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PubMed2002

HUGE: a database for human large proteins identified in the Kazusa cDNA sequencing project.

We have been developing a HUGE database to summarize results from the sequence analysis of human novel large (>4 kb) cDNAs identified in the Kazusa cDNA sequencing project, systematically designated KIAA plus a four-digit number. HUGE currently contains nearly 2000 gene/protein characteristic tables harboring the results of the computer-assisted analysis of the cDNA and the predicted protein sequences together with those of expression profiling and chromosomal mapping. In the updated version of HUGE, we made it possible to compare each KIAA cDNA sequence with the corresponding entry in the human draft genome sequence that was published recently. Approximately 90% of KIAA cDNAs in HUGE can be localized along the human genome for at least half or more of the cDNA's length. Any nucleotide differences between the cDNA and the corresponding genomic sequences are also presented in detail. This new version of HUGE greatly helps us evaluate the completeness of cDNA clones and the accuracy of cDNA/genomic sequences. More interestingly, in some cases, the ability to compare cDNA with genomic sequences allows us to identify candidate sites of RNA editing. HUGE is available on the World Wide Web at http://www.kazusa.or.jp/huge.

Amino Acid Sequence↗
PubMed1977

Purification of cDNA complementary to sea urchin histone mRNA.

Complementary DNA (cDNA) was transcribed from a polyadenylated sea urchin histone mRNA preparation isolated by density gradient centrifugation. By hybridization, this cDNA was shown to be extensively contaminated (85% of hybridizable cDNA) with DNA complementary to RNA derived from the large ribosomal subunit. Purification of a mRNA specific cDNA fraction was achieved by hybridization of purified rRNA to cDNA followed by fractionation on hydroxyapatite. After further purification to remove nonhybridizable cDNA our purified cDNA showed only 8% hybirdization to rRNA.

Animals↗
PubMed1999

Structural characterization of the gene for human histidine-rich glycoprotein, reinvestigation of the 5'-terminal region of cDNA and a search for the liver specific promoter in the gene.

Genomic DNA libraries were screened for the human histidine-rich glycoprotein (HRG) gene and a sequence of 15,499 nucleotides was determined. The gene is composed of 7 exons and 6 introns, and all the exon-intron boundaries match the consensus GT/AG sequence for donor and acceptor splice sites. Each of cystatin-like domains I and II of HRG is encoded by three exons, exons I to III and exons IV to VI, respectively, like those of other members of the cystatin superfamily. The entire C-terminal half of the molecule is encoded by the largest exon, VII. The first 103 nucleotides of the cDNA sequence reported for human HRG [Koide, T., Foster, D., Yoshitake, S. , and Davie, E.W. (1986) Biochemistry 25, 2220-2225] could not be found in the determined gene sequence. A homology search of this sequence against a database showed the complete matching to a part of the yeast mitochondrial DNA encoding 21S ribosomal RNA. Rapid amplification of cDNA 5' ends (5'-RACE) analysis revealed that the cDNA has multiple 5'-ends and that a possible starting point is nucleotide 104 of the reported cDNA sequence. These results suggest that the first 103 nucleotides of the cDNA sequence reported for human HRG originated from yeast mitochondrial DNA and were incidentally incorporated into the HRG cDNA in the process of the construction of a cDNA library. Various fragments obtained on restriction endonuclease digestion of the 5'-noncoding region of the HRG gene were ligated to the chloramphenicol acetyltransferase (CAT) gene and then transfected into HepG2 and 293 cells to analyze the promoter activity. The sequence between -262 and -21 from the putative translation initiation site supported the expression of CAT in HepG2 cells but not in 293 cells, suggesting that this segment promotes the liver-specific transcription of the human HRG gene.

5' Untranslated Regions↗
PubMed1991

Hamster cytochrome P-450 IA gene family, P-450IA1 and P-450IA2 in lung and liver: cDNA cloning and sequence analysis.

Two cDNA clones, 2C19 and 4C1, were isolated from a lung cDNA library of 3-methylcholanthrene (MC)-treated hamster by using rat P-450c cDNA as a probe. The cDNA determined from 2C19 and 4C1 was 2,916 bp long and contained an entire coding region for 524 amino acids with a molecular weight of 59,408. The deduced amino acid sequence showed a 85% identity with that of rat P-450c indicating 2C19 and 4C1 encode the hamster P-450IA1 protein. Another cDNA clone, designated H28, was isolated from a MC-induced hamster liver cDNA library by using the hamster lung 2C19 or 4C1 cDNA clone as a probe. H28 was 1,876 bp long and encoded a polypeptide of 513 amino acids with a molecular weight of 58,079. The N-terminal 20 residues deduced from nucleotide sequence of H28 were identical to those determined by sequence analysis of purified hamster hepatic P-450MCI. The high similarity of the nucleotide and deduced amino acid sequences between H28 and P-450IA2 of other species indicated that H28 encoded a P-450 protein which belongs to the P-450IA2 family. Northern blot analysis revealed that the mRNAs for hamster P-450IA1 and IA2 were about 2.9 and 1.9 kb long, respectively. Hamster P-450IA1 mRNA was induced to the same level in lungs as in livers by MC treatment, whereas hamster P-450IA2 mRNA was induced and expressed only in hamster liver.

Amino Acid Sequence↗
PubMed1994

Purification and cDNA cloning of human liver CYP4A fatty acid omega-hydroxylase.

Laurate omega-hydroxylase activity of human liver microsomes was strongly inhibited by an antibody against rabbit fatty acid omega-hydroxylase P450 4A5, and Western blot analysis with this antibody showed the presence of two immunochemically related proteins with apparent molecular weights of approximately 50 and 52 kDa in all of 14 human liver specimens examined. A fatty acid omega-hydroxylase (designated P450HL omega) was purified to a specific content of 15 nmol of P450/mg of protein from microsomes of a single human liver on the basis of its laurate omega-hydroxylase activity and its reactivity with the P450 4A5 antibody. This P450HL omega showed an apparent molecular weight of 52 kDa on SDS-PAGE. Furthermore, a cDNA clone (designated HL24) has been isolated from a human liver cDNA library by using the cDNA for P450 4A5 as a probe. The sequence of residues 5 through 25 deduced from cDNA HL24 was identical to the NH2-terminal amino acid sequence of P450HL omega except for one undetermined residue. This cDNA encoded a protein of 519 amino acids with a molecular weight of 59,347. The amino acid sequence predicted from the cDNA showed 82% identity with that of P450 4A5. Northern blot analysis showed that the mRNA hybridized to the cDNA is expressed in the human liver and kidney. (ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Rapid cDNA sequencing in combination with RNA expression studies in mice identifies a large number of male germ cell-specific sequence tags.

A large number of cDNA clones were isolated from an adult mouse testis cDNA library and partially sequenced. Sequence comparisons revealed that many of them displayed similarities to genes previously identified only in invertebrates and lower eukaryotes, including, e.g., one cDNA clone related to the Drosophila melanogaster suppressor of forked gene. Other cDNA clones were found to be related, but not identical, to rodent genes involved in a variety of cellular activities, e.g., signal transduction and tumor development. The RNA expression patterns of 258 cDNA clones were analyzed through use of Northern blot methodology. Forty-two novel cDNA clones were found to be expressed only in male germ cells, the majority of them during spermiogenesis. One male germ cell-specific mouse cDNA clone was found to be similar to tektin A1, a protein known to interact with the flagellar microtubules of sea urchin sperm.

Amino Acid Sequence↗
PubMed1998

Lumbar spine fusion by local gene therapy with a cDNA encoding a novel osteoinductive protein (LMP-1).

STUDY DESIGN: A posterior arthrodesis animal model using local expression of a newly discovered osteoinductive protein delivered in bone marrow cells. OBJECTIVE: To introduce the concept of local gene therapy and determine its feasibility for achieving lumbar spine fusion using a gene for a novel osteoinductive protein: LIM Mineralization Protein-1 (LMP-1). SUMMARY OF BACKGROUND DATA: Extensive work is currently underway to improve the healing success and morbidity associated with the gold standard bone-grafting material of autogenous iliac crest. As a result, alternative osteoinductive proteins and new delivery methods warrant investigation. The authors' laboratory recently identified a novel gene that had osteoinductive capacity in vitro and is therefore a candidate for a new in vivo osteoinductive agent. METHODS: Single-level posterior lumbar and thoracic arthrodesis was attempted in 14 athymic rats. The graft material, which consisted of a devitalized bone matrix (no osteoinductive activity) soaked with 0.75 to 1.5 million bone marrow cells, was inserted with the dorsal spine exposed. In each rat, one site received marrow cells transfected with the cDNA encoding a novel osteoinductive protein. At the other site for a control, the marrow cells were transfected with the reverse copy of the cDNA that did not express any protein. Transfection of marrow cells for 2 hours was accomplished using the mammalian expression vector pCMV2. Rats were killed after 4 weeks, and the spines were evaluated by manual palpation, radiographs, and nondecalcified histology. RESULTS: In the pivotal experiment, successful spine fusion was obtained in 9/9 (100%) of the sites receiving marrow cells transfected with the active LMP-1 cDNA and in 0/9 (0%) of the sites receiving marrow cells transfected with the reverse (inactive) LMP-1 cDNA. Radiographs and histology confirmed the manual palpation results, demonstrating controlled new bone formation in the carrier and marrow transfected with the active LMP-1 cDNA and essentially no bone induction in the sites treated with marrow cells that did not express the protein. CONCLUSIONS: These data confirm that local delivery of the novel LMP-1 cDNA using bone marrow cells is feasible in vivo. Furthermore, these results demonstrate that posterior thoracic or lumbar spine fusion can be achieved in rats by local delivery of the LMP-1 cDNA.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Isolation and characterization of a new rat P450 (CYP3A18) cDNA encoding P450(6)beta-2 catalyzing testosterone 6 beta- and 16 alpha-hydroxylations.

A cytochrome P450 cDNA, encoding a new form of CYP3A protein, was isolated from a liver cDNA library of a male rat using anti-P450(6)beta-1 and anti-P450(6)beta-2 antibodies and the CYP3A2 cDNA. The cDNA (CYP3A18 cDNA) consisted of 1987 nucleotides, in which were contained an open reading frame of 1491 bp (corresponding to 497 amino acids), 5'-(59 bp) and 3'-noncoding regions (437 bp). The deduced amino acid sequence of CYP3A18 cDNA was completely identical in the first 27 N-terminal residues of P450(6)beta-2 previously isolated by us (Nagata et al. J Biochem 1990: 107, 718-725) from livers of rats treated with dexamethasone, and also shared higher extents of similarity with hamster CYP3A10 (78.5%) than with rat CYP3As previously sequenced (66.3-69.3%). Northern blot analyses indicated a male-dominant expression of this new CYP3A mRNA and enhanced expression in dexamethasone-or pregnenolone-16 alpha-carbonitrile (PCN)-treated, but not phenobarbital-or 3-methylcholanthrene-treated rats. Expressed CYP3A18 protein in COS-1 cells migrated at a position identical to that of purified P450(6)beta-2 on sodium dodecyl sulfate-acrylamide gel electrophoresis and catalyzed 16 beta- and 6 alpha-hydroxylations of testosterone. In contrast to CYP3A1 and CYP3A2, cytochrome b5 was not essential for maximal catalytic activities of recombinant CYP3A18 protein. These results, together with ontogenic profiles of CYP3A18 mRNA and P450(6)beta-2 protein, indicate that the newly isolated CYP3A18 cDNA encodes P450(6)beta-2 in rat liver.

Amino Acid Sequence↗
PubMed2002

Characterization of a human immunodeficiency virus type 1 pre-integration complex in which the majority of the cDNA is resistant to DNase I digestion.

The human immunodeficiency virus type 1 (HIV-1) pre-integration complex (PIC) is a cytoplasmic nucleoprotein structure derived from the core of the virion and is responsible for reverse transcription of viral RNA to cDNA, transport to the nucleus and integration of the cDNA into the genome of the infected target cell. Others have shown by Mu phage-mediated PCR footprinting that only the LTRs of the cDNA of PICs isolated early in infection are protected by bound protein, while the rest of the genome is susceptible to nuclease attack. Here, using DNase I footprinting, we confirmed that the majority of the cDNA of PICs isolated at 8.5 h after infection with cell-free virus was sensitive to digestion with DNase I and that only part of the LTRs (approximately 6% of the total cDNA) was protected. However, PICs isolated 90 min later (at 10 h post-infection) were very different in that the majority (approximately 90%) of cDNA was protected from nuclease degradation. These late PICs were integration active in vitro. We conclude that HIV-1 has at least two types of PIC, an early PIC characterized by protein bound only at the LTRs, and a late, and possibly more mature form, in which protein is bound along the length of the cDNA.

Cell Nucleus↗
PubMed2000

Detection of spurious interruptions of protein-coding regions in cloned cDNA sequences by GeneMark analysis.

cDNA is an artificial copy of mRNA and, therefore, no cDNA can be completely free from suspicion of cloning errors. Because overlooking these cloning errors results in serious misinterpretation of cDNA sequences, development of an alerting system targeting spurious sequences in cloned cDNAs is an urgent requirement for massive cDNA sequence analysis. We describe here the application of a modified GeneMark program, originally designed for prokaryotic gene finding, for detection of artifacts in cDNA clones. This program serves to provide a warning when any spurious split of protein-coding regions is detected through statistical analysis of cDNA sequences based on Markov models. In this study, 817 cDNA sequences deposited in public databases by us were subjected to analysis using this alerting system to assess its sensitivity and specificity. The results indicated that any spurious split of protein-coding regions in cloned cDNAs could be sensitively detected and systematically revised by means of this system after the experimental validation of the alerts. Furthermore, this study offered us, for the first time, statistical data regarding the rates and types of errors causing protein-coding splits in cloned cDNAs obtained by conventional cloning methods.

Alternative Splicing↗
PubMed1994

Cloning and sequence analysis of a cDNA encoding ferric leghemoglobin reductase from soybean nodules.

A cDNA encoding soybean (Glycine max [L.] Merr) ferric leghemoglobin reductase (FLbR), an enzyme that is postulated to play an important role in maintaining leghemoglobin in its functional ferrous state, has been cloned and characterized. A group of highly degenerate oligonucleotides deduced from the N-terminal amino acid sequence of FLbR was used to prime the polymerase chain reaction (PCR) on soybean nodule mRNA and cDNA. A full-length clone of FLbR cDNA was isolated by screening a lambda gt11 soybean nodule cDNA library using the specific PCR-amplified FLbR cDNA fragment as a probe. The cDNA contained about 1.8 kb and had a coding sequence for 523 amino acids with a predicted molecular mass of 55,729 D, which included a putative 30-residue signal peptide and a 493-residue mature protein. Computer-aided analysis of the deduced FLbR amino acid sequence showed considerable homology (varied from 20-50% with enzymes and species) to dihydrolipoamide dehydrogenase (EC 1.8.1.4), glutathione reductase (EC 1.6.4.2), mercuric reductase (EC 1.16.1.1), and trypanothione reductase (EC 1.6.4.8) in a superfamily of pyridine nucleotide-disulfide oxidoreductases from various organisms. Northern blot analysis using FLbR cDNA as a probe showed that the FLbR gene was expressed in soybean nodules, leaves, roots, and stems, with a greater level of expression in nodules and leaves than in roots and stems. Southern blot analysis of the genomic DNA showed the presence of two homologous FLbR genes in the soybean genome.

Amino Acid Sequence↗
PubMed1990

Characterization of a cDNA Encoding Lens culinaris Glycolate Oxidase and Developmental Expression of Glycolate Oxidase mRNA in Cotyledons and Leaves.

mRNA obtained from green leaves of lentil (Lens culinaris) was used to construct a cDNA library in phage lambdagt11. The cDNA library was screened with antibodies raised against lentil glycolate oxidase and catalase. Clone CL 1 containing the full-length sequence complementary to glycolate oxidase mRNA was characterized and sequenced. In addition, a 800-base pair catalase cDNA clone was sequenced. To prove the correlation of cDNA insert in CL 1 with glycolate oxidase, the cDNA was transcribed in vitro. The mRNA was translated in vitro yielding a 43 kilodalton protein immunoprecipitable with anti-glycolate oxidase serum. Nucleotide sequences of lentil cDNA and spinach cDNA were 86% identical. Lentil glycolate oxidase was characterized by a C-terminal sequence -P-R-A-L-P-R-L. The expression of glycolate oxidase mRNA in cotyledons, leaves and roots was compared with that of catalase. In leaves, the relative amount of glycolate oxidase mRNA increased during the first 2 days of greening, but decreased later, and was hardly detectable during senescence. In cotyledons of germinating seeds, the level of glycolate oxidase mRNA was markedly lower than the catalase mRNA.

Journal Article↗
PubMed1990

Partial cDNA cloning of the 230-kD mouse bullous pemphigoid antigen by use of a human monoclonal anti-basement membrane zone antibody.

A cDNA clone with the coding sequence for the 230-kD bullous pemphioid (BP) antigen was isolated from a mouse cDNA expression library by using an anti-basement membrane zone human monoclonal antibody (MoAb-5E). The lambda gt11 cDNA expression library was constructed from poly(A)+RNA from the mouse epidermal cell line, Pam cells, by random priming. 1.5 X 10(5) recombinant clones were screened by immunostaining with MoAb-5E and one positive clone (BPM1) was obtained. All of the ten BP sera but none of the five normal or seven pemphigus sera tested reacted with the fusion protein produced by BPM1. The size of the cDNA was 3.2 kb. Northern blot analysis indicated that BPM1 cDNA hybridized to a mRNA of about 9 kb, which is large enough to encode for a 230-kD protein. DNA sequencing demonstrated a 2,991-bp open reading frame encoding a peptide of 115 kD. Sequence comparison between mouse and human cDNA clones revealed that the 230-kD BP antigen was well conserved during evolution except for the carboxyl terminus. Highly conserved and hydrophilic regions in the molecule were considered to be good candidates for antigenic determinants. This cDNA clone will be useful not only for diagnosis of BP, e.g., enzyme-linked immunosorbent assay using recombinant proteins or synthetic peptides as antigens, but also for pathophysiologic study in which mouse models of BP might be used.

Amino Acid Sequence↗
PubMed1996

Cloning and sequencing of a dextranase-encoding cDNA from Penicillium minioluteum.

A cDNA from Penicillium minioluteum HI-4 encoding a dextranase (1,6-alpha-glucan hydrolase, EC 3.2.1.11) was isolated and characterized. cDNA clones corresponding to genes expressed in dextran-induced cultures were identified by differential hybridization. Southern hybridization and restriction mapping analysis of selected clones revealed four different groups of cDNAs. The dextranase cDNA was identified after expressing a cDNA fragment from each of the isolated groups of cDNA clones in the Escherichia coli T7 system. The expression of a 2 kb cDNA fragment in E. coli led to the production of a 67 kDa protein which was recognized by an anti-dextranase polyclonal antibody. The cDNA contains 2109 bp plus a poly(A) tail, coding for a protein of 608 amino acids, including 20 N-terminal amino acid residues which might correspond to a signal peptide. There was 29% sequence identity between the P. minioluteum dextranase and the dextranase from Arthrobacter sp. CB-8.

Amino Acid Sequence↗
PubMed1999

Rescue of Newcastle disease virus from cloned cDNA: evidence that cleavability of the fusion protein is a major determinant for virulence.

A full-length cDNA clone of Newcastle disease virus (NDV) vaccine strain LaSota was assembled from subgenomic overlapping cDNA fragments and cloned in a transcription plasmid between the T7 RNA polymerase promoter and the autocatalytic hepatitis delta virus ribozyme. Transfection of this plasmid into cells that were infected with a recombinant fowlpoxvirus that expressed T7 RNA polymerase, resulted in the synthesis of antigenomic NDV RNA. This RNA was replicated and transcribed by the viral NP, P, and L proteins, which were expressed from cotransfected plasmids. After inoculation of the transfection supernatant into embryonated specific-pathogen-free eggs, infectious virus derived from the cloned cDNA was recovered. By introducing three nucleotide changes in the cDNA, we generated a genetically tagged derivative of the LaSota strain in which the amino acid sequence of the protease cleavage site (GGRQGR downward arrowL) of the fusion protein F0 was changed to the consensus cleavage site of virulent NDV strains (GRRQRR downward arrowF). Pathogenicity tests in day-old chickens showed that the strain derived from the unmodified cDNA was completely nonvirulent (intracerebral pathogenicity index [ICPI] = 0.00). However, the strain derived from the cDNA in which the protease cleavage site was modified showed a dramatic increase in virulence (ICPI = 1.28 out of a possible maximum of 2.0). Pulse-chase labeling of cells infected with the different strains followed by radioimmunoprecipitation of the F protein showed that the efficiency of cleavage of the F0 protein was greatly enhanced by the amino acid replacements. These results demonstrate that genetically modified NDV can be recovered from cloned cDNA and confirm the supposition that cleavage of the F0 protein is a key determinant in virulence of NDV.

Amino Acid Sequence↗
PubMed2000

Retroviral cDNA integration: stimulation by HMG I family proteins.

To replicate, a retrovirus must synthesize a cDNA copy of the viral RNA genome and integrate that cDNA into a chromosome of the host. We have investigated the role of a host cell cofactor, HMG I(Y) protein, in integration of human immunodeficiency virus type 1 (HIV-1) and Moloney murine leukemia virus (MoMLV) cDNA. Previously we reported that HMG I(Y) cofractionates with HIV-1 preintegration complexes (PICs) isolated from freshly infected cells. PICs depleted of required components by treatment with high concentrations of salt could be reconstituted by addition of purified HMG I(Y) in vitro. Here we report studies using immunoprecipitation that indicate that HMG I(Y) is associated with MoMLV preintegration complexes. In mechanistic studies, we show for both HIV-1 and MoMLV that each HMG I(Y) monomer must contain multiple DNA binding domains to stimulate integration by HMG I(Y)-depleted PICs. We also find that HMG I(Y) can condense model HIV-1 or MoMLV cDNA in vitro as measured by stimulation of intermolecular ligation. This reaction, like reconstitution of integration, depends on the presence of multiple DNA binding domains in each HMG I(Y) monomer. These data suggest that binding of multivalent HMG I(Y) monomers to multiple cDNA sites compacts retroviral cDNA, thereby promoting formation of active integrase-cDNA complexes.

DNA, Complementary↗
PubMed2004

Lv1 inhibition of human immunodeficiency virus type 1 is counteracted by factors that stimulate synthesis or nuclear translocation of viral cDNA.

Human immunodeficiency virus type 1 (HIV-1) cDNA synthesis is inhibited in cells from some nonhuman primates by an activity called Lv1. Sensitivity to restriction by Lv1 maps to a region of the HIV-1 CA required for interaction with the cellular protein cyclophilin A. A similar antiviral activity in mammalian cells, Ref1, inhibits reverse transcription of murine leukemia virus (MLV), but only with viral strains bearing N-tropic CA. Disruption of the HIV-1 CA-cyclophilin A interaction inhibits Lv1 restriction in some cells and, paradoxically, seems to render HIV-1 sensitive to Ref1. Lv1 and Ref1 activities are overcome by high-titer infection and are saturable with nonreplicating, virus-like particles encoded by susceptible viruses. Two compounds that disrupt mitochondrial membrane potential, As(2)O(3) and m-Cl-CCP, reduce Ref1 activity. Here we show that these drugs, as well as a third compound with similar effects on mitochondria, PK11195, attenuate Lv1 activity in rhesus macaque and African green monkey cells. Effects of PK11195 and virus-like particles on HIV-1 infectivity in these cells were largely redundant, each associated with increased HIV-1 cDNA. Comparison of acutely infected macaque and human cells suggested that, in addition to effects on cDNA synthesis, Lv1 inhibits the accumulation of nuclear forms of HIV-1 cDNA. Disruption of the HIV-1 CA-cyclophilin A interaction caused a minimal increase in total viral cDNA but increased the proportion of viral cDNA in the nucleus. Consistent with a model in which Lv1 inhibits both synthesis and nuclear translocation of HIV-1 cDNA, complete suppression of macaque or African green monkey Lv1 was achieved by the additive effect of factors that stimulate both processes.

Active Transport, Cell Nucleus↗
PubMed1996

The human clotting factor VIII cDNA contains an autonomously replicating sequence consensus- and matrix attachment region-like sequence that binds a nuclear factor, represses heterologous gene expression, and mediates the transcriptional effects of sodium butyrate.

Expression of the human blood-clotting factor VIII (FVIII) cDNA is hampered by the presence of sequences located in the coding region that repress transcription. We have previously identified a 305-bp fragment within the FVIII cDNA that is involved in the repression (R.C. Hoeben, F.J. Fallaux, S.J. Cramer, D.J.M. van den Wollenberg, H. van Ormondt, E. Briet, and A.J. van der Eb, Blood 85:2447-2454, 1995). Here, we show that this 305-bp region of FVIII cDNA contains sequences that resemble the yeast (Saccharomyces cerevisiae) autonomously replicating sequence consensus. Two of these DNA elements coincide with AT-rich sequences that are often found in matrix attachment regions or scaffold-attached regions. One of these elements, consisting of nucleotides 1569 to 1600 of the FVIII cDNA (nucleotide numbering is according to the system of Wood et al. (W.I. Wood, D.J. Capon, C.C. Simonsen, D.L. Eaton, J. Gitschier, D. Keyt, P.H. Seeburg, D.H. Smith, P. Hollingshead, K.L. Wion, et al., Nature [London] 312:330-337,1984), binds a nuclear factor in vitro but loses this capacity after four of its base pairs have been changed. A synthetic heptamer of this segment can repress the expression of a chloramphenicol acetyltransferase (CAT) reporter gene and also loses this capacity upon mutation. Furthermore, we demonstrate that repression by FVIII sequences can be relieved by sodium butyrate. We demonstrate that the synthetic heptamer (FVIII nucleotides 1569 to 1600), when placed upstream of the Moloney murine leukemia virus long terminal repeat promoter that drives the CAT reporter, can render the CAT reporter inducible by butyrate. This effect was absent when the same element was mutated. The stimulatory effect of butyrate could not be attributed to butyrate-responsive elements in the studied long terminal repeat promoters. Our data provide a functional characterization of the sequences that repress expression of the FVIII cDNA. These data also suggest a link between transcriptional repression by FVIII cDNA elements and the stimulatory effect of butyrate on FVIII cDNA expression.

Base Sequence↗