Multiple Transcription Initiation Sites Research Articles

Characterization of the immediate-early 2 protein of human herpesvirus 6, a promiscuous transcriptional activator

In the present work we report the cloning of a full-length cDNA encoding the immediate-early (IE) 2 protein from human herpesvirus 6 (HHV-6) variant A (GS strain). The transcript is 4690 nucleotides long and composed of 5 exons. Translation initiation occurs within the third exon and proceeds to the end of U86. Kinetic studies indicate that the 5.5-kb IE2 mRNA is expressed under IE condition, within 2–4 h of infection. IE2 transcripts from both variants A and B are expressed under similar kinetics with IE2 transcripts accumulating up to 96 h postinfection. Although several large transcripts (>5.5 kb) hybridized with the IE2 probe, suggesting multiple transcription initiation sites, a single form of the IE2 protein, in excess of 200 kDa, was detected by Western blot. Within cells, the IE2 protein was detected (8–48 h) as intranuclear granules while at later time points (72–120 h), the IE2 protein coalesced into a few large immunoreactive patches. Transfection of cells with an IE2 expression vector (pBK-IE2A) failed to reproduce the patch-like distribution, suggesting that other viral proteins are necessary for this process to occur. Last, IE2 was found to behave as a promiscuous transcriptional activator. Cotransfection experiments in T cells indicate that IE2 can induce the transcription of a complex promoter, such as the HIV-LTR, as well as simpler promoters, whose expression is driven by a unique set of responsive elements (CRE, NFAT, NF-kB). Moreover, minimal promoters having a single TATA box or no defined eukaryotic regulatory elements were significantly activated by IE2, suggesting that IE2 is likely to play an important role in initiating the expression of several HHV-6 genes. In all, the work presented represents the first report on the successful cloning, expression, and functional characterization of the major regulatory IE2 gene/protein of HHV-6.

Cloning and Characterization of the Rat α1a-Adrenergic Receptor Gene Promoter: DEMONSTRATION OF CELL SPECIFICITY AND REGULATION BY HYPOXIA

Recent studies reveal important and distinct roles for cardiac alpha(1a) adrenergic receptors (alpha(1a)ARs). Surprisingly, given their importance in myocardial ischemia/reperfusion, hypoxia, and hypertrophy as well as frequent use of rat cardiomyocyte model systems, the rat alpha(1a)AR gene promoter has never been characterized. Therefore, we isolated 3.9 kb of rat alpha(1a)AR 5'-untranslated region and 5'-regulatory sequences and identified multiple transcription initiation sites. One proximal (P1) and several clustered upstream distal promoters (P2, P3, and P4) were delineated. Sequences surrounding both proximal and distal promoters lack typical TATA or CCAAT boxes but contain cis-elements for multiple myocardium-relevant nuclear regulators including Sp1, GATA, and CREB, findings consistent with enhanced cardiac basal alpha(1a)AR expression seen in Northern blots and reporter constructs. Promoter analysis using deletion reporter constructs reveals, in addition to a powerful upstream enhancer, a key region (-558/-542) important in regulating all alpha(1a)AR promoters with hypoxic stress. Gel shift analysis of this 14-bp region confirms a hypoxia-induced shift independent of direct hypoxia-inducible factor binding. Mutational analysis of this sequence identifies a novel 9-bp hypoxia response element, the loss of which severely attenuates hypoxia-mediated repression of alpha(1a)AR transcription. These findings for the alpha(1a) gene should facilitate elucidation of alpha(1)AR-mediated mechanisms involved in distinct myocardial pathologies.

Structure and promoter activity of the human glia maturation factor-gamma gene: a TATA-less, GC-rich and bidirectional promoter

Human glia maturation factor-gamma (hGMFG) was recently identified as a gene that is homologous to glia maturation factor-beta (GMFB). In this study, we determined the organization of the 9.5-kb hGMFG gene and characterized its promoter activity. The 5′-flanking region of the first exon has putative elements for binding transcription factors Sp-1, GATA-1, AML-1a, Lyf-1 and Ets-1, but there were no TATA or CAAT boxes within a 226-bp sequence upstream from the initiation codon. Primer extension analysis and 5′RACE (rapid amplification of cDNA 5′ ends) identified multiple transcription initiation sites within the region −84 to −70 nucleotides from the first ATG codon in a Kozak consensus sequence. A core promoter region was determined by transfecting a series of deletion constructs with a dual luciferase reporter system into rat astrocyte-derived ACT-57 cells. We found that 226 bp of the core promoter region exhibited bidirectional promoter activity.

Widely Spaced Alternative Promoters, Conserved between Human and Rodent, Control Expression of the Opitz Syndrome Gene

Mutations in the gene MID1 are responsible for the X-linked form of Opitz syndrome, a genetic disorder that primarily affects the development of midline structures. Several mRNA isoforms with variant 5′ ends have been reported for MID1, suggesting the presence of alternative transcription initiation sites. However, the genomic organization and expression pattern of the heterogeneous MID1 5′-untranslated regions (UTRs), as well as the promoter regions regulating their transcription, have not been determined. We now report the characterization of alternative MID1 first exons and their associated promoters in human and other species. Multiple transcription initiation sites, dispersed over 250 kb on Xp22, were found to give rise to five MID1 isoforms in human that differed in their 5′-UTRs, but contained identical coding sequence. These alternative MID1 transcripts showed distinct expression patterns with the most 5′ first exon being adipose-specific and another exon being placenta-selective, whereas other 5′-UTRs seemed to be ubiquitous. Consistent with the expression data, the putative promoter regions upstream of the variant first exons also varied in their strength and tissue specificity in transfection experiments. Similar to the human gene, heterogeneous Mid1 isoforms were also identified in mouse and rat by 5′-RACE, which revealed that three of the five alternative first exons isolated in human are also used in rodent. Comparison of MID1 promoter regions from several mammals indicated a high level of identity suggesting evolutionary conservation. Together, these results suggest that alternative promoters are important in the complex transcriptional regulation of MID1 expression in several species.

Widely Spaced Alternative Promoters, Conserved between Human and Rodent, Control Expression of the Opitz Syndrome Gene MID1

Mutations in the gene MID1 are responsible for the X-linked form of Opitz syndrome, a genetic disorder that primarily affects the development of midline structures. Several mRNA isoforms with variant 5′ ends have been reported for MID1, suggesting the presence of alternative transcription initiation sites. However, the genomic organization and expression pattern of the heterogeneous MID1 5′-untranslated regions (UTRs), as well as the promoter regions regulating their transcription, have not been determined. We now report the characterization of alternative MID1 first exons and their associated promoters in human and other species. Multiple transcription initiation sites, dispersed over 250 kb on Xp22, were found to give rise to five MID1 isoforms in human that differed in their 5′-UTRs, but contained identical coding sequence. These alternative MID1 transcripts showed distinct expression patterns with the most 5′ first exon being adipose-specific and another exon being placenta-selective, whereas other 5′-UTRs seemed to be ubiquitous. Consistent with the expression data, the putative promoter regions upstream of the variant first exons also varied in their strength and tissue specificity in transfection experiments. Similar to the human gene, heterogeneous Mid1 isoforms were also identified in mouse and rat by 5′-RACE, which revealed that three of the five alternative first exons isolated in human are also used in rodent. Comparison of MID1 promoter regions from several mammals indicated a high level of identity suggesting evolutionary conservation. Together, these results suggest that alternative promoters are important in the complex transcriptional regulation of MID1 expression in several species.

Characterization of the Gene Encoding Mouse Retinoblastoma Binding Protein-7, a Component of Chromatin-Remodeling Complexes

RBBP7 is a highly conserved WD-repeat protein that interacts with histone deacetylases and is a component of several co-repressor complexes. The mouse gene Rbbp7 spans ∼20 kb, consists of at least 12 exons, and contains a C/T polymorphism in the 3′ splice acceptor region of intron 3. We found that Rbbp7 contains a TATA-less promoter with multiple transcription initiation sites. In transient transfection assays, we identified potential positive regulatory elements upstream of the proximal promoter at −668 to −1710. RBBP7 protein is detectable from at least day 9.5 of embryogenesis and is strongly expressed in the developing kidney and brain. Consistent with its association with co-repressor complexes, we demonstrate that RBBP7 represses the c-FOS transactivation domain in response to mitogen stimulation. We have also excluded human RBBP7 as a candidate gene in six patients that exhibit X-linked mental retardation, a heterogeneous developmental disorder that has been linked in some cases to mutations in genes involved in chromatin remodeling.

Analysis of human neuropeptide FF gene expression.

As an initial step to study the function of the gene encoding the human neuropeptide FF (NPFF), we cloned a 4.7-kb sequence from the promoter region. Primer extension and 5'-rapid amplification of cDNA ends revealed multiple transcription initiation sites. Northern blot analysis of the mRNA expression revealed a specific signal only in poly(A) + RNA from medulla and spinal cord. Chimeric luciferase reporter gene constructs were transiently transfected in A549, U-251 MG, SK-N-SH, SK-N-AS and PC12 cells. The promoter activity was directly comparable with the level of endogenous NPFF mRNA as determined by real-time quantitative RT-PCR. The highest promoter activity was measured when a region from - 552 to - 830 bp of the 5'-flanking region was fused to the constructs, and a potential silencer element was localized between nucleotides -220 and -551. A twofold increase in NPFF mRNA was observed after 72 h of nerve growth factor stimulation of PC12 cells and the region between - 61 and - 214 bp of the 5'-flanking region was found to be responsive to this stimulation. We postulate that control of human NPFF gene expression is the result of both positive and negative regulatory elements and the use of multiple transcription initiation sites.

Molecular cloning and characterization of the mouse thymocyte protein gene

Chicken thymocyte protein (cThy28) has recently been identified and implicated to be involved in apoptosis of avian lymphocytes, while its functional role remains undefined. To elucidate the role of Thy28, we have molecularly cloned the mouse Thy28 ( mThy28) cDNA and clarified its genomic organization in the present study. The mThy28 cDNA encodes a 226 amino acid protein, whose sequence is highly conserved among bacteria, yeast and plants as well as vertebrates. Northern blot analysis revealed abundant expression of approximately 1 kb mRNA in testis, liver, brain and kidney with lower levels of the expression in thymus, spleen, heart and stomach. The mThy28 gene spans at least 7 kb of genomic DNA, and contains eight exons and seven introns. The 5′-flanking region of the mThy28 gene does not contain a typical TATA box, but contains a putative CCAAT box and presumably multiple transcription initiation sites. Potential Sp1, GATA-1, -2, -3, AP-1, and p300 binding sites were found in the 5′-flanking region of the mThy28 gene. The gene was mapped to mouse chromosome 9.

The 5'-flanking region of the rat GABA(A) receptor alpha2-subunit gene (Gabra2).

The GABA(A) receptor alpha2-subunit gene (Gabra2) has a specific spatial and temporal pattern of expression in rat brain. As a first step towards understanding the molecular mechanism underlying this regulation, we have investigated the structural properties of the 5'- flanking region of the rat Gabra2 gene. We identified six alpha2 transcript isoforms, each of which differs only in the 5'-untranslated region (UTR). Alignment of cDNA and genomic DNA sequences revealed that six 5'-UTRs are generated from three alternative first exons by alternative splicing using internal and terminal 5'-splice donor sites present in these exons. Promoter regions containing multiple transcription initiation sites were identified in the 5' proximity of each first exon. Two of these promoters lack TATA and CCAAT sequences. Finally, we have shown that differential activation of alternative promoters is used for the expression of the alpha2 mRNA isoforms during brain development, and that the diversity at the 5'-end of these transcripts affects GABA(A) receptor expression. Taken together, these results suggest that the expression of the Gabra2 gene can be influenced at both the transcriptional and post-transcriptional levels.

The use of multiple transcription starts causes the dual targeting of Arabidopsis putative monodehydroascorbate reductase to both mitochondria and chloroplasts.

Monodehydroascorbate reductase (MDAR) isoforms exist in mitochondria, chloroplasts, cytosol and microbodies. Two putative MDAR sequences with an extended N-terminal region are found in Arabidopsis. They differ in the length of the extension by 21 bp. We have shown that these two isoforms arise from a single gene by the use of multiple transcription starts. Green fluorescent protein was fused to each extension, revealing that the longer and shorter fusion proteins were imported into mitochondria and chloroplasts, respectively. These results demonstrate that putative MDAR is a dual-targeting protein transported into both mitochondria and chloroplasts. Although there have been several reports of dual targeting of proteins to mitochondria and chloroplasts, this is the first example in which the dual targeting of the protein to mitochondria and chloroplasts is achieved by the use of multiple transcription initiation sites.

Full-length messenger RNA sequences greatly improve genome annotation.

Annotation of eukaryotic genomes is a complex endeavor that requires the integration of evidence from multiple, often contradictory, sources. With the ever-increasing amount of genome sequence data now available, methods for accurate identification of large numbers of genes have become urgently needed. In an effort to create a set of very high-quality gene models, we used the sequence of 5,000 full-length gene transcripts from Arabidopsis to re-annotate its genome. We have mapped these transcripts to their exact chromosomal locations and, using alignment programs, have created gene models that provide a reference set for this organism. Approximately 35% of the transcripts indicated that previously annotated genes needed modification, and 5% of the transcripts represented newly discovered genes. We also discovered that multiple transcription initiation sites appear to be much more common than previously known, and we report numerous cases of alternative mRNA splicing. We include a comparison of different alignment software and an analysis of how the transcript data improved the previously published annotation. Our results demonstrate that sequencing of large numbers of full-length transcripts followed by computational mapping greatly improves identification of the complete exon structures of eukaryotic genes. In addition, we are able to find numerous introns in the untranslated regions of the genes.

Multiple Transcription Initiation Sites, Alternative Splicing, and Differential Polyadenylation Contribute to the Complexity of Human Neurofibromatosis 2 Transcripts

Northern blot analysis has shown that the human neurofibromatosis type 2 (NF2) cDNA hybridizes to multiple RNA species. To examine whether these hybridizing RNA species represent NF2 transcripts, we cloned the complete NF2 cDNA by a combination of techniques: 5′ and 3′ rapid amplification of cDNA ends, RT–PCR, and searching and sequencing the NF2-related cDNA clones from the IMAGE consortium. We showed that human NF2 transcripts initiate at multiple positions. Analogous to those reported previously, NF2 transcripts undergo alternative splicing in the coding exons. We isolated eight alternatively spliced NF2 cDNA isoforms, including one that contains a new exon termed exon 2′, which potentially could encode proteins of different sizes. We assembled the overlapping cDNA fragments, and the longest NF2 cDNA, containing all 17 exons, consists of 6067 nucleotides, which is consistent with the size of the major RNA species hybridized to the NF2 probe. The cDNA has a 425-nucleotide 5′ untranslated region upstream from the ATG start codon, and a long 3′ untranslated region of 3869 nucleotides. We also isolated two shorter NF2 cDNAs that were terminated by different polyadenylation signal sequences, which indicates that differential usage of multiple polyadenylation sites also contributes to the complexity of human NF2 transcripts. By reference to the transcription initiation site mapped, we analyzed the 5′ flanking sequence of the human NF2 gene. Transient transfection analysis in human 293 kidney, SK-N-AS neuroblastoma, and NT2/D1 teratocarcinoma cells with NF2 promoter-luciferase chimeric constructs revealed a core promoter region extending 400 base pairs from the major transcription initiation site. Although multiple regions are required for full promoter activity, a site-directed mutagenesis experiment identified a GC-rich sequence (position −58 to −46), which could be bound by transcription factor Sp1, as a positive cis-acting regulatory element. Cotransfection studies in Drosophila melanogaster SL2 cells showed that Sp1 could activate the NF2 promoter through the GC-rich sequence.

Multiple Messenger Ribonucleic Acid Transcripts and Revised Gene Organization of the Human TSH Receptor.

Northern blot analysis of human TSH receptor (hTSHR) messenger ribonucleic acid (mRNA) expression has previously demonstrated multiple species of transcripts in the thyroid gland, suggesting the presence of multiple transcription initiation sites, alternatively spliced forms or alternate polyadenylation (poly(A)) sites. The first two have already been reported elsewhere. To clarify alternate poly(A) sites in the hTSHR gene, the present study was designed to characterize three full-length hTSHR cDNAs with distinct poly(A) signals that we have previously cloned. The comparison of the nucleotide sequencing data on the 3'UTR of these three clones to the Draft Human Genome in NCBI database revealed that the 3' segment of exon 10 of hTSHR gene contains three tandem repeats of the poly(A) sites, from which are expressed three full-length TSHR mRNAs with distinct 3'UTR length. The longest one appears to be a predominant transcript. From these data, together with (i) the previously reported organization of hTSHR genome and (ii) use of the Draft Human Genome to localize the unidentified sequence in the alternatively spliced form of truncated hTSHR, we propose the complete structure of hTSHR gene. Rather than 10 exons, our analysis suggests that hTSHR gene seems to contain 13 exons and 12 introns. At least three full-length TSHR mRNAs with distinct poly(A) sites and five alternatively spliced forms of TSHR mRNAs are expressed from the single hTSHR gene.

Genomic structure and regulation of a novel human gene, Klp1

Klp1 (K562 cells-derived leucine zipper-like protein 1) is a transcription factor which binds to the coproporphyrinogen oxidase promoter regulatory element (GGACTACAG). In order to clarify the function of Klp1, we determined the complete human Klp1 genomic structure and regulatory element in the promoter region. The gene spans about 2.4 kb and has three exons. Its promoter region has multiple GC boxes, E2F binding site, one cAMP response element (CRE), and no TATA box with multiple transcription initiation sites, which is characteristic of housekeeping and growth regulating genes. Promoter analysis showed that the promoter was more active in K562 cells entered into the cell cycle by serum stimulation than quiescent cells. Further promoter analysis revealed that CRE at −42 is essential for full promoter activity, and c-Jun and activation transcription factor 1/cAMP response element binding protein 1 proteins bind to this element. These structural characteristics and the promoter function suggest that Klp1 may play a role in cell cycle regulation.

Identification and Characterization of the Human Serotonin-4 Receptor Gene Promoter

The human serotonin-4 (5-HT4) receptor gene expression is highly regulated in various tissues. We isolated the human 5-HT4 receptor gene containing the 5′-flanking region and characterized its promoter. By 5′-RACE (5′-rapid amplification of the cDNA ends) and inverse PCR, multiple transcription initiation sites were identified. The most 5′ one (assigned to +1) was 5135 bp upstream to the translation start site. The 500-bp 5′-flanking region contained potential binding sites for transcription factor Sp-1, AP-2, AP-4, and GATA. However, this region lacked TATA- and CAAT-boxes. Transient transfection analyses in human choriocarcinoma T3M-3 (5-HT4 receptor-positive) and HepG2 (5-HT4 receptor-negative) cells revealed that the region (−210 to −105) is necessary for the basic and cell-type specific 5-HT4 receptor gene expression. In addition, untranslated exon 1 contained negative (+112 to +182) as well as positive (+1 to +111) modulators, indicating that exon 1 plays a regulatory role in the 5-HT4 receptor gene expression.

The human tuftelin gene: cloning and characterization

Tuftelin has been suggested to play an important role during the development and mineralization of enamel. We isolated the full-length human tuftelin cDNA using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (5′ RACE and 3′ RACE) methods. Sequence analysis of the tuftelin cDNA revealed an open reading frame of 1170 bp encoding a 390 amino acid protein with a molecular mass of 44.3 kDa and an isoelectric point of 5.7. The human tuftelin protein shares 89 and 88% amino acid sequence identity with the bovine and mouse tuftelin, respectively. It contains a coiled-coil region, recently reported to be involved with tuftelin self-assembly and with the interaction of tuftelin with TIP39 (a novel tuftelin interacting protein). Detailed DNA analysis of the cloned genomic DNA revealed that the human tuftelin gene contains 13 exons and is larger than 26 kb. Two alternatively spliced tuftelin mRNA transcripts have now been identified in the human tooth bud, one lacking exon 2, and the other lacking exon 2 and exon 3. Primer extension analysis, corroborated by RT-PCR and DNA sequencing, revealed multiple transcription initiation sites. The cloned 1.6 kb promoter region contained several GC boxes and several transcription factor binding sites such as those for activator protein 1 and stimulatory protein 1. Our blast search of the human and mouse expressed sequence tag data bases, as well as our RT-PCR and DNA sequencing results, and a previous study using Northern blot analysis revealed that tuftelin cDNA sequences are also expressed in normal and cancerous non-mineralizing soft tissues, suggesting that tuftelin has a universal function. We have now identified and characterized different alternatively spliced mouse tuftelin mRNAs in several non-mineralizing tissues. These results provide an important baseline for future understanding of the biological role of tuftelin.

Nucleosomes are translationally positioned on the active allele and rotationally positioned on the inactive allele of the HPRT promoter.

Differential chromatin structure is one of the hallmarks distinguishing active and inactive genes. For the X-linked human hypoxanthine phosphoribosyltransferase gene (HPRT), this difference in chromatin structure is evident in the differential general DNase I sensitivity and hypersensitivity of the promoter regions on active versus inactive X chromosomes. Here we characterize the nucleosomal organization responsible for the differential chromatin structure of the active and inactive HPRT promoters. The micrococcal nuclease digestion pattern of chromatin from the active allele in permeabilized cells reveals an ordered array of translationally positioned nucleosomes in the promoter region except over a 350-bp region that is either nucleosome free or contains structurally altered nucleosomes. This 350-bp region includes the entire minimal promoter and all of the multiple transcription initiation sites of the HPRT gene. It also encompasses all of the transcription factor binding sites identified by either dimethyl sulfate or DNase I in vivo footprinting of the active allele. In contrast, analysis of the inactive HPRT promoter reveals no hypersensitivity to either DNase I or a micrococcal nuclease and no translational positioning of nucleosomes. Although nucleosomes on the inactive promoter are not translationally positioned, high-resolution DNase I cleavage analysis of permeabilized cells indicates that nucleosomes are rotationally positioned over a region of at least 210 bp on the inactive promoter, which coincides with the 350-bp nuclease-hypersensitive region on the active allele, including the entire minimal promoter. This rotational positioning of nucleosomes is not observed on the active promoter. These results suggest a model in which the silencing of the HPRT promoter during X chromosome inactivation involves remodeling a transcriptionally competent, translationally positioned nucleosomal array into a transcriptionally repressed architecture consisting of rotationally but not translationally positioned nucleosomal arrays.

Structure of the 5′ region of the Hst70 gene transcription unit: presence of an intron and multiple transcription initiation sites

The rat Hst70 gene and its mouse counterpart Hsp70.2 belong to the family of Hsp70 heat shock genes and are specifically expressed in male germ cells. Previous studies regarding the structure of the 5′ region of the transcription unit of these genes as well as localization of the ‘cis’ elements conferring their testis-specific expression gave contradictory results [Widłak, Markkula, Krawczyk, Kananen and Huhtaniemi (1995) Biochim. Biophys. Acta 1264, 191–200; Dix, Rosario-Herrle, Gotoh, Mori, Goulding, Barret and Eddy (1996) Dev. Biol. 174, 310–321]. In the present paper we solve these controversies and show that the 5′ untranslated region (UTR) of the Hst70 gene contains an intron which is localized similar to that of the mouse Hsp70.2 gene. Reverse transcriptase-mediated PCR, Northern blotting and RNase protection analysis revealed that the transcription initiation of both genes starts at two main distant sites, and one of them is localized within the intron. As a result two populations of Hst70 gene transcripts with similar sizes but different 5′ UTR structures can be detected in total testicular RNA. Functional analysis of the Hst70 gene promoter in transgenic mice and transient transfection assays proved that the DNA fragment of approx. 360bp localized upstream of the ATG transcription start codon is the minimal promoter required for testis-specific expression of the HST70/chloramphenicol acetyltransferase transgene. These experiments also suggest that the expression of the gene may depend on ‘cis’ regulatory elements localized within exon 1 and the intron sequences.

Conserved Structure and Promoter Sequence Similarity in the Mouse and Human Genes Encoding the Zinc Finger Factor BERF-1/BFCOL1/ZBP-89

We have characterized the genomic structure of the mouse Zfp148 gene encoding Beta-Enolase Repressor Factor-1 (BERF-1), a Kruppel-like zinc finger protein involved in the transcriptional regulation of several genes, which is also termed ZBP-89, BFCOL1. The cloned Zfp148 gene spans 110 kb of genomic DNA encompassing the 5′-end region, 9 exons, 8 introns, and the 3′-untranslated region. The promoter region displays the typical features of a housekeeping gene: a high G+C content and the absence of canonical TATA and CAAT boxes consistent with the multiple transcription initiation sites determined by primary extension analysis. Computer-assisted search in the human genome database allowed us to determine that the same genomic structure with identical intron–exon organization is conserved in the human homologue ZNF 148. Functional analysis of the 5′-flanking sequence of the mouse gene indicated that the region from nucleotide −205 to +144, relative to the major transcription start site, contains cis-regulatory elements that promote basal expression. Such sequences and the overall promoter architecture are highly conserved in the human gene. Furthermore, we show that the complex transcription pattern of the Zfp148 gene might be due to a combination of alternative splicing and differential polyadenylation sites utilization.

Structural Diversity Despite Strong Evolutionary Conservation in the 5′-Untranslated Region of the P-Type Dystrophin Transcript

Analysis of the 5′-flanking regions of the Purkinje (P-) dystrophin genes and mRNAs in different species revealed strong sequence conservation but functional diversity. Multiple transcription initiation sites were identified in cerebella and muscles, tissues expressing P-dystrophin. The predominant initiation site was conserved, with another muscle-specific site located upstream. Despite sequence homology, significant tissue- and species-specific structural diversity in the P-type 5′-ends exists, including alternative splicing within the 5′-untranslated region combined with alternative splicing of intron 1. One amino terminus is conserved in mammals and, to a lesser extent, in chicken. However, alternative usage of ATG codons may result in a choice of N-termini or translation of short upstream ORFs in different species. Promoter activity of a fragment upstream of the cap site was shown by transient expression in myoblasts and in vivo following intramuscular injection. It is tissue- and developmentally regulated. Analysis of promoter deletions suggests the existence of negative regulatory elements in the proximal region.