Characterization of a fusion cDNA (RARA/myl) transcribed from the t(15;17) translocation breakpoint in acute promyelocytic leukemia.

A nonrandom chromosomal translocation breakpoint, t(15;17)(q22;q21), is found in almost all patients with acute promyelocytic leukemia (APL). Most of these breakpoints occur within the second intron of the retinoic acid receptor-alpha (RARA) gene. We screened a cDNA library of APL and have identified and sequenced a cDNA transcribed from the t(15;17) translocation breakpoint. The 5' end of cDNA p1715 consists of 503 bp of the RARA exon II sequence. A 1.76-kb cDNA without homology to any known gene available in GenBank was found truncated downstream. This cDNA sequence was assigned to chromosome 15 by dot blot hybridization of the flow cytometry-sorted chromosomes. We designate this fusion cDNA RARA/myl, which is different from myl/RARA reported by de The et al. (H. de The, C. Chomienne, M. Lanotte, L. Degos, and A. Dejean, Nature (London) 347:558-561, 1990). This result demonstrates that the two different types of hybrid mRNA can be transcribed from this breakpoint. We screened a non-APL cDNA library and identified a 2.8-kb myl cDNA. This cDNA is able to encode a polypeptide with a molecular weight of 78,450. Alternative splicing of the myl gene which resulted in myl proteins with different C terminals was found. Southern blot analysis of the genomic DNA isolated from 17 APL patients by using the myl DNA probe demonstrated that the myl gene in 12 samples was rearranged. Northern (RNA) blot analysis of RARA gene expression in two APL RNA samples showed abnormal mRNA species of 4.2 and 3.2 kb in one patient and of 4.8 and 3.8 kb in another patient; these were in addition to the normal mRNA species of 3.7 and 2.7-kb. The myl DNA probe detected a 2.6-kb abnormal mRNA in addition to the normal mRNA species of 3.2, 4.2, and 5.5 kb. Using the polymerase chain reaction, we demonstrated that both RARA/myl and myl/RARA were coexpressed in samples from three different APL patients. From this study, we conclude that the t(15;17) translocation breakpoint results in the transcription of two different fusion transcripts which are expected to be translated into fusion proteins.

Cell-cycle-specific chromosome damage following treatment of cultured Chinese hamster cells with 4'-[(9-acridinyl)-amino]methanesulphon-m-anisidide-HCl.

The induction of chromosome damage in Chinese hamster (line CHO) cells by 4'-[(9-acridinyl)-amino]methanesulphon-m-anisidide-HCl (MAC) (NSC-141549) was studied in cell populations growing exponentially and at various stages of the cell cycle following release from isoleucine-deficient G1-arrest. Autoradiographic analysis demonstrated that cells in S-phase at time of drug addition (2 microgram MAC/ml for 2 hr) were delayed 8 hours before entering mitosis. Cells in G1 at the time of MAC treatment were not as severely delayed, which resulted in a rather sharp increase and decrease in a percent labeled mitosis curve. Chromosome damage occurred differentially during the cell cycle. Cells in late G2 during MAC treatment contained incompletely condensed chromosomes with occasional chromosome interchanges at the next mitosis. Early G2 cells were severely damaged (greater than 20 breaks/cell). Damage to cells in S or G1 at the time of MAC addition was less severe, whereas cells in G1-S traverse had intermediate levels of chromosome breaks. Thus MAC appeared to be particularly effective at times when chromatin was undergoing structural modifications (G1-S and S-G2 boundaries). Low concentrations of MAC (0.05 microgram/ml) increased the rate of sister chromatid exchange to almost eight times the background rate. The cellular effects of MAC were compared with previously reported studies of other antitumor agents.

Gene structure and differential expression of acidic fibroblast growth factor mRNA: identification and distribution of four different transcripts.

We have isolated four cDNA clones coding for human acidic fibroblast growth factor (aFGF) containing alternative 5' untranslated exons. Using RNAase protection analyses, we demonstrated the presence of at least four upstream, untranslated exons that are alternatively spliced to the first protein-coding exon. We designate these four untranslated exons, -1A, -1B, -1C and -1D. Splicing of these exons to the first coding exon will generate mRNA 1.A, 1.B, 1.C and 1.D respectively. Expression of these transcripts is regulated in a tissue-specific manner, as the major aFGF transcript in human brain frontal cortex differs from that in kidney. Furthermore, the pattern of aFGF transcripts in several glioblastoma cell lines tested is different from that in normal brain tissue. We isolated nine overlapping genomic clones containing these four upstream, untranslated exons. These four exons were localized on these clones by Southern hybridization and nucleotide sequence analysis. The overlapping clones are shown to be contiguous with our previously isolated genomic clones that contain the three aFGF-coding exons. The sizes of the four introns are 82.9, 71.1, 29.3 and 6.9 kbp. The transcriptional start sites of the two most upstream exons (-1A and -1B) have been mapped using RNAase protection and primer extension analyses. The sequences upstream of the start sites for aFGF 1.B mRNA do not contain a consensus TATA box. In contrast, the canonical CCAAT and TATA sequences are located at the proper distances from the transcription start site of aFGF 1.A mRNA.

Cystosine-rich strands of the insulin minisatellite adopt hairpins with intercalated cytosine+.cytosine pairs.

Previously, we reported the high resolution NMR structure of the hairpin G-quartet structure formed by the G-rich strand of the insulin minisatellite of repeat sequence, (ACAG4TGTG4/TGTC4ACAC4) located upstream of the human insulin gene. Here, we report structural studies on the C-rich strand of this insulin minisatellite. First, we show by high resolution NMR that (C4TGTC4) forms a hairpin dimer with intercalated C+.C pairs (referred to as the hairpin i-motif); 340 NOE distance constraints uniquely define the nature of hairpin folding and the pattern of C+.C intercalation. Second, we show by one-dimensional NMR spectroscopy and molecular modeling studies that (C4TGTC4ACA4TGTC4) forms an intramolecularly folded hairpin with intercalated C+.C pairs. Third, we demonstrate by in vitro replication studies that several such hairpin i-motifs are present in long (C4TGTC4ACA)n (n>/=6) sequences, even in the presence of their complementary strands. Finally, we discuss structural and biological significance of the hairpin i-motifs formed by the C-rich strands of the insulin minisatellite.

Transcriptional regulation and chromosomal assignment of the mammalian calbindin-D28k gene.

To determine whether 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] regulates transcription of the rat renal calbindin-D28k gene, the rate of calbindin-D28k mRNA synthesis was measured directly in nuclei using the in vitro nuclear transcription assay. Nuclei were prepared from kidneys of vitamin D-deficient rats at various times after a single ip injection of 1,25-(OH)2D3, and transcription was allowed to proceed in vitro in the presence of [32P]UTP for 30 min at 29 C, at which time the incorporation of UTP into trichloroacetic acid-precipitable material was optimal. Incorporation of UTP was decreased by 64.6% by alpha-amanitin, which selectively inhibits polymerase II. Purified [32P]RNA was analyzed for newly synthesized calbindin-D-28k gene transcripts by hybridization to calbindin-D28k cDNA immobilized on nitrocellulose filters. Using this assay we found that the first significant increase in calbindin-D28k gene transcription occurred at 1 h, and the peak of transcriptional activity occurred at 2 h. Within 12 h of 1,25-(OH)2D3 treatment, calbindin-D28k gene transcription returned to control levels. Using Northern blot analysis, a significant increase in calbindin-D RNA was first observed 2 h after hormone administration, reaching a maximum at 12 h. Renal calbindin-D28k protein levels are significantly increased by 3 h and reach a maximum value 48 h after hormone administration. Our results suggest that the early increase in renal calbindin-D28k may be due to transcriptional regulation. The long time lag between transcription and the peak of calbindin mRNA and calbindin protein accumulation may reflect the involvement of post-transcriptional mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Human platelet glycoprotein IX. Characterization of cDNA and localization of the gene to chromosome 3.

Overlapping cDNAs encoding human platelet glycoprotein (Gp)IX were cloned from a human erythroleukemia cell lambda gt11 library. The possibly 'full-length' cDNA of 896 base pairs (bp) includes an open reading frame (528 bp), both 5' (222 bp) and 3' (146 bp) noncoding regions, and a poly(A) tail. Translation predicts a signal peptide of 16 amino acids and a mature protein of 160 amino acids that includes a 24 amino acid leucine-rich glycoprotein (LRG) segment. Southern blot analysis suggests the presence of a single copy of the Gp IX gene, and hybridization of Gp IX cDNA to sorted human chromosomes localizes the Gp IX gene to chromosome 3.

Human release factor eRF1: structural organisation of the unique functional gene on chromosome 5 and of the three processed pseudogenes.

In lower and higher eukaryotes, a family of tightly related proteins designated eRF1 (for eukaryotic release factor 1) catalyses termination of protein synthesis at all three stop codons. The human genome contains four eRF1 homologous sequences localised on chromosomes 5, 6, 7 and X. We report here the cloning and the structural analysis of the human eRF1 gene family. It appears that the gene located on chromosome 5 alone is potentially functional, whereas the other three sequences resemble processed pseudogenes. This is the first description of the structural organisation of the human eRF1 gene, which has been remarkably conserved during evolution and which is essential in the translation termination process.

A cDNA from the ovarian cancer critical region of deletion on chromosome 17p13.3.

Chromosome 17p13.3 is frequently deleted in human ovarian carcinoma, and the 15 kb critical region of deletion may contain a tumor suppressor gene. A 2.3 kb cDNA has been identified which spans 17 kb of genomic DNA, including 8.1 kb within the critical region, and thus is a candidate tumor suppressor gene. This highly conserved gene has significant sequence similarity to a yeast gene of unknown function and to one of the yeast enzymes in the diphthamide synthetic pathway, DPH2, that has a role in global protein synthesis regulation. This gene, named DPH2L (diphthamide biosynthesis protein 2-like), is expressed in multiple tissues and stages of development.

Biological effectiveness of neutrons from Hiroshima bomb replica: results of a collaborative cytogenetic study.

The effectiveness of neutrons from a facsimile of the Hiroshima bomb was determined cytogenetically. The "Little-Boy" replica (LBR), assembled at Los Alamos as a controlled nuclear reactor for detailed physical dosimetry, was used. Of special interest, the neutron energy characteristics (including lineal energy) measured 0.74 m from the LBR were remarkably similar to those calculated for the 1945 Hiroshima bomb at 1 to 2 km from the hypocenter, as shown in a companion dosimetric paper (Straume, et al., Radiat. Res. 128, 133-142 (1991)). Thus we examine here the effectiveness of neutrons closely resembling those that the A-bomb survivors received at Hiroshima. Chromosome aberration frequencies were determined in human blood lymphocytes exposed in vitro to graded doses of LBR radiation (97% neutrons, 3% gamma rays). Vials of blood suspended in air at distances up to 2.10 m from the center of the LBR uranium core received doses ranging from 0.02 to 2.92 Gy. The LBR neutrons (E approximately 0.2 MeV) produced 1.18 dicentrics and rings per cell per Gy. They were more effective than the higher-energy fission neutrons (E approximately 1 MeV) commonly used in radiobiology. The maximum RBE (RBEM) of LBR neutrons at low doses is estimated to be 60 to 80 compared to 60Co gamma rays and 22 to 30 compared to 250-kVp X rays. These results provide a quantitative measurement of the biological effectiveness of Hiroshima-like neutrons.

In vivo cytogenetic effects of oil shale retort process waters.

The induction of cytogenetic effects by oil shale retort process waters from 3 types of pilot plant retorts were examined in murine bone marrow. Each of the process waters induced increased frequencies of structural aberrations in mice treated with 3 daily intraperitoneal injections of the waters. The same treatment had no effect on the frequency of sister chromatid exchanges. Mice given a 1% solution of an above-ground retort water ad libitum for 8 weeks consumed about 1 ml/kg per day of the process water and had a frequency of aberrations comparable to mice given the same dose intraperitoneally for 3 days. Transplacental exposure of C3H mouse embryos indicated that clastogenic compounds in the above-ground retort process water can cross the placenta and induce chromosomal aberrations in embryonic tissues.

Identification of two Fas-associated phosphatase-1 (FAP-1) promoters in human cancer cells.

Fas-associated phosphatase-1 (FAP-1) has been reported as a negative regulator of Fas-mediated signal transduction in human cancer cells. To obtain insights into the potential carcinogenesis of the FAP-1 gene, we investigated its transcriptional regulation in normal and cancerous cells. To identify the FAP-1 promoter sequences, we first isolated P1 and cosmid clones that contained the regulatory region upstream from the FAP-1 gene by using the PCR products of 5' rapid amplification of cDNA end (5'-RACE) as probes. Genomic analysis of positive clones revealed that the major FAP-1 mRNA was transcribed from its proximal promoter (pPRM) in all human cancer cell lines tested, but 1 additional large transcript derived from its distal promoter (dPRM) was found in the human colon cancer cell line DLD-1. This suggests that the FAP-1 gene may be aberrantly dysregulated in some types of human cancers, including colon carcinoma. Sequence analysis of the region upstream from the FAP-1 gene strongly suggests that the transcript of the FAP-1 gene may be controlled by a variety of transcriptional regulatory elements, including NF-kappa B, NF-IL6, and p53 in its 2 promoters. These results imply that the FAP-1 gene may be a target gene under the control of important apoptosis-related nuclear factors in human cancers.

Analysis of a 69-kb contiguous genomic sequence at a putative tumor suppressor gene locus on human chromosome 6q27.

Multiple neoplasias including B-cell non-Hodgkin's lymphoma, breast carcinoma, and ovarian carcinoma, have been associated with frequent deletions of the distal region on the long arm of human chromosome 6, suggesting the presence of one or more tumor suppressor gene(s) at this locus. Loss of heterozygosity analysis of breast and ovarian tumors has further restricted the minimal region of loss within 6q27. To further characterize this genomic region for gene content including putative tumor suppressor genes as well as other elements that may contribute to tumorigenesis, a 68940-bp contiguous sequence, encompassing markers D6S193 and D6S297, was generated by random shotgun sequencing of a cosmid, P1, and PAC contig. In addition, exon trapping was performed utilizing a subset of these clones. Sixteen trapped exons, ranging in size from 44 to 399 bp, span this approximately 69-kb region. Many other putative exons have been identified computationally. Further analysis has identified 13 potential promoters and 13 putative polyadenylation sites in the region. Northern analysis identified a transcript mapping within this interval that is expressed in ovarian, breast, and lymphoid-derived tumor cell lines. Consideration of these data, together with the demonstration of several regions of high CpG content, suggests the possibility of several genes at this locus.

General human health risks associated with the use of chemicals.

Health risks to man associated with the use of chemicals include carcinogenesis, mutagenesis, and systems damage. Adverse effects from chemical exposures are determined by the nature and amount of the chemical, the type and length of exposure, and individual susceptibility to the chemical. A series of short- or long-term tests have been devised to predict human risk from suspect chemicals. These tests have provided enough information to establish guidelines for human safety, but they are not capable of providing sufficient information for unequivocal, scientifically valid standards for exposure limits. New methods now under development promise to provide more detailed information on early effects and cumulative damage to individuals. Developing countries should use existing data and regulatory experiences to the greatest possible extent to establish exposure limits according to local needs. When more ideal methods for the detection of chemical damage to man are available, these approaches and the data derived from their use can be incorporated into the programs in developing countries.

Factors affecting the induction of chromosomal aberrations by cadmium in Chinese hamster cells.

Chinese hamster cells (line CHO) were examined for cadmium-induced growth-rate pertubations and chromosomal aberrations. The threshold concentration of Cd++ for the induction of chromosomal aberrations in CHO cells cultured in F-10 medium supplemented with 15% newborn calf serum was determined to be 1 X 10(-6) M. CHO cells grown in the presence of nontoxic levels of Cd++ (2 X 10(-7) M) for 12 wk became resistant to toxic levels (2 X 10(-6) M) after this exposure. Metaphase cells in these resistant populations contained only background levels of chromosomal aberrations. CHO cells grown in medium supplemented with several types of serum (fetal calf, newborn calf, and human) at concentrations commonly used for in vitro culture (5-20% v/v) or found in mammalian circulatory systems (50% v/v) differed markedly in Cd++ tolerance. Cells grown in medium containing 1 X 10(-6) M Cd++ and supplemented with human or newborn calf serum were slightly protected against Cd++ damage at high serum concentrations (30 and 50% v/v) and accumulated approximately 90 microgram Cd++/10(9) cells in 48 h. Cells growing at low or high concentrations of fetal calf serum were completely protected from 1 X 10(-6) M Cd++ and accumulated approximately 12 microgram Cd/10(9) cells in 48 h. The threshold Cd++ concentration for euploid Chinese hamster cells derived from skin, lung, and kidney tissues was determined to be 2 X 10(-5) M. Cadmium does not induce elevated sister chromatid exchange levels. These results demonstrate the necessity for standardized protocols for cytogenetic investigations of Cd++ toxicity and may help to explain the discrepancies between studies of chromosome damage in patients with high Cd++ blood levels.

Cytogenetic effects of shale-derived oils in murine bone marrow.

The cytogenetic effects of exposure of mice to shale-derived oils by either skin painting or intraperitoneal injection were examined. Skin painting with 40 mg crude oil every other day for five weeks had no effect on the frequency of chromosomal aberrations observed in the bone marrow. Three daily intraperitoneal injections of 0.5-2.0 ml/kg per day of a crude shale oil from an aboveground retort induced a dose-related increase in the frequency of aberrations observed. A hydrotreated sample of this oil produced a similar pattern of aberration induction, but at lower frequencies. Crude shale oil from a modified in situ retort induced the highest frequency of chromosomal aberrations at the 0.5 ml/kg dose; lower frequencies were induced at the 1.0 ml and 2.0 ml/kg doses. Of the three shale oils tested, only the crude oil from the aboveground retort induced increased frequencies of sister chromatid exchanges and only at doses that also induced structural aberrations. These studies indicate that structural aberration analysis is a more sensitive test than sister chromatid exchange analysis for the type of DNA damage induced by shale-derived oils in murine bone marrow cells.

Construction of gene libraries for each human chromosome.

We describe the construction of two complete sets of small insert, complete digest DNA libraries for each of the 24 human chromosomal types by the National Laboratory Gene Library Project. Flow sorting was used to purify the chromosomes which provided the DNA for cloning. One set of libraries was cloned into the HindIII site of the lambda vector Charon 21A, and the other set was cloned into the EcoRI site of the same vector. Characterization information from both in-house experiments and user feedback is presented. These chromosome-specific libraries are available to the general scientific community from a repository at the American Type Culture Collection, Rockville, MD. The second phase of the project, the construction of large insert, partial digest libraries in both lambda and cosmid vectors, is underway.

Dephosphorylation of histones H1 and H3 during the isolation of metaphase chromosomes.

Histones have been extracted from isolated metaphase chromosomes prepared by the method of Wray and Sutbblefield [Exp. Cell Res 59, 469-478 (1970)] and by a Nonidet P-40 detergent procedure based on the method of Wigler and Axel [Nucleic Acids Res. 3, 1463-1471 (1976)]. Analysis of the densitometer profiles of long polyacrylamide gels shows that the mitotic phosphorylations of histone H1 (H1M) and histone H3 are extensively depleted during chromosome isolation. These data indicate that CHO metaphase chromosomes prepared by standard methodologies do not represent in vivo chromosomes with respect to their histone phosphorylations; therefore, current chemical and structural studies of isolated metaphase chromosomes may require further clarification.