Minor Transcription Start Sites Research Articles

Implications of differential transcription start site selection on chronic myeloid leukemia and prostate cancer cell protein expression.

The relevance of minor transcription start sites in broad promoters is not well understood. We have studied AGAP2 expression in prostate cancer and chronic myeloid leukemia (CML), showing transcription is initiated from alternative transcription start sites (TSSs) within a single TSS cluster, producing cancer-type-specific AGAP2 mRNAs with small differences in their 5' UTR length. Interestingly, in the CML cell lines where the 5' UTR is longer, AGAP2 protein levels are lower. We demonstrate that the selection of an upstream TSS involved the formation of a G quadruplex in the 5' UTR, decreasing polysome formation. After developing a bioinformatics pipeline to query data from the FANTOM project and the NCl-60 human tumor cell lines screen, we found HK1 expression can also be regulated by the same mechanism. Overall, we present compelling data supporting TSS selection within a TSS cluster play a role in protein expression and should not be ignored.

A motif within a complex promoter from the oomycete Phytophthora infestans determines transcription during an intermediate stage of sporulation

Sporulation in Phytophthora infestans is associated with a major remodeling of the transcriptome. To better understand promoter structure and how sporulation-specific expression is determined in this organism, the Pks1 gene was analyzed. Pks1 encodes a protein kinase that is induced at an intermediate stage of sporulation, prior to sporangium maturation. Major and minor transcription start sites mapped throughout the promoter, which contains many T-rich stretches and Inr-like elements. Within the T-rich region are several motifs which bound nuclear proteins in EMSA. Tests of modified promoters in transformants implicated a CCGTTG located 110-nt upstream of the transcription start point as a major regulator of sporulation-specific transcription. The motif also bound a sporulation-specific nuclear protein complex. A bioinformatics analysis indicated that the motif is highly over-represented within co-expressed promoters, in which it predominantly resides 100–300-nt upstream of transcription start sites. Other sequences, such as a CATTTGTT motif, also bound nuclear proteins but did not play an essential role in spore-specific expression.

Constitutive Expression of Murine Decay-Accelerating Factor 1 Is Controlled by the Transcription Factor Sp1

The complement regulatory protein decay-accelerating factor (DAF or CD55) protects host tissue from complement-mediated injury by inhibiting the classical and alternative complement pathways. Besides its role in complement regulation, DAF has also been shown to be a key player in T cell immunity. Modulation of DAF expression could therefore represent a critical regulatory mechanism in both innate and adaptive immune responses. To identify and characterize key transcriptional regulatory elements controlling mouse Daf1 expression, a 2.5-kb fragment corresponding to the 5' flanking region of the mouse Daf1 gene was cloned. Sequence analysis showed that the mouse Daf1 promoter lacks conventional TATA and CCAAT boxes and displays a high guanine and cytosine content. RACE was used to identify one major and two minor transcription start sites 47, 20, and 17 bp upstream of the translational codon. Positive and negative regulatory regions were identified by transiently transfecting sequential 5'deletion constructs of the 5'flanking region into NIH/3T3, M12.4, and RAW264.7 cells. Mutational analyses of the promoter region combined with Sp1-specific ELISA showed that the transcription factor Sp1 is required for basal transcription and LPS-induced expression of the Daf1 gene. These findings provide new information on the regulation of the mouse Daf1 promoter and will facilitate further studies on the expression of Daf1 during immune responses.

Regulation of the ribonucleotide reductase small subunit (R2) in the yellow fever mosquito, Aedes aegypti

Ribonucleotide reductase (RNR) catalyzes the formation of deoxyribonucleotides, a rate limiting step in DNA synthesis. Class I RNR is a tetramer that consists of two subunits, R1 and R2; enzymatic activity requires association of R1 with R2. The R2 subunit is of special interest because it dictates the interaction with R1 that is required for enzymatic activity expression, and it is expressed only during the S phase of the cell cycle. We previously sequenced an R2 cDNA clone from the yellow fever mosquito, Aedes aegypti. We found the message was upregulated by blood feeding. We now report the sequence of an R2 genomic clone. The gene consists of 4 introns and 5 exons. Both major and minor transcriptional start sites have been identified, and their use differs in sugar-fed versus blood-fed females. The gene contains putative cis-regulatory sites for E2F, Caudal (Cdx) and Dearolf (Dfd). The mosquito R2 gene contains iron-specific regulatory elements immediately upstream of the minimal promoter region. Binding of a factor to the distal putative Cdx site in the − 400 region is altered by iron treatment of cells. Further, following blood feeding, R2 message is significantly induced in mosquito ovaries (tissues that are involved in oogenesis—a process requiring DNA synthesis).

PHOX2B Regulates Its Own Expression by a Transcriptional Auto-regulatory Mechanism

The specification of neuronal identity is a result of interactions between the following two distinct classes of determinants: extrinsic factors that include secreted or cell membrane-associated signals in the local environment, and intrinsic factors that generally consist of ordered cascades of transcription factors. Little is known about the molecular mechanisms underlying the interplay between these extrinsic and intrinsic factors and the transcriptional processes that establish and maintain a given neuronal phenotype. Phox2b is a vertebrate homeodomain transcription factor and a well established intrinsic factor in developing autonomic ganglia, where its expression is triggered by the bone morphogenic proteins secreted by the dorsal aorta. In this study we characterized its proximal 5'-regulatory region and found that it contained five putative DNA sites that potentially bind homeodomain proteins, including PHOX2B itself. Chromatin immunoprecipitation assays showed that PHOX2B could bind its own promoter in vivo, and electromobility gel shift assays confirmed that four of the five sites could be involved in PHOX2B binding. Functional experiments demonstrated that 65% of the transcriptional activity of the PHOX2B promoter in neuroblastoma cells depends on this auto-regulatory mechanism and that all four sites were required for full self-transactivation. Our data provide a possible molecular explanation for the maintenance of PHOX2B expression in developing ganglia, in which initially its expression is triggered by bone morphogenic proteins, but may become independent of external stimuli when it reaches a certain nuclear concentration and sustains its own transcription.

Cloning, gene organization and identification of an alternative splicing process in lecithin:retinol acyltransferase cDNA from human liver

Lecithin:retinol acyltransferase (LRAT) catalyzes the synthesis of retinyl esters in many tissues and is crucial for the transport and intracellular storage of vitamin A. LRAT expression is highly regulated in the liver. In this study, we have cloned and sequenced the full-length LRAT mRNA from human liver and identified its 5′- and 3′-ends. Full-length LRAT mRNA comprises 5023 nt with a predicted ORF of 230 amino acids, a short 5′UTR, and a relatively long 3′UTR of 4 kb containing several polyadenylation signals and AU-rich regions. Based on alignment of this mRNA with human genomic DNA in the GenBank database, the human LRAT gene spans about 9.1 kbp and consists of two exons and a relatively long 4-kbp intron. Further analysis of normal liver revealed a minor alternative splicing variant which lacks a 103 nt polynucleotide contained in the 5′UTR of the full-length LRAT transcript. This variant predicts that the LRAT gene is organized into three exons and two introns, as reported for LRAT cloned from retinal pigment epithelium (RPE) cells. These two LRAT mRNA variants are also present in testis, which is known to express LRAT and contain retinyl esters. Major and minor transcription start sites for human liver LRAT mRNA were identified and the sequence of the upstream proximal promoter region was retrieved from the GenBank database and physically analyzed for the presence of putative cis-acting elements essential for basal transcription. This region contains a TATA box, CCAAT box and Sp1 site, which are apparently conserved in mouse and rat LRAT genes. Our results provide evidence that multiple LRAT mRNA transcripts, which are expressed in a tissue-specific manner, may result from several mechanisms including differential splicing of the 5′UTR region and the use of multiple polyadenylation signals in the 3′UTR.

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.

Mining the Giardia lamblia Genome for New Cyst Wall Proteins

The Giardia lamblia cyst wall (CW), which is required for survival outside the host and infection, is a primitive extracellular matrix. Because of the importance of the CW, we queried the Giardia Genome Project Database with the coding sequences of the only two known CW proteins, which are cysteine-rich and contain leucine-rich repeats (LRRs). We identified five new LRR-containing proteins, of which only one (CWP3) is up-regulated during encystation and incorporated into the cyst wall. Sequence comparison with CWP1 and -2 revealed conservation within the LRRs and the 44-amino-acid N-flanking region, although CWP3 is more divergent. Interestingly, all 14 cysteine residues of CWP3 are positionally conserved with CWP1 and -2. During encystation, C-terminal epitope-tagged CWP3 was transported to the wall of water-resistant cysts via the novel regulated secretory pathway in encystation-secretory vesicles (ESVs). Deletion analysis revealed that the four LRRs are each essential to target CWP3 to the ESVs and cyst wall. In a deletion of the most C-terminal region, fewer ESVs were stained in encysting cells, and there was no staining in cysts. In contrast, deletion of the 44 amino acids between the signal sequence and the LRRs or the region just C-terminal to the LRRs only decreased the number of cells with CWP3 targeting to ESVs and cyst wall by approximately 50%. Our studies indicate that virtually every portion of the CWP3 protein is needed for efficient targeting to the regulated secretory pathway and incorporation into the cyst wall. Further, these data demonstrate the power of genomics in combination with rigorous functional analyses to verify annotation.

Characterization of the promoter for the mouse alpha 3 integrin gene.

The alpha 3 beta 1 integrin is an adhesion receptor for extracellular matrix proteins including isoforms of laminin, and the changes of its expression level in various cancer cells are thought to cause their malignant phenotypes. We have cloned an approximately 4 kb DNA fragment of the 5'-flanking region of the murine alpha 3 integrin gene and analyzed its promoter activity. Transfection of MKN1 gastric carcinoma cells with serially truncated segments of the 5'-flanking region linked to a luciferase gene indicated that a 537-bp SalI/SacI fragment upstream of exon 1 was sufficient to promote high level gene expression. By 5'-rapid amplification of cDNA ends (5'-RACE) using a cap site-labeled cDNA library, we determined one major and one minor transcription start sites in this region. The murine alpha 3 integrin gene was found to contain a CCAAT box, but to lack a TATA box. Luciferase assay following transfection with a series of deletion constructs of the SalI/SacI fragment revealed that the sequence between positions -260 and -119 bp (relative to the major transcription start site) is required for efficient transcription in gastric carcinoma cells. The sequence analysis of this segment showed the presence of several consensus sequences for transcription factors including Ets, GATA and MyoD/E-box binding factors. The introduction of mutation in one of the Ets-binding sequences greatly decreased its promoter activity, suggesting that the transcription of the alpha 3 integrin gene in these cells is regulated by the Ets-family of transcription factors.

The Rod cGMP-phosphodiesterase β-Subunit Promoter Is a Specific Target for Sp4 and Is Not Activated by Other Sp Proteins or CRX

The beta-subunit of cGMP-phosphodiesterase (beta-PDE) is a key protein in phototransduction expressed exclusively in rod photoreceptors. It is necessary for visual function and for structural integrity of the retina. beta-PDE promoter deletions showed that the -45/-23 region containing a consensus Crx-response element (CRE) was necessary for low level transcriptional activity. Overexpressed Crx modestly transactivated this promoter in 293 human embryonic kidney cells; however, mutation of CRE had no significant effect on transcription either in transfected Y79 retinoblastoma cells or Xenopus embryonic heads. Thus, Crx is unlikely to be a critical beta-PDE transcriptional regulator in vivo. Interestingly, although the beta/GC element (-59/-49) binds multiple Sp transcription factors in vitro, only Sp4, but not Sp1 or Sp3, significantly enhanced beta-PDE promoter activity. Thus, the Sp4-mediated differential activation of the beta-PDE transcription defines the first specific Sp4 target gene reported to date and implies the importance of Sp4 for retinal function. Further extensive mutagenesis of the beta-PDE upstream sequences showed no additional regulatory elements. Although this promoter lacks a canonical TATA box or Inr element, it has the (T/A)-rich beta/TA sequence located within the -45/-23 region. We found that it binds purified TBP and TFIIB in gel mobility shift assays with cooperative enhancement of binding affinity.

Characterization of the Mouse Matrilin-4 Gene: A 5′ Antiparallel Overlap with the Gene Encoding the Transcription Factor RBP-L

We have isolated and characterized the gene encoding mouse matrilin-4 (Matn4), an extracellular matrix protein present in a broad spectrum of tissues. The gene spanned 16 kb, consisted of 12 exons, and localized to chromosome 2. As in all known matrilin genes, the last intron, separating the exons coding for the coiled-coil domain, did not follow the GT-AG rule and belonged to the subgroup of introns having AT-AC at the ends. Matn4 contained two exons in the 5′ UTR that could be alternatively spliced. We localized a major and a minor transcription start site to two different untranslated exons: exon 0a and exon 0b. Matn4 divergently overlapped 5′ with the gene encoding RBP-L (for recombining binding protein suppressor of hairless-like; Rbpsuhl), a transcription factor with homology to RBP-JK. Exon 1 of Rbpsuhl was located in the second intron of Matn4, whereas exon 0a, the first exon of Matn4, was located in the second intron of Rbpsuhl. The second exons of the respective genes overlapped in an antisense orientation. We mapped the major transcription start of Rbpsuhl to a position approximately 150 nt upstream of the splice acceptor site of the first intron, leading to the synthesis of a truncated variant of RBP-L probably missing the amino-terminal 121 amino acid residues. We analyzed the expression of the different Matn4 and Rbpsuhl transcripts by quantitative RT-PCR; this showed the highest expression for both genes in lung and brain. In situ hybridization of brain sections showed a partially overlapping expression pattern for the two genes.

Genomic organization, transcription start sites, and chromosomal location of the Drosophila cortactin gene.

An actin filament binding protein cortactin was initially identified as a major phosphotyrosine-containing protein in v-Src-transformed chicken embryo fibroblast cells. The mouse, human, and Drosophila homologs were independently identified as a signaling molecule involved in a mitogenic response, as a product of a putative oncogene EMS1, and as a molecule interacting with a scaffolding protein ZO-1, respectively. In this report, we describe the cloning of the Drosophila cortactin gene, which consists of four exons and three introns, covering 3 kilobases in length. All exon-intron junctions are well matched with the GT/AG consensus sequence. S1 nuclease mapping revealed one major and several minor transcription start sites. The cytological location of the Drosophila cortactin gene is between chromosome segments 93B3 and 93B7.

The major transcription initiation site of the p27Kip1 gene is conserved in human and mouse and produces a long 5'-UTR

BackgroundThe cyclin-dependent kinase inhibitor p27Kip1 is essential for proper control of cell cycle progression. The levels of p27Kip1 are regulated by several mechanisms including transcriptional and translational controls. In order to delineate the molecular details of these regulatory mechanisms it is important to identify the transcription initiation site within the p27Kip1 gene, thereby defining the promoter region of the gene and the 5'-untranslated region of the p27Kip1 mRNA. Although several previous studies have attempted to map p27Kip1 transcription start sites, the results vary widely for both the mouse and human genes. In addition, even though the mouse and human p27Kip1 gene sequences are very highly conserved, the reported start sites are notably different.ResultsIn this report, using a method that identifies capped ends of mRNA molecules together with RNase protection assays, we demonstrate that p27Kip1 transcription is initiated predominantly from a single site which is conserved in the human and mouse genes. Initiation at this site produces a 5'-untranslated region of 472 nucleotides in the human p27Kip1 mRNA and 502 nucleotides in the mouse p27Kip1 mRNA. In addition, several minor transcription start sites were identified for both the mouse and human genes.ConclusionsThese results demonstrate that the major transcription initiation sites in the mouse and human p27Kip1 genes are conserved and that the 5'-UTR of the p27Kip1 mRNA is much longer than generally believed. It will be important to consider these findings when designing experiments to identify elements that are involved in regulating the cellular levels of p27Kip1.

The Mouse Vitamin D Receptor Is Mainly Expressed through an Sp1-Driven Promoter in Vivo

The availability of the mouse vitamin D receptor (mVDR) gene has allowed a characterization of a TATA-less promoter containing a cluster of four Sp1 sites named Sp1-1, Sp1-2, Sp1-3, and Sp1-4 (F. Jehan and H. F. DeLuca, 1997, Proc. Natl. Acad. Sci. USA 94, 10138–10143). By means of primer extension analysis, S1 nuclease mapping and ribonuclease protection assay, the start site has been deduced, as has the existence of other minor transcription start sites. Initiation of transcription at the major site is located 4 bp upstream of the 5′ end of the mVDR cDNA sequence and very close to the putative Sp1 sites. A second minor promoter might exist between exon 1 and exon 2 of the mVDR gene. The nucleotide sequence of the Sp1 region is well conserved between the mouse, the human, and the chicken VDR genes, suggesting an important role for these Sp1 sites. Gel shift analysis of the four Sp1 sites of the mVDR promoter has confirmed specific binding complexes to Sp1-1, Sp1-2, and Sp1-4 (Sp1-3 rather binds an unknown complex that is unable to bind the canonical Sp1 GGGGCGGGGC). Deletion or mutation of all the Sp1 sites eliminates promoter activity. However, mutation or deletion of individual Sp1 sites did not dramatically change the promoter activity, except for mutation of Sp1-3 that increases promoter activity. We, therefore, conclude that the mVDR promoter is controlled by the Sp1 sites and is the main VDR promoter in intestine and kidney.

Structure and mapping of the mouse matrilin-3 gene (Matn3), a member of a gene family containing a U12-type AT-AC intron.

The gene for murine matrilin-3, an extracellular matrix protein present in cartilage, was isolated and further characterized. The gene spans 23.4 kb and comprises 8 exons; with one exception, this reflects the modular structure of the protein. The major and a minor transcription start site were determined by RNase protection assays to positions approximately 72 nt and 87 nt upstream of the ATG codon, respectively. The promoter contains a TATA-like box 32 bp upstream of the main transcription start as well as several potential binding sites for eukaryotic transcription factors. As in all known matrilin genes, the last intron, separating the exons coding for the coiled-coil domain, does not follow the GT-AG rule and belongs to the subgroup of introns having AT-AC at the ends that are spliced by the U12-type spliceosome. The mouse matrilin-3 gene does not contain hidden exon sequences coding for the second vWFA-like domain present in all other matrilins. The intron that could possibly contain such sequences instead shows 75% repetitive sequences, indicating an evolutionary process that has led to the loss of sequences coding for vWFA2. Single-strand conformation polymorphism analysis was used to map the Matn3 gene to the proximal end of Chr 12, linked to the genes Synd1, Apob, Dntb, and Kif3c.

Structural analysis and promoter characterization of the human membrane-type matrix metalloproteinase-1 (MT1-MMP) gene

Membrane type-1 matrix metalloproteinase (MT1-MMP) degrades extracellular matrix components directly and indirectly by activation of other matrix metalloproteinases (MMPs). In the present study, we have isolated and characterized the human MT1-MMP gene and its promoter. The gene consists of 10 exons and nine introns spanning more than 10 kilobases (kb). The locations of two exon–intron splicing sites are distinct from the preserved positions among other known MMP genes. Primer extension and RNAse and S1 nuclease protection analyses indicated that there are four major and several minor transcription start sites. The 5′-flanking sequence of the gene contains putative regulatory elements, including one Sp-1 site and four CCAAT-boxes, whereas there is no TATA-box. The Sp-1 binding site was functional, as shown by gel shift and supershift analyses. Transfection studies with promoter constructs containing 0.1 to 7.2 kb of 5′-flanking sequence coupled to a luciferase reporter gene indicated that the promoter contains additional positive and negative regulatory sequences. Deletion of the Sp-1 binding site by site-directed mutagenesis reduced luciferase activity by about 90%, demonstrating the crucial role of this element in maintaining MT1-MMP transcription. Our findings indicate that the human MT1-MMP promoter has distinctive structural and functional features compared with other MMP genes, which may lead to a unique expression pattern and regulation during physiological and pathological processes.

Isolation and Functional Characterization of the Human 90K Promoter

90K is a secreted protein thought to be involved in the body's defense against pathogens and cancer. To elucidate its transcriptional regulation, the promoter of human 90K (HGMW-approved symbol LGAL S3BP) was isolated and characterized. Analysis of the 3.3-kb 5′-flanking region revealed that it is a TATA-less promoter, but neither GC-rich nor dependent on SP1 sites. RNase protection assays detected one major transcription start site (+1) and several minor transcription start sites upstream and downstream. Deletion studies defined a minimal promoter (−103 → −49) and indirectly suggested positive synergism between different elements within it. Consistent with the proposed function of 90K, its promoter activity could be stimulated by poly(I) · poly(C), mimicking viral infection. Two regions mediating induction by poly(I) · poly(C) (−171 → −112, −32 → 46) were identified by deletion mutants. A small region around the minimal promoter (−99 → −12) was highly homologous between human and mouse. While both human and mouse minimal promoters contained an interferon-responsive element (IRF-E), the human minimal promoter was not inducible by poly(I) · poly(C) in contrast to that of the mouse. Point mutations 30 bp upstream of the IRF-E, however, conferred inducibility to the human minimal promoter, suggesting interaction between different promoter elements.

The chick and human collagen alpha1(XII) gene promoter--activity of highly conserved regions around the first exon and in the first intron.

A single gene encodes collagen XII, an extracellular matrix protein with three large fibronectin-related subunits connected via a short collagen triple helix. Since collagen XII is a component of a specific subset of collagen fibrils in tissues bearing high tensile stress, we are interested to know how its restricted expression is regulated. To this aim, we have isolated the region around the first exon of both the chick and human collagen alpha1(XII) gene. The upstream sequences of the two genes share common features but are not related. Strong similarity starts about 100 bp 5' of the first exon and ends 100 bp into the first intron. In addition, two large conserved regions (56-63% similarity) were found in the first intron. A single major and two clusters of minor transcription start sites were identified in both the chick and human gene. To test for promoter activity, conserved fragments from the chick gene were cloned into reporter plasmids for transient transfection of fibroblasts. A 70-bp stretch containing a conserved nuclear factor-1 binding sequence just upstream of the first transcription start site was found to work as a basal promoter. An adjacent, but nonoverlapping short segment including the more downstream start sites and a conserved TATTAA sequence exhibited independent promoter activity. GC-rich sequences just 5' and 3' of the minimal promoter fragments were required for full activity. In contrast, inclusion of more upstream sequences (up to 2.4 kb) had no effect. The two conserved regions in the first intron showed no promoter activity on their own but modulated activity when linked to autologous or heterologous promoters. Specifically, one of these intronic regions might contain enhancer element(s) that respond to mechanical stress acting on the fibroblasts. We conclude that the collagen XII gene is driven by a basal promoter with two halves that can act independently; conserved control regions are located around the first exon and in the first intron.

Gene and cDNA Structures of Flounder Insulin-Like Growth Factor-I (IGF-I): Multiple mRNA Species Encode a Single Short Mature IGF-I

To understand the comprehensive mechanisms of gene expression and processing for insulin-like growth factor-I (IGF-I) in vertebrates, we have investigated the gene organization, promoter and transcriptional initiation sites, alternative splicing and polyadenylating sites, and the cDNA structures of this gene in the Japanese flounder, Paralichthys olivaceus. The flounder IGF-I gene was found to be composed of five exons and four introns spanning 17.5 kb. By Northern blot analysis, two major mRNA classes of 4.7 kb and 2.9 kb were found in the liver. cDNA cloning and reverse transcription polymerase chain reaction (RT-PCR) analysis indicated that these two mRNA classes result from two different-sized 3'-noncoding regions generated by alternative usage of two polyadenylating signals. Further analysis by RT-PCR and sequencing revealed that these mRNA classes both contain two subclasses of mRNA encoding two forms of IGF-I prepropeptide, preproIGF-I-1 and preproIGF-I-2. The two forms of preproIGF-I share the identical signal peptide and mature IGF-I domain but contain different E domains as a result of alternative splicing in exon 3. The mature form of flounder IGF-I was found to comprise 68 amino acid residues, showing a small molecular weight, 7486. In the 5'-flanking region, one major and four minor transcription start sites have been identified by ribonuclease protection assay between -230 and -130 from the translation initiation codon, but no canonical TATA box or GC box was detected in their upstream regions up to -724. The results suggest that some unknown transcription initiation factors are functioning in the promotion of IGF-I gene expression.

Promoter characterization and constitutive expression of the Escherichia coli gcvR gene.

The Escherichia coli glycine cleavage repressor protein (GcvR) negatively regulates expression of the glycine cleavage operon (gcv). In this study, the gcvR translational start site was determined by N-terminal amino acid sequence analysis of a GcvR-LacZ fusion protein. Primer extension analysis of the gcvR promoter region identified a primary transcription start site 27 bp upstream of the UUG translation start site and a minor transcription start site approximately 100 bp upstream of the translation start codon. The -10 and -35 promoter regions upstream of the primary transcription start site were defined by mutational analysis. Expression of a gcvR-lacZ fusion was unaltered in the presence of glycine or inosine, molecules known to induce or repress expression of gcv, respectively. In addition, it was shown that gcvR-lacZ expression is neither regulated by the glycine cleavage activator protein (GcvA) nor autogenously regulated by GcvR. From DNA sequence analysis, it was predicted that the translation start codon of the downstream bcp gene overlaps the gcvR stop codon, suggesting that these genes may form an operon. However, a down mutation in the -10 promoter region of gcvR had no effect on the expression of a downstream bcp-lacZ fusion, and primer extension analysis of the bcp promoter region demonstrated that bcp has its own promoter within the gcvR coding sequence. These results show that gcvR and bcp do not form an operon. Furthermore, the deletion of bcp from the chromosome had no effect on gcv-lacZ expression.