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PubMed1991

Host-derived 5' ends and overlapping complementary 3' ends of the two mRNAs transcribed from the ambisense S segment of Uukuniemi virus.

Two mRNAs, coding for the N and NSS proteins, are transcribed from the small (S) Uukuniemi virus RNA segment by an ambisense strategy (J. F. Simons, U. Hellman, and R. F. Pettersson, J. Virol. 64:247-255, 1990). In this report, we describe the analysis of the 5' and 3' ends of the two mRNAs. Primer extension as well as cloning and sequencing of individual mRNAs showed that the 5' ends of both mRNAs contained nonviral sequences ranging from 7 to 25 residues in length (mean, 12 residues), indicating a cap-snatching mechanism similar to the one originally described for priming of influenza virus mRNA synthesis. In 35% of the cases, the first virion-specified nucleotide (an A residue) was substituted with a G residue. Between the translation termination codons of N and NSS, there is a 74-residue-long noncoding intergenic region (Simons et al., J. Virol. 64:247-255, 1990). Nuclease protection assays using both RNA and DNA hybridization probes showed that the 3' ends of the N and NSS mRNAs overlap each other by about 100 nucleotides. The 3' end of the NSS mRNA extends into the coding sequence of the N mRNA, whereas the N mRNA is terminated just prior to the stop codon of NSS. To our knowledge, this is the first example of overlapping complementary mRNAs in viruses with an ambisense coding strategy. No obvious transcription termination sequence was identified. However, because of a short palindromic sequence in the intergenic region, the 3' ends of both mRNAs (and consequently also the template RNAs) can be folded into an A/U-rich hairpin structure. It remains to be determined whether this structure plays any role in transcription termination.

Amino Acid Sequence↗

PubMed2005

The cellular response to ectopic overexpression of the tuberous sclerosis genes, TSC1 and TSC2: a proteomic approach.

The genes, TSC1 on chromosome 9q34, encoding hamartin, and TSC2 on chromosome 16p13.3, encoding tuberin, are responsible for tuberous sclerosis (TSC). TSC is an autosomal dominant tumor suppressor gene syndrome affecting about 1 in 6000 individuals. It is characterized by mental retardation and epilepsy. A variety of tumors characteristically occur in different organs of TSC patients and are believed to result from defects in cell cycle/cell size control. Hamartin and tuberin form a complex providing a tentative explanation for the similar disease phenotype in TSC patients with mutations in either of these genes. Beside overlap in many features of patients with TSC1 and TSC2 mutations, data accumulated providing evidence for specific clinical differences. In this study, we performed a proteomic approach of two-dimensional gel electrophoresis with subsequent mass spectrometrical identification of protein spots after ectopic overexpression of human TSC1 or TSC2. We found the protein levels of the calumenin precursor; the complement component 1; heterogeneous nuclear ribonucleoproteins, C1/C2; heterogeneous nuclear ribonucleoprotein, C1-like protein; nascent polypeptide-associated complex-alpha; proteasome subunit alpha type 5; reticulocalbin 1 precursor; translationally-controlled tumor protein; UV excision repair protein, RAD23 homolog B; elongation factor 1-delta; and the eukaryotic initiation factors, eIF-4A-like NUK-34 and eIF-6; to be deregulated upon ectopic TSC gene expression. These findings suggest that deregulation of the control of these new target proteins might contribute to the development of tubers/hamartomas in tuberous sclerosis patients. The data are presented and discussed in the context of the published literature on proteomic approaches for the identification of targets of the TSC genes.

Electrophoresis, Gel, Two-Dimensional↗

PubMed1995

Differential activation of the rat phenylethanolamine N-methyltransferase gene by Sp1 and Egr-1.

The rat phenylethanolamine N-methyltransferase (PNMT) gene contains overlapping consensus elements for the Sp1 and Egr-1 transcription factors located at -45 bp and -165 bp in the PNMT promoter. In the present study, we show that Sp1 and Egr-1 can specifically bind to these overlapping elements, that this binding appears to be mutually exclusive, and that binding site occupancy is dependent upon the concentration of each factor and its binding affinity for each site. Egr-1 binds to the -165 bp site with relatively high affinity (IC50 = 14 nM) and to the -45 bp site with relatively low affinity (IC50 = 1360 nM), whereas Sp1 binds to both sites with intermediate affinities (IC50 = 210 and 140 nM, respectively). Consistent with the DNA-binding data, Egr-1 stimulates PNMT promoter activity primarily through interaction with the -165 bp site, while Sp1 stimulates PNMT promoter activity by interacting with both the -45 bp and the -165 bp sites. These results show that Sp1 and Egr-1 are capable of differentially activating PNMT gene expression, thereby suggesting that different stimuli may control the activity of the PNMT gene by selectively regulating Sp1 and/or Egr-1.

Animals↗

PubMed2001

The Human Achaete Scute Homolog 2 gene contains two promotors, generating overlapping transcripts and encoding two proteins with different nuclear localization.

Placental development involves control by the basic helix-loop-helix transcription factor Mash2. Transcript analysis of the Human Achaete Scute Homolog 2 (HASH2) mRNA revealed the presence of two overlapping transcripts in first trimester placentae. The two transcripts (2.6 and 1.5 kb) are generated by two promotors which are separated by 1.1 kb, generating transcripts 1 and 2, respectively. Surprisingly, in transcript 1 which shows a broad expression, a second potential coding region, tentatively called Human Achaete Scute Associated Protein (HASAP) was present. Transcript 2 contains the HASH2 encoding region only. Analysis of protein expression from both transcripts by transfection studies with eGFP fusion proteins, revealed that both coding regions are translated from their endogenous translation initiation site and showed that both proteins are transported to the nucleus. HASH2 is distributed throughout the nucleus but the HASAP protein is transported into nuclear compartments, the nucleoli. In addition, the HASAP protein lacks the bHLH domain and bears no homology to known proteins. Moreover, allele-specific RT-PCR showed the human gene not to be subject to imprinting, possibly reflecting the biallelic expression of one of both transcripts. Our data indicate a species-specific difference between mouse and human expression of the Achaete Scute Homolog 2 and suggests a dual function of the human homologue.

Alleles↗

PubMed1989

An oligonucleotide hybridization approach to DNA sequencing.

We have proposed a DNA sequencing method based on hybridization of a DNA fragment to be sequenced with the complete set of fixed-length oligonucleotides (e.g., 4(8) = 65,536 possible 8-mers) immobilized individually as dots of a 2-D matrix [(1989) Dokl. Akad. Nauk SSSR 303, 1508-1511]. It was shown that the list of hybridizing octanucleotides is sufficient for the computer-assisted reconstruction of the structures for 80% of random-sequence fragments up to 200 bases long, based on the analysis of the octanucleotide overlapping. Here a refinement of the method and some experimental data are presented. We have performed hybridizations with oligonucleotides immobilized on a glass plate, and obtained their dissociation curves down to heptanucleotides. Other approaches, e.g., an additional hybridization of short oligonucleotides which continuously extend duplexes formed between the fragment and immobilized oligonucleotides, should considerably increase either the probability of unambiguous reconstruction, or the length of reconstructed sequences, or decrease the size of immobilized oligonucleotides.

Base Sequence↗

PubMed1996

Population evolution on a multiplicative single-peak fitness landscape.

A theory for evolution of either gene sequences or molecular sequences must take into account that a population consists of a finite number of individuals with related sequences. Such a population will not behave in the deterministic way expected for an infinite population, nor will it behave as in adaptive walk models, where the whole of the population is represented by a single sequence. Here we study a model for evolution of population in a fitness landscape with a single fitness peak. This landscape is simple enough for finite size population effects to be studied in detail. Each of the N individuals in the population is represented by a sequence of L genes which may either be advantageous or disadvantageous. The fitness of an individual with k disadvantageous genes is Wk = (1-s)k, where s determines the strength of selection. In the limit L-->infinity, the model reduces to the problem of Muller's Ratchet: the population moves away from the fitness peak at a constant rate due to the accumulation of disadvantageous mutations. For finite length sequences, a population placed initially at the fitness peak will evolve away from the peak until a balance is reached between mutation and selection. From then on the population will wander through a spherical shell in sequence space at a constant mean Hamming distance from the optimum sequence. We give an approximate theory for the way depends on N, L, s, and the mutation rate u. This is found to agree well with numerical simulation. Selection is less effective on small populations, so increases as N decreases. Our simulations also show that the mean overlap between gene sequences separated by a time of t generations is of the form Q(t) = Q infinity + (Q0-Q infinity)exp(-2ut), which means that the rate of evolution within the spherical shell is independent of the selection strength. We give a simplified model which can be solved exactly for which Q(t) has precisely this form. We then consider the limit L-->infinity keeping U = uL constant. We suppose that each mutation may be favourable with probability p, or unfavourable with probability 1-p. We show that for p less than a critical value pc, the population decreases in fitness for all values of U, whereas for pc < p < 1/2, the population increases in fitness for small U and decreases in fitness for large U. In this case there is an optimum non-zero value of U at which the fitness increases most rapidly, and natural selection will favour species with non-zero mutation rates.

Animals↗

PubMed1993

Bovine leukemia virus RNA sequences involved in dimerization and specific gag protein binding: close relation to the packaging sites of avian, murine, and human retroviruses.

In vitro detection of a specific complex of the bovine leukemia virus (BLV) MA(p15) protein and the 5'-terminal RNA dimer led to the hypothesis that the NH2-terminal domain of retrovirus gag protein precursor is involved in the selective viral RNA packaging mechanism. Here we describe mapping of the BLV RNA for dimer-forming and MA(p15)-binding abilities by a simple cDNA probing method followed by mutation analyses with the reactive U5-5' gag RNA. The RNA dimerization is mediated by the region harboring U5, the primer binding site (PBS), and the 30 bases immediately downstream of PBS. This conclusion is supported by computer-assisted RNA secondary-structure analysis which predicted a multibranched stem-loop folding throughout the dimer region determined. Another region from PBS to the 5'-terminal 60 residues of the gag gene, partially overlapping the dimer region, likely provides essential elements for the MA(p15) binding reaction, although the presence of either the 3' or 5' neighboring sequences increases the complex-forming efficiency significantly, and each of the substructures predicted within the core region has, if any, only very weak affinity to MA(p15). These in vitro characterizations of the BLV RNA may reflect general features of the specific protein-RNA interaction in the packaging events of various retroviruses. 5'-terminal folded structures of retroviral RNA molecules and their biological activities are discussed.

Base Sequence↗

PubMed1995

c-otx2 is expressed in two different phases of gastrulation and is sensitive to retinoic acid treatment in chick embryo.

We cloned the chick homologue of the mouse Otx2 gene, c-otx2, and analyzed its expression pattern during gastrulation. During mouse embryogenesis, Otx2 expression is first detected in the entire epiblast and after the formation of the primitive streak becomes confined to the most anterior region of the embryo corresponding to presumptive fore- and mid-brain. Similarly, two distinct phases of c-otx2 expression were observed in the chick. c-otx2 transcripts were first detected in the unincubated egg and up to stage XIII, in all epiblast, and forming hypoblast and mesoblast cells. During primitive streak progression, c-otx2 expression becomes progressively restricted to anterior regions and is mainly associated with Hensen's node. When the extension of the streak is maximal, transcripts are only found in Hensen's node. A second phase of c-otx2 expression starts during streak regression. c-otx2 transcripts are lost from the node and present in higher abundance in anterior neuroectoderm and mesendoderm, with the exception of forming notochord and floor plate. The first phase of expression bears strong similarity with that of c-gsc, a gene shown to be a marker for cells that have organizer activity in the chick. Therefore, we compared the expression of the two genes by double staining on the same embryo. This analysis demonstrated that c-otx2 is transcribed first and its expression in the hypoblast precedes that of c-gsc. On the other hand, c-gsc is an earlier marker of primitive streak cells. The expression domains of the two genes transiently overlap in Hensen's node and anterior mesendoderm, whereas only c-otx2 is expressed in neuroectodermal areas. The second phase of c-otx2 expression is sensitive to an early treatment with retinoic acid. This treatment abolishes c-otx2 expression in mesendoderm and restricts it to most anterior regions in the forming neural plate. In conclusion, our results suggest that c-otx2 expression is first associated with cells with an anterior mesendoderm fate and subsequently extends to anterior neuroectoderm.

Amino Acid Sequence↗

PubMed1983

The external transcribed spacer and preceding region of Xenopus borealis rDNA: comparison with the corresponding region of Xenopus laevis rDNA.

We report sequence data from a cloned rDNA unit from Xenopus borealis, extending leftwards from the 18S gene to overlap a region previously sequenced by R. Bach, B. Allet and M. Crippa (Nucleic Acids Research 9, 5311-5330). Comparison with data from other species of Xenopus leads to the inference that the transcription initiation site in X.borealis is in the newly sequenced region and not, as was previously thought, in the region sequenced earlier. The X.borealis external transcribed spacer thus defined is some 612 nucleotides long, about 100 nucleotides shorter than in X.laevis. The X.borealis and X.laevis external transcribed spacers show a pattern of extensive but interrupted sequence divergence, with a large conserved tract starting about 100 nucleotides downstream from the transcription initiation site and shorter conserved tracts elsewhere. The regions in between the conserved tracts differ in length between the respective external transcribed spacers indicating that insertions and deletions have contributed to their divergence, as previously inferred for the internal transcribed spacers. Much of the overall length difference is in the region flanking the 18S gene, where there are also length microheterogeneities in X.laevis rDNA. As in X.laevis, the transcribed spacer sequences flanking the 18S gene in X.borealis contain no major tracts of mutual complementarity. The accumulated data on transcribed spacers in Xenopus render it unlikely that processing of ribosomal precursor RNA involves interaction between the regions flanking 18S RNA.

Animals↗

PubMed2026

Genome-wide association studies for feed efficiency, production and feeding behavior traits in Canadian purebred Duroc pigs.

This study aimed to identify potential genetic variants and candidate genes associated with feed efficiency (FE), production, and feeding behavior traits in Canadian purebred Duroc pigs. Genome-wide association studies (GWAS) were conducted using 8,861 individuals and an imputed Affymetrix PigGen Canada 50K panel v2.0 using a linear mixed model (LMM) and a Bayesian B model. This analysis used an adjusted P-value threshold (ranging from 6.6 × 10-5 to 1.3 × 10-4) using a false-discovery rate to determine significance. The number of significant SNPs identified for each trait was as follows: average daily gain (ADG, 48), daily feed intake (DFI, 85), feed conversion ratio (FCR, 101), residual feed intake (RFI, 37), residual gain (RG, 64), residual intake and gain (RIG, 55), backfat thickness (BF, 100), loin depth (LD, 6), Kleiber's ratio (KR, 0), total time spent eating per day (TPD, 7), and number of visits to the feeder per day (NVD, 6). Several traits (BF, DFI, FCR, RFI, RG, and RIG) showed strong overlapping signals on chromosomes 7 and 10 with 24 shared significant SNPs, indicating potential shared genetic mechanisms. These traits also had 71 overlapping candidate genes, such as PACSIN1, PTCH1, ADIPOR1, and ITPR3, associated with glucose, lipid, and cholesterol metabolism. Well-known candidate genes in literature associated with growth and fatness such as MC4R and CDH20 were also identified to be associated with ADG, BF, FCR, and DFI in this study. Gene ontology enrichment analysis revealed that a set of the candidate genes were involved in the gonadotropin-releasing hormone (GnRH) and the platelet-derived growth factor (PDGF) signaling pathways. Overall, this study contributed to understanding the genetic architecture and provided a biological foundation for improving FE, production, and feeding behavior traits in Canadian Duroc pigs, facilitating the selection of more efficient pigs.

Sus scrofa↗

PubMed2002

Genome profiles of familial/bilateral and sporadic testicular germ cell tumors.

In order to investigate the genetics of testicular germ cell tumors (TGCTs), we examined 33 TGCTs, including 15 familial/bilateral and 18 sporadic tumors, using comparative genomic hybridization. The frequencies of the histological subtypes were comparable between the two groups. Gains of the whole or parts of chromosome 12 were found in 30 tumors (91%). Furthermore, increased copy number of the whole or parts of chromosomes 7, 8, 17, and X, and decreased copy number of the whole or parts of chromosomes 4, 11, 13, and 18 were observed in > or = 50% of the tumors. Sixteen smallest regions of overlapping changes were delineated on 12 different chromosomes. The chromosomal copy numbers of familial/bilateral and sporadic TGCTs were comparable, suggesting similar genetic pathways to disease in both groups. However, significant differences were observed between the two main histological subgroups. Gains from 15q and 22q were associated with seminomas (P = 0.005 and P = 0.02, respectively), whereas gain of the proximal 17q (17q11.2-21) and high-level amplification from chromosome arm 12p, and losses from 10q were associated with nonseminomas (P < 0.001, P = 0.04, and P = 0.03, respectively).

Adult↗

PubMed2006

Association evidence of schizophrenia with distal genomic region of NOTCH4 in Taiwanese families.

Evidence for association with schizophrenia has been reported for NOTCH4, although results have been inconsistent. Previous studies have focused on polymorphisms in the 5' promoter region and first exon of NOTCH4. Our aim was to test the association of the entire genomic region of NOTCH4 in 218 families with at least two siblings affected by schizophrenia in Taiwan. We genotyped seven single nucleotide polymorphisms (SNPs) of this gene, with average intermarker distances of 5.3 kb. Intermarker linkage disequilibrium (LD) was calculated using gold software, and single-locus and haplotype association analyses were performed using transmit software. We found that the T allele of SNP rs2071285 (P= 0.035) and the G allele of SNP rs204993 (P= 0.0097) were significantly preferentially transmitted to the affected individuals in the single-locus association analysis. The two SNPs were in high LD (D' > 0.8). Trend for overtransmission was shown for the T-G haplotype of the two SNPs to affected individuals (P= 0.053), with the A-A haplotype significantly undertransmitted (P= 0.034). The associated region distributed across the distal portion of the NOTCH4 gene and overlapped with the genomic region of the G-protein signaling modulator 3 and pre-B-cell leukemia transcription factor 2. In summary, we found modest association evidence between schizophrenia and the distal genomic region of NOTCH4 in this Taiwanese family sample. Further replication for association with the distal genomic region of NOTCH4 is warranted.

Gene Frequency↗

PubMed1994

Targeted disruption within the CD3 zeta/eta/phi/Oct-1 locus in mouse.

To elucidate the role of the CD3 eta subunit of the T cell receptor (TCR) in thymic development, a CD3 eta -/- mouse was generated by gene targeting. Insertion of a neomycin resistance gene into exon 9 of the CD3 zeta/eta/phi locus disrupted expression of CD3 eta and CD3 phi without affecting the expression of CD3 zeta. Little difference was observed between wild type and CD3 eta -/- mice with regard to cellularity or subset composition in thymus and peripheral lymphoid organs. Furthermore, neither alloproliferative responses nor cytotoxic T lymphocyte generation and effector function was affected by the mutation. The effect of the CD3 eta -/- mutation on thymic selection was examined by crossing the CD3 eta knockout animals with anti-HY TCR transgenic animals: the absence of the CD3 eta subunit altered neither positive nor negative selection. Thus, CD3 eta is not required for thymic selection. Of note, the birth rate of the CD3 eta -/- animals was significantly lower than that of wild type or heterozygous animals (P = 0.041-0.002). This unexpected result is probably the consequence of an alteration in mRNA expression of the Oct-1 nuclear transcription factor in CD3 eta -/- animals. The CD3 zeta/eta/phi locus partially overlaps the gene encoding Oct-1 whose transcription is dysregulated by the CD3 eta -/- mutation. Our results clearly underscore the value of characterizing all products of a genetic locus disrupted by gene targeting.

Alleles↗

PubMed2000

Early and dynamic expression of cSfrp1 during chick embryo development.

Secreted frizzled related proteins (SFRPs) are a new class of signalling molecules that appear to antagonise the activity of the Wnt proteins. Here we report the dynamic expression pattern of cSfrp1, a new member of this family, at early stages of chick embryo development. cSfrp1 transcripts are first detected at pre-streak stages throughout the chick blastula but, during early primitive streak formation, expression is restricted to the anterior primitive streak and later to the blastoderm anterior to the Hensen' s node. This pattern of expression overlaps with that of Otx2 and is complementary to that of cWnt8c. During neural plate formation cSfrp1 mRNAs are abundantly localized only to the anterior domain of the embryo but, as neural tube closes, the expression extends caudally. Later, the main sites of expression in the neural tissue are the telencephalic vesicles, the epiphysis, the developing eyes and the ventral hindbrain and neural tube. Additionally, cSfrp1 transcripts were found in the axial and lateral mesoderm, the otic placode, the trigeminal ganglia, the mesoderm of the branchial arches, the developing limb buds, as well as in the mesodermal component of the developing kidney.

Animals↗

PubMed2006

Disentangling information flow in the Ras-cAMP signaling network.

The perturbation of signal-transduction molecules elicits genomic-expression effects that are typically neither restricted to a small set of genes nor uniform. Instead there are broad, varied, and complex changes in expression across the genome. These observations suggest that signal transduction is not mediated by isolated pathways of information flow to distinct groups of genes in the genome. Rather, multiple entangled paths of information flow influence overlapping sets of genes. Using the Ras-cAMP pathway in Saccharomyces cerevisiae as a model system, we perturbed key pathway elements and collected genomic-expression data. Singular value decomposition was applied to separate the genome-wide transcriptional response into weighted expression components exhibited by overlapping groups of genes. Molecular interaction data were integrated to connect gene groups to perturbed signaling elements. The resulting series of linked subnetworks maps multiple putative pathways of information flow through a dense signaling network, and provides a set of testable hypotheses for complex gene-expression effects across the genome.

Cyclic AMP↗

PubMed2004

Characterization of the Drosophila sphingosine kinases and requirement for Sk2 in normal reproductive function.

Sphingosine kinase is a highly conserved enzyme that catalyzes the synthesis of sphingosine 1-phosphate and reduces cellular levels of sphingosine and ceramide. Although ceramide is pro-apoptotic and sphingosine is generally growth-inhibitory, sphingosine 1-phosphate signaling promotes cell proliferation, survival, and migration. Sphingosine kinase is thus in a strategic position to regulate important cell fate decisions which may contribute to normal animal development. To facilitate studies examining the potential role of sphingosine kinase and long chain base metabolism in Drosophila development, we characterized two putative Drosophila sphingosine kinase genes, Sk1 and Sk2. Both genes functionally and biochemically complement a yeast sphingosine kinase mutant, express predominantly cytosolic activities, and are capable of phosphorylating a range of endogenous and non-endogenous sphingoid base substrates. The two genes demonstrate overlapping but distinct temporal and spatial expression patterns in the Drosophila embryo, and timing of expression is consistent with observed changes in long chain base levels throughout development. A null Sk2 transposon insertion mutant demonstrated elevated long chain base levels, impaired flight performance, and diminished ovulation. This is the first reported mutation of a sphingosine kinase in an animal model; the associated phenotypes indicate that Sk1 and Sk2 are not redundant in biological function and that sphingosine kinase is essential for diverse physiological functions in this organism.

Amino Acid Sequence↗

PubMed2003

Role of RopB in growth phase expression of the SpeB cysteine protease of Streptococcus pyogenes.

The Rgg family of transcription regulators is widely distributed among gram-positive bacteria; however, how the members of this family control transcription is poorly understood. In the pathogen Streptococcus pyogenes, the Rgg family member RopB is required for transcription of the gene that encodes the secreted SpeB cysteine protease. Expression of the protease follows distinct kinetics that involves control of transcription in response to the growth phase. In this study, the contribution of RopB to growth phase control was examined. The gene encoding the protease (speB) and ropB are transcribed divergently from a 940-bp intergenic region. Primer extension analyses, in conjunction with reporter fusion studies, revealed that the major region controlling the transcription of both speB and ropB is adjacent to ropB and that the promoters for the two genes likely overlap. Furthermore, it was found that RopB is a DNA-binding protein that specifically binds to sequences in this control region. The interrelationship between ropB and speB expression was further reflected in the observation that transcription of ropB itself is subject to growth phase control. However, while expression of ropB from a promoter expressed during the early logarithmic phase of growth could complement a ropB deletion mutant, ectopic expression of ropB did not uncouple the expression of speB from its growth phase signal. These data implicate other factors in growth phase control and suggest that regulation of ropB expression itself is not the central mechanism of control.

Bacterial Proteins↗

PubMed1996

Promoters for synthesis of the pre-C and pregenomic mRNAs of human hepatitis B virus are genetically distinct and differentially regulated.

Two similar, yet functionally distinct genomic RNAs are transcribed from the DNA genome of the human hepatitis B virus. The pre-C RNAs encode the precore protein which is proteolytically processed to yield e antigen. The pregenomic RNAs encode both the nucleocapsid protein and reverse transcriptase and serve as the templates for viral DNA replication. To determine whether synthesis of these two RNAs is directed from a single or a closely spaced pair of promoters, we introduced point and insertion mutations into the basal elements of the promoter that directs their synthesis. Transcription from these mutants was examined both in cell-free transcription systems derived from hepatoma (HepG2) and nonliver (HeLa) cell lines and by transient transfection of hepatoma cell lines (Huh7 and HepG2). The data from these experiments indicated that synthesis of the pre-C and pregenomic RNAs is directed by two distinct promoters and that the basal elements of these two promoters partially overlap, yet are genetically separable, with each consisting of its own transcriptional initiator and a TATA box-like sequence situated approximately 25 to 30 bp upstream of its sites of initiation. A 15-bp insertion was found to be sufficient to physically separate these two promoters. Furthermore, these two promoters can be differentially regulated, with the transcriptional activator Sp1 specifically activating transcription from the pregenomic promoter and the hepatocyte nuclear factor 4 specifically repressing transcription from the pre-C promoter. Thus, we conclude that the promoters used in synthesis of the pre-C and pregenomic mRNAs are genetically distinct and separately regulated.

Base Sequence↗