Homologous recombination of monkey alpha-satellite repeats in an in vitro simian virus 40 replication system: possible association of recombination with DNA replication.

To study homologous recombination between repeated sequences in an in vitro simian virus 40 (SV40) replication system, we constructed a series of substrate DNAs that contain two identical fragments of monkey alpha-satellite repeats. Together with the SV40-pBR322 composite vector encoding Apr and Kmr, the DNAs also contain the Escherichia coli galactokinase gene (galK) positioned between two alpha-satellite fragments. The alpha-satellite sequence used consists of multiple units of tandem 172-bp sequences which differ by microheterogeneity. The substrate DNAs were incubated in an in vitro SV40 DNA replication system and used to transform the E. coli galK strain DH10B after digestion with DpnI. The number of E. coli galK Apr Kmr colonies which contain recombinant DNAs were determined, and their structures were analyzed. Products of equal and unequal crossovers between identical 172-bp sequences and between similar but not identical (homeologous) 172-bp sequences, respectively, were detected, although those of the equal crossover were predominant among all of the galK mutant recombinants. Similar products were also observed in the in vivo experiments with COS1 cells. The in vitro experiments showed that these recombinations were dependent on the presence of both the SV40 origin of DNA replication and SV40 large T antigen. Most of the recombinant DNAs were generated from newly synthesized DpnI-resistant DNAs. These results suggest that the homologous recombination observed in this SV40 system is associated with DNA replication and is suppressed by mismatches in heteroduplexes formed between similar but not identical sequences.

Characterization of DNA synthesis and DNA-dependent ATPase activity at a restrictive temperature in temperature-sensitive tsFT848 cells with thermolabile DNA helicase B.

A temperature-sensitive mutant defective in DNA replication, tsFT848, was isolated from the mouse mammary carcinoma cell line FM3A. In mutant cells, the DNA-dependent ATPase activity of DNA helicase B, which is a major DNA-dependent ATPase in wild-type cells, decreased at the nonpermissive temperature of 39 degrees C. DNA synthesis in tsFT848 cells at the nonpermissive temperature was analyzed in detail. DNA synthesis measured by incorporation of [3H]thymidine decreased to about 50% and less than 10% of the initial level at 8 and 12 h, respectively. The decrease in the level of thymidine incorporation correlated with a decrease in the number of silver grains in individual nuclei but not with the number of cells with labeled nuclei. DNA fiber autoradiography revealed that the DNA chain elongation rate did not decrease even after an incubation for 10 h at 39 degrees C, suggesting that initiation of DNA replication at the origin of replicons is impaired in the mutant cells. The decrease in DNA-synthesizing ability coincided with a decrease in the level of the DNA-dependent ATPase activity of DNA helicase B. Partially purified DNA helicase B from tsFT848 cells was more heat sensitive than that from wild-type cells. Inactivation of DNA-dependent ATPase activity of DNA helicase B from mutant cells was considerably reduced by adding DNA to the medium used for preincubation, indicating that the DNA helicase of mutant cells is stabilized by binding to DNA.

Characterization of chromosome aberrations induced by incubation at a restrictive temperature in the mouse temperature-sensitive mutant tsFT20 strain containing heat-labile DNA polymerase alpha.

tsFT20 cells derived from a mouse mammary carcinoma cell line, FM3A, which has temperature-sensitive DNA polymerase alpha activity (Y. Murakami, H. Yasuda, H. Miyazawa, F. Hanaoka, and M. Yamada, Proc. Natl. Acad. Sci. USA, 82:1761-1765, 1985) were rapidly committed to death after temperature upshift to 39 degrees C. tsFT20 cells synchronized in S phase were more sensitive to the restrictive temperature than exponentially growing cells. In order to gain insight into the processes from the interruption of DNA synthesis to cell death, we analyzed chromosome aberrations induced in tsFT20 cells which had been incubated for 2 or 4 h at the restrictive temperature and then cultured at the permissive temperature. The majority of metaphase cells showed extensive chromosome aberrations such as chromatid gaps, breaks, and exchanges; chromosome pulverizations; their mixed types; and ring chromosomes. Analyses with the use of cell synchronization and autoradiography revealed that chromosome aberrations were induced only in the cells which synthesized DNA during incubation at 39 degrees C. We classified the chromosome aberrations into five types: gap or break type; exchange type; pulverization type; complex type; and ring type. The temporal order of the appearance of these types of chromosome aberrations was found to be the above described order. It was further found that cycloheximide dramatically repressed the induction of chromosome aberrations, and metaphases with many chromosome aberrations exhibited a large number of sister chromatid exchanges. These results indicate that abnormal cessation of DNA replication in tsFT20 cells at the restrictive temperature due to the inactivation of DNA polymerase alpha results in cell death via induction of double-strand breaks which lead to chromosome aberrations as well as sister chromatid exchanges.

Genomic structure, chromosomal localization and identification of mutations in the xeroderma pigmentosum variant (XPV) gene.

The xeroderma pigmentosum variant (XP-V) is one of the most common forms of this cancer-prone syndrome. XP groups A through G are characterized by defective nucleotide excision repair, whereas the XP-V phenotype is proficient in this pathway. The XPV gene encodes DNA polymerase eta, which catalyzes an accurate translesion synthesis, indicating that the XPV gene contributes tumor suppression in normal individuals. Here we describe the genomic structure and chromosomal localization of the XPV gene, which includes 11 exons covering the entire coding sequence, lacks a TATA sequence in the upstream region of the transcription-initiation, and is located at the chromosome band 6p21.1-6p12. Analyses of patient-derived XP-V cell lines strongly suggested that three of four cell lines carried homozygous mutations in the XPV gene. The fourth cell line, XP1RO, carried heterozygous point mutations in the XPV gene, one of which was located at the splice acceptor site of exon 2, resulting in the omission of exon 2 from the mature mRNA. These findings provide a basis for diagnosis and therapy of XP-V patients.

Synthesis of acyl phosphatidylglycerol from phosphatidylglycerol in Escherichia coli K-12. Evidence for the participation of detergent-resistant phospholipase A and heat-labile membrane-bound factor(s).

The conversion of phosphatidylglycerol to acyl phosphatidylglycerol by extracts of Escherichia coli K-12 strains was examined under various conditions. The maximum rate of conversion was observed at pH 7.2 in the presence of 50% (v/v) diethyl ether and 10 mM CaCl2. This conversion was found to involve two sequential reactions: (1) The formation of 2-acyl glycerophosphoglycerol and 2-acyl glycerophosphoethanolamine from phosphatidylglycerol and phosphatidylethanolamine, respectively, by detergent-resistant phospholipase A in the presence of Ca2+ and (2) transfer of the acyl group of 2-acyl lysophospholipid to phosphatidylglycerol by a heat-labile factor(s) in the presence of diethyl ether. Neither fatty, acid acyl-CoA nor 1-acyl lysophospholipid could act as an acyl donor for phosphatidylglycerol. The heat-labile factor(s) was found in both the inner membrane and supernatant fractions.

Assignment of human membrane-type matrix metalloproteinase-2 (MT2-MMP) gene to 16q12 by FISH and PCR-based human/rodent cell hybrid mapping panel analysis.

A new group of matrix metalloproteinase with a potential membrane domain was reported as membrane-type matrix metalloproteinases (MT-MMPs), and the gene coding for one of them, MT2-MMP was recently cloned from a human lung cDNA library. To predict its physiological functions by the relation to the genetic disorders mapped on the chromosomes, the chromosomal location of the human MT2-MMP gene was examined by fluorescence in situ hybridization (FISH) and PCR-based analysis with human/rodent hybrid cell mapping panels, and it was assigned to 16q12.

Identification of the coding region of Saccharomyces cerevisiae chromosome VI using the computer program GenMark.

We searched the nucleotide sequence of budding yeast Saccharomyces cerevisiae chromosome VI (270 kb) for candidate coding regions, using the computer program GenMark. One hundred and twenty-nine putative genes were identified, which is almost the same as the number of ORFs on this chromosome. Nineteen new putative genes were identified through the GenMark analysis. Most large ORFs were also correctly identified (87% of the predicted putative genes identified by the GenMark (110 of 127) matched the reported ORFs). The new coding regions were mostly small but they were distinguished from the more than 2000 ORFs identified by Genetyx. GenMark did not predict 17 ORFs that were over 300 bp long. As these ORFs include known genes, their sequence context may differ somewhat from that of typical yeast genes. These analyses revealed the high potential of GenMark to identify putative genes from numerous short ORFs and will produce information on the likelihood of their being actual genes.

Poly (ADP-ribose) polymerase inhibits DNA replication by human replicative DNA polymerase alpha, delta and epsilon in vitro.

The influence of poly (ADP-ribose) polymerase (PARP) and poly ADP-ribosylation on DNA synthesis supported by human replicative DNA polymerase (DNA pol) alpha, delta, and epsilon has been examined using the replication system containing poly(dA)4500-oligo(dT)12-18 as the template primer. PARP alone inhibited the pol activities in a dose-dependent manner even in the presence of the accessory factors for DNA pol delta, proliferating cell nuclear antigen (PCNA) and activator 1 (Al; RF-C). Both DNA pol alpha and epsilon activities were decreased approximately 10-fold under the poly ADP-ribosylating condition. In contrast, DNA synthesis by DNA pol delta holoenzyme was not affected by poly ADP-ribosylation like prokaryotic DNA pol's. The analysis of poly(dT) formed by DNA pol alpha and epsilon indicated that poly ADP-ribosylation mainly reduced the frequency of replication. These observations suggest a possibility that PARP acts as a negative regulator for the initiation of DNA replication upon cellular DNA damage.

Efficient replication of polyomavirus DNA in a cell-free system supplemented with Escherichia coli single-stranded DNA binding protein, which exhibits species-specificity in the requirement for DNA polymerase alpha-primase.

We established a modified cell-free system for polyomavirus (PyV) DNA replication, which was supplemented with Escherichia coli single-stranded DNA binding protein (SSB). DNA synthesis in this system was enhanced by 1.4- to over 15-fold depending upon the amount of cell extracts contained in the reaction mixture. By supplementing with E. coli SSB, we were able to reduce the amount of cell extracts in the reaction mixture, and to lower the concentrations of creatine phosphate and Tris, rendering this system more resistant to salts than the conventional PyV DNA replication system. The modified system was characterized using mutant cell extracts which had heat-inactivated DNA polymerase alpha. DNA synthesis in the system was dependent on PyV T antigen, the PyV origin of DNA replication, mutant cell extracts, and DNA polymerase alpha-primase complex purified from wild-type cells. The DNA polymerase alpha-primase complex was not replaced by DNA polymerase alpha, indicating that this system requires a functional DNA polymerase alpha-primase complex. This system exhibited species-specificity in the requirement for DNA polymerase alpha-primase; only mouse DNA polymerase alpha-primase but not human DNA polymerase alpha-primase functioned in this system.

Purification and characterization of lysophospholipase L2 of Escherichia coli K-12.

Lysophospholipase L2, which is bound to the inner membrane of Escherichia coli K-12, was produced in a large amount in cells bearing its cloned structural gene. Starting from these cells, the lysophospholipase L2 was purified approximately 700-fold to near homogeneity by solubilization with KCl, ammonium sulfate fractionation, chromatofocusing in the presence of a zwitterionic detergent, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), and heparin-Sepharose affinity column chromatography. The final preparation showed a single protein band with a molecular weight of 38,500 daltons in SDS-polyacrylamide gel electrophoresis. The amino acid sequence of the NH2-terminal portion of the purified enzyme was determined. It was in complete agreement with that deduced from the nucleotide sequence of the structural gene, pldB [Kobayashi, T., Kudo, I., Karasawa, K., Mizushima, H., Inoue, K., & Nojima, S. (1985) J. Biochem. 98, 1017-1025.] The purified enzyme hydrolyzes 2-acyl glycerophosphoethanolamine (GPE) and 2-acyl glycerophosphocholine (GPC) more effectively than 1-acyl GPE and 1-acyl GPC, but does not attack diacylphospholipids. The enzyme also catalyzes the transfer of an acyl group from lysophospholipid to phosphatidylglycerol for formation of acyl phosphatidylglycerol. The acyl group was more effectively transferred from 2-acyl lysophospholipid than from the 1-acyl derivative. This enzyme was heat-labile and was inactivated at 55 degrees C within 5 min. The present paper shows clearly that lysophospholipase L2 is a different enzyme protein from lysophospholipase L1 which was formerly purified from the supernatant of the wild strain of E. coli K-12 homogenates [Doi, O. & Nojima, S. (1975) J. Biol. Chem. 250, 5208-5214].

Cloning and characterization of novel gene, DCRR1, expressed from Down's syndrome critical region of human chromosome 21q22.2.

The new gene, DCRR1, from the proximal part of the Down's syndrome critical region (DCR) was identified by the GRAIL analysis of the 97-kb nucleotide sequence of two P1 DNAs and the cDNA for DCRR1 gene was cloned. A 7.36-kb cDNA encodes the imcompleted open reading frame composed of 1941 amino acid residues (220.2 kDa). The deduced amino acid sequence contains the conserved domain for protein phosphatases at the N-terminus. The domain encoding the rod-like tail of a myosin heavy chain was also found near the C-terminal region besides the signature for an actin binding protein, profilin, suggesting its possible role as a microtuble-associated protein. Two different sizes (7.9 and 9.0 kb) of mRNAs were detected in the poly(A)+ RNA from abundant tissues by the Northern analysis. The smaller transcript was only transcribed at a high level in the testis. The imbalance of the DCRR1 gene dosage may contibute to the pathogenesis of Down's syndrome.

1.8-megabases fine physical map encompassing IFNAR and AML1 loci on human chromosome 21q22.1.

A long-range restriction map of the 1.8-megabases (mb) region encompassing the area between the interferon-alpha receptor and the acute myelogenous leukemia loci on human chromosome 21q22.1 was constructed after analysis of both the contiguous yeast artificial chromosome (YAC) clones and genomic DNA. Analysis of pulsed-field gel electrophoresis of lymphoblastoid DNA digested with three rare-cutting enzymes, Not I, Mlu I, and Nru I, revealed the positions of 17 markers on each restriction map. The 1.8-mb YAC contig that covers this region was obtained through YAC walking mediated by sequence-tagged sites (STSs), with 29 STSs including 12 newly generated YAC end-specific STSs. The consensus restriction map from 15 overlapping YACs and the positioning of the STS markers on each clone allowed 24 markers including 4 Not I-linking STSs to be ordered and mapped physically. Comparison of the maps revealed that the proximal region contains more unmethylated CpG islands than the distal region, which suggests that many expressed genes are in the proximal region. This fine consensus physical map will be informative and useful for construction of contigs of cosmid, P1, or BAC clones for further large-scale sequencing in this gene-rich region.

Influence of poly(ADP-ribose) polymerase on the enzymatic synthesis of SV40 DNA.

The influence of poly(ADP-ribose) polymerase (PARP) on the replication of DNA containing the SV40 origin of replication has been examined. Extensive replication of SV40 DNA can be carried out in the presence of T antigen, topoisomerase I, the multimeric human single strand DNA-binding protein (HSSB), and DNA polymerase alpha-DNA primase (pol alpha-primase) complex (the monopolymerase system). In the monopolymerase system, both small products (Okazaki fragments), arising from lagging strand synthesis, and long products, arising from leading strand synthesis, are formed. The synthesis of long products requires the presence of relatively high levels of pol alpha-primase complex. In the presence of PARP, the synthesis of long products was blocked and only small Okazaki fragments accumulated, arising from the replication of the lagging strand template. The inhibition of leading strand synthesis by PARP can be effectively reversed by supplementing the monopolymerase system with the multimeric activator 1 protein (A1), the proliferating cell nuclear antigen (PCNA) and PCNA-dependent DNA polymerase delta (the dipolymerase system). The inhibition of leading strand synthesis in the monopolymerase system was caused by the binding of PARP to the ends of DNA chains, which blocked their further extension by pol alpha. The selective accumulation of Okazaki fragments was shown to be due to the coupled synthesis of primers by DNA primase and their immediate extension by pol alpha complexed to primase. PARP had little effect on this coupled reaction, but did inhibit the subsequent elongation of products, presumably after pol alpha dissociated from the 3'-end of the DNA fragments. PARP inhibited several other enzymatic reactions which required free ends of DNA chains. PARP inhibited exonuclease III, DNA ligase, the 5' to 3' exonuclease, and the elongation of primed DNA templates by pol alpha. In contrast, PARP only partly competed with the elongation of primed DNA templates by the pol delta elongation system which required SSB, A1, and PCNA. These results suggest that the binding of PARP at the ends of nascent DNA chains can be displaced by the binding of A1 and PCNA to primer ends. HSSB can be poly(ADP-ribosylated) in vivo as well as in vitro. However, the selective effect of PARP in blocking leading strand synthesis in the monopolymerase system was shown to depend primarily on its DNA binding property rather than on its ability to synthesize poly(ADP-ribose).

Studies on the initiation of simian virus 40 replication in vitro: RNA primer synthesis and its elongation.

DNA primase-dependent synthesis of oligoribonucleotides 10-15 nucleotides long was observed in the presence of ATP, UTP, GTP, and CTP by using the purified components of the simian virus 40 (SV40) DNA replication system. The DNA primase-catalyzed reaction required the SV40 large tumor antigen (T antigen), DNA polymerase alpha (pol-alpha), the three-subunit human single-stranded DNA binding protein (HSSB), and topoisomerase I. The synthesis of small RNAs was unaffected by the addition of activator 1, proliferating cell nuclear antigen, and DNA polymerase delta, proteins that can support extensive leading-strand synthesis. The RNA primers were derived predominantly from transcription of the lagging-strand template, even after prolonged incubation, indicating that the leading strand did not serve as a template. When the four dNTPs were added after oligoribonucleotide synthesis, pol-alpha extended the RNA primers hybridized to SV40 DNA. Pulse-chase experiments revealed that the small RNA chains were elongated to Okazaki-sized products. T7 DNA polymerase was also shown to rapidly extend oligoribonucleotide primers in the presence of aphidicolin or antibodies against pol-alpha, conditions under which pol-alpha was markedly inhibited. These findings suggest that interactions between T antigen, pol-alpha-primase, and HSSB position the pol-alpha-primase complex on the lagging-strand template for RNA primer synthesis.

Studies on the initiation and elongation reactions in the simian virus 40 DNA replication system.

The synthesis of oligoribonucleotides by DNA primase in the presence of duplex DNA containing the simian virus 40 (SV40) origin of replication was examined. Small RNA chains (10-15 nucleotides) were synthesized in the presence of the four common ribonucleoside triphosphates, SV40 large tumor antigen (T antigen), the human DNA polymerase alpha (pol alpha)-DNA primase complex, the human single-stranded DNA-binding protein (HSSB), and topoisomerase I isolated from HeLa cells. The DNA primase-catalyzed reaction showed an absolute requirement for T antigen, HSSB, and pol alpha. The requirement for HSSB was not satisfied by other SSBs that can support the T-antigen-catalyzed unwinding of DNA containing the SV40 origin of replication. Oligoribonucleotide synthesis occurred with a lag that paralleled the lag observed in DNA synthesis. These results indicate that the specificity for the HSSB in the SV40 replication reaction is due to the pol alpha-primase-mediated synthesis of the Okazaki fragments. In contrast to this specificity, the elongation of Okazaki fragments can be catalyzed by a variety of different DNA polymerases, including high levels of pol alpha, the polymerase delta holoenzyme, T4 polymerase holoenzyme, the Escherichia coli polymerase III holoenzyme, and other polymerases. These observations suggest that leading-strand synthesis in the in vitro SV40 replication system can be nonspecific.

Characterization of factors that suppress linear DNA replication in SV40 in vitro replication system.

The in vitro simian virus 40 (SV40) replication system has been developed as a model system of cellular DNA replication, because the replication initiated from the replication origin of SV40 and replication fork proceeds bidirectionally. In this system, SV40 T-antigen (TAg) is the only factor provided by viral genes, while all other factors are supplied by the host cells. A suppression of replication has been observed in the linear template containing SV40 replication origin, compared with the closed circular template in the SV40 in vitro replication system using a crude extract of HeLa cells. However in the in vitro replication system reconstituted from partially purified factors, less preference was observed for the replication of the closed circular DNA over the linear DNA. In a mono-polymerase system supplemented by crude extracts, a suppression of replication in a linear template was also observed, when compared with a closed circular template. This suppression effect of crude extract was abolished by heat treatment, suggesting that the suppression was induced by some protein factors. A crude extract of HeLa cells was fractionated by stepwise elution with buffers containing 0.2 M, 0.4 M, 0.6 M and 1 M NaCl on a phosphocellulose column, and characterization of factors that suppress linear DNA replication has been done. Both fractions that were eluted at 0.4 M and 0.6 M from phosphocellulose were necessary to suppress linear DNA replication efficiently. The factors in the 0.6 M fraction that suppressed linear DNA replication synergistically with the 0.4 M fraction were partially purified by successive chromatography with heparin-sepharose and dsDNA-cellulose followed by glycerol gradient centrifugation. These results suggested that multiple factors are required to suppress DNA replication of the linear template.

Synthesis of DNA containing the simian virus 40 origin of replication by the combined action of DNA polymerases alpha and delta.

Proliferating-cell nuclear antigen (PCNA) mediates the replication of simian virus 40 (SV40) DNA by reversing the effects of a protein that inhibits the elongation reaction. Two other protein fractions, activator I and activator II, were also shown to play important roles in this process. We report that activator II isolated from HeLa cell extracts is a PCNA-dependent DNA polymerase delta that is required for efficient replication of DNA containing the SV40 origin of replication. PCNA-dependent DNA polymerase delta on a DNA singly primed phi X174 single-stranded circular DNA template required PCNA, a complex of the elongation inhibitor and activator I, and the single-stranded DNA-binding protein essential for SV40 DNA replication. DNA polymerase delta, in contrast to DNA polymerase alpha, hardly used RNA-primed DNA templates. These results indicate that both DNA polymerase alpha and delta are involved in SV40 DNA replication in vitro and their activity depends on PCNA, the elongation inhibitor, and activator I.

The replication of DNA containing the simian virus 40 origin by the monopolymerase and dipolymerase systems.

The influence of DNA polymerase (pol) alpha and DNA primase on SV40 DNA replication was examined in both the monopolymerase and dipolymerase systems. The synthesis of oligoribonucleotides in the monopolymerase and dipolymerase systems, followed by pulse labeling with deoxynucleoside triphosphates, yielded short Okazaki fragments approximately 35 nucleotides in length that were chased into full-length Okazaki fragments with time. In the presence of activator 1 and proliferating cell nuclear antigen (PCNA), but no pol delta, these short fragments hardly increased in size with time. DNA fragments of similar size (approximately 35 nucleotides) were previously observed in SV40 replication reactions carried out with crude extracts of HeLa cells in the presence of antibodies directed against PCNA (Bullock, P. A., Seo, Y.S., and Hurwitz, J. (1991) Mol. Cell. Biol. 11, 2350-2361). Thus, the pol alpha-primase complex appears to act processively for only a short distance. At high levels of pol alpha and primase, both short and long DNA products were formed in both systems. In the presence of limiting amounts of pol alpha and excess primase, the monopolymerase system inefficiently yielded longer length Okazaki fragments than those formed with excess pol alpha and primase, whereas the dipolymerase system yielded both short and long DNA fragments. In the presence of limiting amounts of primase and excess pol alpha, long products were formed in both systems, and virtually no short products accumulated. Thus, the ratio between the polymerase and primer ends available controls the size of the nascent product DNA strands. We examined whether PCNA, the T4 phage-encoded gene product 45 (T4 gp45), and the Escherichia coli beta subunit of DNA polymerase III (dnaN gene product) supported SV40 DNA replication and the elongation of single-stranded DNA-binding protein-coated singly primed DNA in reactions catalyzed by pol delta, T4 DNA pol, and E. coli DNA pol III*, respectively. In the presence of T4 gp44/62 and T4 gp32 (but not human single-stranded DNA-binding protein isolated from HeLa cells), T4 DNA pol was weakly activated by PCNA and the beta subunit in lieu of T4 gp45 in the elongation of singly primed phi X174 DNA. However, the other systems were specific for their analogous auxiliary factors. This specificity indicates the importance of protein-protein interactions.

Characterization and chromosomal mapping of the human thromboxane A2 receptor gene.

The human thromboxane A2 gene, present as a single copy, spans over 15 kilobases (kb) and contains 3 exons divided by 2 introns. The first intron, intron 1, exists in the 5'-noncoding region, 83 base pairs upstream from the ATG start site and is 6.3 kb long. Intron 2 with a length of 4.3 kb is located at the end of the sixth transmembrane region, thereby separating it from the downstream coding sequences including the seventh transmembrane region and the 3'-untranslated region. By rapid amplification of 5'-cDNA ends, transcription initiation sites starting in two different putative promoter regions were determined. In the 5'-flanking region of these transcription initiation sites, no typical TATA box exists. The major promoter, the promoter region I, contains four potential SP-1 binding sites and several potential binding sites for other transcription factors. By polymerase chain reaction analysis, a small portion of the RNA transcribed from this gene was shown to contain an additional 115-base pair long noncoding exon, exon 1b, which is located in intron 1. No additional exon in intron 2 was detectable, indicating that a single type of thromboxane A2 receptor protein is encoded by this gene. Chromosomal localization was carried out by fluorescence in situ hybridization of cloned genomic DNA to the metaphase chromosome. The gene was assigned to 19p13.3 of human chromosome.