viral replication. We expressed and purified the recombinant NS5B of an HCV genotype 3a from Esherichia coli, and we investigated its ability to bind to the viral RNA and its enzymatic activity. The results presented here demonstrate that NS5B interacts strongly with the coding region of positive-strand RNA, although not in a sequence-specific manner. It was also determined that more than two molecules of polymerase bound sequentially to this region with the direction 3' to 5'. Also, we attempted to determine the initiation site(s) of de novo synthesis by NS5B on X RNA, which contains the last 98 nucleotides of HCV positive-strand RNA. The initiation site(s) on X RNA was localized in the pyrimidine-rich region of stem I. However, when more than five of the nucleotides of stem I in X RNA were deleted from the 3' end, RNA synthesis initiated at another site of the specific ribonucleotide. Our study also showed that the efficiency of RNA synthesis, which was directed by X RNA, was maximized by the GC base pair at the penultimate position from the 3' end of the stem. These results will provide some clues to understanding the mechanism of HCV genomic RNA replication in terms of viral RNA-NS5B interaction and the initiation of de novo RNA synthesis.

The evolution of an alternatively spliced exon in the alphaA-crystallin gene.

The evolutionary aspects of alternative splicing, as a mechanism to increase the diversity of gene products, are poorly understood. Here we analyse the evolution of a 69-bp exon that is alternatively spliced in the primary transcript of the gene for the mammalian eye lens protein alphaA-crystallin. In rodents, the skipping of this exon 2 is attributed to the presence of a non-consensus 5' splice site GC, and results in the expression of 10-20% of alphaA(ins)-crystallin, with an insert of 23 residues, as compared with normal alphaA-crystallin. alphaA(ins)-crystallin is also expressed in some non-rodent mammals, including kangaroo, while lacking in others. We now demonstrate that the alternatively spliced exon 2 is present in mammals from different orders that do not express alphaA(ins)-crystallin. The expression of this exon has thus been silenced independently in various lineages. Sequence comparison in 16 species reveals that--whether or not alphaA(ins)-crystallin is expressed--exon 2 is always flanked by the non-consensus donor splice site GC, while a consensus branch point sequence and 3' pyrimidine-rich region are hardly detectable in the downstream intron. Increased numbers of amino acid replacements in the peptide encoded by exon 2 indicate that it is subject to much lower selective constraints than the exons that code for normal alphaA-crystallin. The absence of any apparent advantage at the protein level may suggest that exon 2 DNA sequences are conserved as cis-acting factors for proper splicing of the alphaA-crystallin transcript.

Expression of p40/Epstein–Barr Virus Nuclear Antigen 1 Binding Protein 2

Nucleolar protein p40/EBP2 is a proliferation-associated antigen that interacts with Epstein–Barr virus nuclear antigen 1 (EBNA1) to maintain the Epstein–Barr virus (EBV) episomes. The yeast p40/EBP2 functions in the processing of 27S-A into 27S-B ribosomal RNA. The present study reports high evolutionary conservation of the cDNA-derived amino acid sequences of p40/EBP2 from frog, chicken, pig, rat, mouse, bovine, and human. p40/EBP2 is ubiquitously expressed in human tissues. It is highly expressed in myelogenous leukemia K-562 compared to other cell lines tested. The human p40/EBP2 gene is located in chromosome 1 with nine exons and eight introns. The minimal promoter region resides 300 nucleotides upstream of a putative ATG initiation codon preceded by a pyrimidine-rich region. These two regions contain eight Sp1 and four c-Ets-1 putative binding sites. Analysis of the p40/EBP2 gene and its promoter region will facilitate studies on the regulation of its expression in EBV-infected and noninfected cells.

Organization of ribosomal RNA genes from a Loofah witches' broom phytoplasma.

Using the technique of integrative mapping with three vectors carrying chromosomal rDNA sequences, one of two rRNA operons of loofah witches' broom (LfWB) phytoplasma was constructed. This is the first complete rRNA operon of a phytoplasma to be reported. The operon has a context of 5'-16S-23S-5S-3' with a tRNA(Ile) gene in the ITS and tRNA(Val) and tRNA(Asn) genes downstream from the 5S rRNA gene. Although the other operon has not been cloned, the DNA sequence of a PCR-amplified product shows that it has no tRNA(Ile) gene in the ITS region. The complete nucleotide sequences of 16S, 23S, and 5S rDNA are 1538, 2864, and 113 bp, respectively. Five -10-like sequences, but no -35 sequences, were found within a 494-bp leader region. There was a TG dinucleotide two nucleotides upstream from each -10-like sequence. The existence of a TG dinucleotide at this position has been reported to enhance the efficiency of a promoter without a -35 region. The regions immediately flanking the 5' and 3' ends of 16S and 23S rDNA can form long basepaired stems that contain sites for processing by RNase III. No obvious sequence for a rho-dependent or rho-independent termination site was found downstream from the tRNA(Asn) gene. The transcription may stop within a pyrimidine-rich region, as has been reported for several polypeptide-encoding genes and rRNA operons of archaeobacteria. The presence of the tRNA genes downstream from the 5S rRNA gene in the rRNA operon of LfWB phytoplasma further supports the hypothesis that phytoplasmas are phylogenetically closer to acholeplasmas than to mycoplasmas. The phylogenetic relatedness of LfWB phytoplasma to other phytoplasmas is discussed on the basis of the nucleotide sequence of rRNA genes and ITS.

Competition Analysis of the Human α2(I) Collagen Promoter Using Synthetic Oligonucleotides

Previous studies have identified four cis-response elements which mediate the basal transcriptional activity of the human alpha2(I) collagen gene. One of these elements, a pyrimidine-rich region (TCCCCC motif), was shown to be a repressor site, and the other three elements were shown to be activator sites. Furthermore, the repressor site and two of the activator sites were found to constitute binding sites for the transcription factors Sp1 and Sp3. In this study, we further determined the affinity and specificity of the binding of Sp1 and Sp3 to the human alpha2(I) collagen promoter and investigated the function of the pyrimidine-rich region which contains the TCCCCC motif. Functional analyses of Sp1 and Sp3 in Drosophila cells confirmed that Sp1 and Sp3 activate the human alpha2(I) collagen promoter via the GC boxes and the TCCTCC motif, but that binding of Sp1 or Sp3 to the repressor site does not activate or repress the collagen promoter activity. Com- petitive analyses using DNA mobility shift assays showed that the TCCCCC motif which constitutes the repressor site abolished the binding of Sp1 or Sp3 to the GC boxes or the TCCTCC motif, but not the binding of CCAAT-binding factor to the fourth cis-response element (CCAAT-binding factor site). Furthermore, the affinity of Sp1 or Sp3 for the TCCTCC motif was shown to be greater than that of the Sp1 consensus oligonucleotide. In vitro transcription analysis revealed that the addition of each activator site oligonucleotide or repressor site oligonucleotide had an inhibitory effect on the transcription of the collagen gene. These results suggest that the repressor site regulates the transcription of the collagen gene by taking away Sp1 or Sp3 from the activator sites.

Phylogenetic analysis of the 5' subterminal region of isolates of Leishmania RNA virus-1.

Leishmania RNA virus-1 (LRV1) is a double-stranded RNA virus present in some Leishmania species. The virus genome consists of a 450-nucleotide, 5' untranslated region (UTR) followed by the coat gene and the RNA-dependent RNA polymerase (RDRP). It has been shown that the 5' end UTR of the genome promotes internal initiation of translation in an in-vitro assay, indicating the presence of an internal ribosomal entry site (IRES) element upstream of the coat gene. The nucleotide sequences of the 5' subterminal regions of six new isolates of LRV1, of different geographical origins, have now been determined. The RNA folding of the 5' subterminal region of LRV1 has been predicted, using a combination of thermodynamic parameters and folding constraints based on nucleotide substitutions. Furthermore, a putative pyrimidine-rich region (a feature unique to all IRES elements), which is complementary to the Leishmania 18S rRNA, has been identified. The significance and relevance of these findings in the context of the function of the 5' UTR of LRV1 as an IRES element are discussed.

Phylogenetic analysis of the 5′ subterminal region of isolates ofLeishmaniaRNA virus-1

Leishmania RNA virus-1 (LRV1) is a double-stranded RNA virus present in some Leishmania species. The virus genome consists of a 450-nucleotide, 5′ untranslated region (UTR) followed by the coat gene and the RNA-dependent RNA polymerase (RDRP). It has been shown that the 5′ end UTR of the genome promotes internal initiation of translation in an in-vitro assay, indicating the presence of an internal ribosomal entry site (IRES) element upstream of the coat gene. The nucleotide sequences of the 5′ subterminal regions of six new isolates of LRV1, of different geographical origins, have now been determined. The RNA folding of the 5′ subterminal region of LRV1 has been predicted, using a combination of thermodynamic parameters and folding constraints based on nucleotide substitutions. Furthermore, a putative pyrimidine-rich region (a feature unique to all IRES elements), which is complementary to the Leishmania 18S rRNA, has been identified. The significance and relevance of these findings in the context of the function of the 5′ UTR of LRV1 as an IRES element are discussed.

Pyrimidine-Rich Region Mutations Compensate for a Stem-Loop V Lesion in the 5′ Noncoding Region of Poliovirus Genomic RNA

Five revertants of a linker-scanning mutation adjacent to the stem-loop V attenuation determinant (X472) in the 5′ noncoding region of poliovirus RNA were independently isolated from neuroblastoma cells and contained RNAs with seven nucleotide changes in the pyrimidine-rich region. Generation of the identical rare second-site mutations suggests the existence of a replicase-dependent mutagenesis mechanism during poliovirus replication. Enzymatic structure probing of the mutated pyrimidine-rich domain identified secondary structure changes between stem-loops V and VI. A consensus secondary structure model is presented for wild-type stem-loops V and VI and the pyrimidine-rich region located in the 5′ noncoding region of poliovirus RNA. A pyrimidine-rich region mutant (X472-R4N) produced large plaques in neuroblastoma cells and small plaques in HeLa cells, but the plaque size differences were not due to cell-type differences in viral translation or RNA replication. Release of X472-R4N from HeLa cells was 10-fold lower than release from neuroblastoma cells, which may explain the small plaque phenotype of X472-R4N in HeLa cells. Wild-type poliovirus was also released more efficiently from neuroblastoma cells (∼4-fold increase compared with release from HeLa cells), indicating that poliovirus neurotropism may be influenced by the cell-type efficiency of virus release. Thermal treatment increased the levels of infectious X472-R4N virions but not wild-type virus particles; thus RNA sequence and structural changes in the mutated 5′ noncoding region of X472-R4N may have altered RNA–protein interactions necessary for virus infectivity.

HnRNP C and polypyrimidine tract-binding protein specifically interact with the pyrimidine-rich region within the 3'NTR of the HCV RNA genome

Like other members of the Flaviviridae family, the 3' non-translated region (NTR) of the hepatitis C virus (HCV) is believed to function in the initiation and regulation of viral RNA replication by interacting with components of the viral replicase complex. To inves-tigate the possibility that host components may also participate in this process, we used UV cross-linking assays to determine if any cellular proteins could bind specifically to the 3'NTR RNA. We demonstrate the specific interaction of two host proteins with the extensive pyrimidine-rich region within the HCV 3'NTR. One host protein migrates as a doublet with a molecular weight of 57 kDa and is immunoreactive with antisera specific for polypyrimidine tract-binding protein (PTB), and the other protein (35 kDa) is recognized by a monoclonal antibody specific for heterogeneous nuclear ribonucleoprotein C (hnRNP C). These results suggest that recognition of the large pyrimidine-rich region by PTB and hnRNP C may play a role in the initiation and/or regulation of HCV RNA replication.

Identification of the Poly(C) Binding Protein in the Complex Associated With the 3′ Untranslated Region of Erythropoietin Messenger RNA

Hypoxia regulates expression of erythropoietin (EPO), a glycoprotein that stimulates erythrocytosis, at the level of transcription and also possibly at the level of messenger RNA (mRNA) stability. A pyrimidine-rich region within the EPO mRNA 3' untranslated region was implicated in regulation of EPO mRNA stability element and shown to bind protein factors. In the present study we wished to identify the protein factor binding to the pyrimidine-rich sequence in the EPO mRNA stability element. Using mobility shift assays, ultraviolet light cross-linking, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and electroelution of protein factors from the gel slices corresponding to the ribonucleoprotein complexes, we found that two isoforms of a 40 kD poly(C) binding protein (PCBP, also known as alphaCP or hnRNPE), PCBP1, and PCBP2 are present in that complex. In Hep3B or HepG2 cells hypoxia induces neither expression of PCBP nor formation of the ribonucleoprotein complex associated with EPO mRNA that involves PCBP.

Function of a bovine papillomavirus type 1 exonic splicing suppressor requires a suboptimal upstream 3' splice site.

Alternative splicing is an important mechanism for the regulation of bovine papillomavirus type 1 (BPV-1) gene expression during the virus life cycle. Previous studies in our laboratory have identified two purine-rich exonic splicing enhancers (ESEs), SE1 and SE2, located between two alternative 3' splice sites at nucleotide (nt) 3225 and nt 3605. Further analysis of BPV-1 late-pre-mRNA splicing in vitro revealed a 48-nt pyrimidine-rich region immediately downstream of SE1 that inhibits utilization of the nt 3225 3' splice site. This inhibitory element, which we named an exonic splicing suppressor (ESS), has a U-rich 5' end, a C-rich central part, and an AG-rich 3' end (Z. M. Zheng, P. He, and C. C. Baker, J. Virol. 70:4691-4699, 1996). The present study utilized in vitro splicing of both homologous and heterologous pre-mRNAs to further characterize the ESS. The BPV-1 ESS was inserted downstream of the 3' splice site in the BPV-1 late pre-mRNA, Rous sarcoma virus src pre-mRNA, human immunodeficiency virus tat-rev pre-mRNA, and Drosophila dsx pre-mRNA, all containing a suboptimal 3' splice site, and in the human beta-globin pre-mRNA, which contains a constitutive 3' splice site. These studies demonstrated that suppression of splicing by the BPV-1 ESS requires an upstream suboptimal 3' splice site but not an upstream ESE. Furthermore, the ESS functions when located either upstream or downstream of BPV-1 SE1. Mutational analyses demonstrated that the function of the ESS is sequence dependent and that only the C-rich region of the ESS is essential for suppression of splicing in all the pre-mRNAs tested.

RNA structure adjacent to the attenuation determinant in the 5'-non-coding region influences poliovirus viability.

In attenuated Sabin strains, point mutations within stem-loop V of the 5'-non-coding region (NCR) reduce neurovirulence and cell-specific cap-independent translation. The stem-loop V attenuation determinants lie within the highly structured internal ribosome entry site. Although stem-loop V Sabin mutations have been proposed to alter RNA secondary structure, efforts to identify such conformational changes have been unsuccessful. A previously described linker-scanning mutation (X472) modified five nucleotides adjacent to the attenuation determinant at nt 480 [for poliovirus (PV) type 1]. Transfection of X472 RNA generated only pseudo-revertants in HeLa (cervical carcinoma) or SK-N-SH (neuroblastoma) cells. Pseudo-revertants from both cell types contained nucleotide changes within the X472 linker. In addition, some neuroblastoma-isolated revertants revealed second site mutations within the pyrimidine-rich region located approximately 100 nt distal to the original lesion. Enzymatic RNA structure probing determined that the X472 linker substitution did not disrupt the overall conformation of stem-loop V but abolished base pairing adjacent to the attenuation determinant. Our analyses correlated increased base pairing proximal to the stem-loop V attenuation determinant with growth of X472 revertant RNAs (measured by northern blot analysis). Potential roles of second site mutations in the pyrimidine-rich region are discussed. In addition, our enzymatic structure probing results are shown on a consensus secondary structure model for stem-loop V of the PV 5'-NCR.

Genomic organization of the murine polysialyltransferase gene ST8SiaIV (PST-1).

Polysialic acid (PSA) is an important regulator of cellular interactions. Two enzymes (ST8SiaII and ST8SiaIV) are capable of synthesizing PSA. In the present study, the gene encoding the murine ST8SiaIV (PST-1) has been isolated and characterized. In contrast to the ST8SiaII (STX) gene which contains six exons and spans about 80 kb, the ST8SiaIV gene comprises only five exons spanning over at least 55 kb. However, alignment of the two genes revealed that exon-intron boundaries of exons 2-5 of ST8SiaIV and exons 3-6 of ST8SiaII are located at identical sites. Differences are restricted to the 5'-region encoded by one exon in the case of ST8SiaIV, whereas the corresponding region of ST8SiaII is interrupted by a very long intron. 5'-RACE analysis of the ST8SiaIV transcript using mRNA from AtT20 cells identified two transcription start sites at positions -324 and -204 relative to the translation start codon. The promoter region of ST8SiaIV lacks TATA- and CAAT-like sequences and is enriched in G+C (60%). The promoter contains putative Sp1, AP-1, AP-2, and PEA3 binding sites, as well as a purine- and a pyrimidine-rich region. Luciferase reporter gene assays demonstrated that the region between nucleotides -443 and -162 is sufficient to direct gene expression. The induction of luciferase activity was 30- and 10-fold in the PSA-positive AtT20 and CHO cells, but only 5- and 7-fold in the PSA-negative NIH-3T3 cells and in a PSA-negative subline of AtT20. Thus, although decreased in activity in PSA-negative cell lines, the basal promoter is not sufficient for the strong cell-type and tissue specific regulation of the ST8SiaIV gene, suggesting regulatory elements in the more upstream 5'-region.

Cloning and characterization of a novel sequence-specific single-stranded-DNA-binding protein.

The promoter region of the chicken alpha2(I) collagen gene contains a pyrimidine-rich element that is well conserved in different mammalian species. This sequence can also form an unusual DNA structure as shown by its sensitivity to SI nuclease in vitro and it lies in a region that is DNase I-hypersensitive only when this promoter is active. We have recently reported that fibroblast nuclear proteins, including chicken Y-box-binding protein 1, bind to this single-stranded pyrimidine-rich sequence. Here we report the isolation, from a chick embryo fibroblast cDNA expression library, of a partial cDNA clone encoding a previously unknown protein, designated SSDP (sequence-specific single-stranded DNA-binding protein), that binds this single-stranded sequence. This clone contains 1199 bp of chicken sequence and has a single long open reading frame that encodes 284 amino acid residues. The affinity-purified recombinant protein encoded by this cDNA binds sequence-specifically to the single-stranded pyrimidine sequence. This cDNA sequence lacks significant similarity to any known gene in the data banks, but it is highly conserved in expressed sequence tags derived from both mouse and human. The corresponding amino acid sequence is remarkably conserved, having 97% identity with mouse and human expressed sequences. The corresponding mRNA is approx. 1800 nt in length and is expressed in both fibroblasts and chondrocytes. The high affinity of this protein for this conserved pyrimidine-rich region suggests that it might be involved in the transcriptional regulation of the alpha2(I) collagen gene.