Characterization of SRp46, a novel human SR splicing factor encoded by a PR264/SC35 retropseudogene.

The highly conserved SR family contains a growing number of phosphoproteins acting as both essential and alternative splicing factors. In this study, we have cloned human genomic and cDNA sequences encoding a novel SR protein designated SRp46. Nucleotide sequence analyses have revealed that the SRp46 gene corresponds to an expressed PR264/SC35 retropseudogene. As a result of mutations and amplifications, the SRp46 protein significantly differs from the PR264/SC35 factor, mainly at the level of its RS domain. Northern and Western blot analyses have established that SRp46 sequences are expressed at different levels in several human cell lines and normal tissues, as well as in simian cells. In contrast, sequences homologous to SRp46 are not present in mice. In vitro splicing studies indicate that the human SRp46 recombinant protein functions as an essential splicing factor in complementing a HeLa cell S100 extract deficient in SR proteins. In addition, complementation analyses performed with beta-globin or adenovirus E1A transcripts and different splicing-deficient extracts have revealed that SRp46 does not display the same activity as PR264/SC35. These results demonstrate, for the first time, that an SR splicing factor, which represents a novel member of the SR family, is encoded by a functional retropseudogene.

Chromosomal assignment of two human B-raf(Rmil) proto-oncogene loci: B-raf-1 encoding the p94Braf/Rmil and B-raf-2, a processed pseudogene.

The B-raf gene is the human homolog of the avian c-Rmil proto-oncogene encoding a 94-kDa serine/threonine kinase detected in avian cells. We have previously shown that this protein contains amino-terminal sequences not found in other proteins of the mil/raf gene family. These sequences are encoded by three exons in the avian genome. We report that these three exons are conserved in the human B-raf gene and that they encode an amino acid sequence similar to that of the avian c-Rmil gene, indicating that in both avian and mammalian species the product of the B-raf/c-Rmil gene is a 94-kDa protein. We also identified two human B-raf loci: B-raf-1, located on chromosome 7q34, which encodes the functional B-raf/Rmil gene product, and B-raf-2, an inactive processed pseudogene located on chromosome Xq13.

Identification of a human guanine nucleotide-releasing factor (H-GRF55) specific for Ras proteins.

A critical step in the activation of cellular Ras is the release of bound GDP. Oligonucleotide primers derived from a mouse cDNA sequence homologous to the Saccharomyces cerevisiae CDC25 gene product were used to screen a human brain cDNA library. The cloning led to the isolation of a 2.8-kb cDNA predicted to encode a protein of 488 amino acids. This protein was produced in Escherichia coli as a glutathione S-transferase fusion protein and functioned in vitro as a specific guanine nucleotide-releasing factor. Polyclonal antibodies raised against the last 281 amino acids of the protein allowed a protein in the molecular weight range of 55 kDa to be identified in human cortex homogenates. Analysis by Northern blotting led to the identification of a 5.5-kb mRNA in brain poly(A)+ RNA. The functionality of the encoded protein was evaluated after expression in different cells: (i) in Saccharomyces cerevisiae the effects of the cdc25.5 and RAS2 Ala-22 mutations were reversed; (ii) in chinese hamster ovary cells, a RAS-responsive element was transactivated as demonstrated by the expression of a CAT reporter gene under the control of the polyomavirus enhancer. Finally, in situ hybridization on of human chromosomes revealed a localization on band 15q2.4.

Isolation, characterization, and chromosomal localization of the human ENSA gene that encodes alpha-endosulfine, a regulator of beta-cell K(ATP) channels.

Human alpha-endosulfine is an endogenous regulator of the beta-cell K(ATP) channels. The recombinant alpha-endosulfine inhibits sulfonylurea binding to beta-cell membranes, reduces cloned K(ATP) channel currents, and stimulates insulin secretion from beta-cells. These properties led us to study the human ENSA gene that encodes alpha-endosulfine. Here, we describe the isolation, the partial characterization, and the chromosomal localization of the ENSA gene. The ENSA gene appears to be a 1.8-kb-long sequence that contains the transcription initiation site located 528 bp upstream of the initiation codon. The ENSA gene is intronless, and a single copy gene seems to be present in the genome. Finally, the ENSA gene co-localizes on human chromosome 14 (14q24.3-q31) with a locus for susceptibility to type 1 diabetes called IDDM11; thus, the ENSA gene represents an IDDM11 candidate.

The OZF gene encodes a protein consisting essentially of zinc finger motifs.

A novel member of the zinc finger Krüppel family was isolated as a cDNA clone from the human mammary cell line HBL100. It contains an open reading frame consisting of 32 amino acid residues followed by ten zinc finger motifs and was accordingly named OZF (Only Zinc Fingers). Assuming that translation initiation starts at the unique methionine codon, 22 codons downstream of the beginning of the open reading frame as suggested by in vitro translation of OZF mRNA, the OZF protein is 292 residues long and has an estimated molecular mass of 33 kDa. The OZF gene is located in band q13.1 of the long arm of human chromosome 19. It is expressed as a single mRNA in liver, skeletal muscle and mammary cells and as two mRNA species in heart muscle. Absent or very low levels of expression were observed in brain, lung, placenta, kidney and human fibroblasts in culture. Thus, the OZF protein, which consists essentially of a DNA/RNA binding domain, may act as a tissue-specific modulator of gene expression.

Chromosomal reallocation of the chicken c-myb locus and organization of 3'-proximal coding exons.

In the course of our studies concerning the tissue-specific expression of the c-myb proto-oncogene, we have established the nucleotide sequence of the chicken c-myb 3'-proximal coding exons. In situ hybridization performed with different genomic DNA probes corresponding to nearly all the c-myb gene allowed us to localize the corresponding locus on the large acrocentric chromosome 3 in chicken. Our sequencing data also indicate that the 3'-proximal noncoding sequences represented in c-myb mRNA species are derived from non-contiguous exons.

Molecular cloning, functional expression, pharmacological characterization and chromosomal localization of the human metabotropic glutamate receptor type 3.

Glutamic acid is the major excitatory amino acid of the central nervous system which interacts with two receptor families, the ionotropic and metabotropic glutamate receptors. The metabotropic glutamate receptors (mGluRs) are coupled to G proteins and can be divided into three subgroups based on their sequence homology, signal transduction pathway and pharmacology. In this study, we describe the cloning of the cDNA encoding the human metabotropic glutamate receptor type 3 (HmGluR3). It was obtained by reverse transcription-polymerase chain reaction (RT-PCR) with degenerate oligonucleotides corresponding to highly conserved sequences between rat mGluRs. The receptor shows 879 amino acids with 96% amino acid sequence identity with rat mGluR3. It is strongly expressed in fetal and adult whole brain, especially in caudate nucleus and corpus callosum. The gene was identified by fluorescence in situ hybridization on chromosome 7 band q22. Activation of the human mGluR3, permanently expressed in Baby Hamster Kidney (BHK) cells, by excitatory amino acid inhibits the forskolin-stimulated accumulation of intracellular cAMP. The rank order of potency is L-glutamic acid > or = (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R)-ACPD) >> ibotenic acid > quisqualic acid. (RS)-alpha-methyl-4-carboxyphenylglycine [(RS)-MCPG, 1 mM] is without effect on inhibition of forskolin-induced cAMP accumulation by L-glutamic acid.

Constitutive amplification of a zinc finger protein gene in cattle.

The human OZF gene encodes a protein consisting essentially of zinc finger motifs of the Krüppel type. Evolutionary studies revealed amplification of the bovine OZF gene in cattle. Domestic cattle includes two major subspecies: taurine (Bos primigenius taurus) and zebu (B. p. indicus). Amplified sequences were found in both subspecies and mapped to the same locus of chromosome X in band q11. No amplification was found in other bovids and in particular in Bison, the closest related genus to the genus Bos. One copy of the amplified locus was cloned and sequenced. It encodes a protein sharing 95% identity with the human OZF protein. The bovine OZF gene was detected in an additional locus on chromosome 18 in both Bos and Bison, in band q23-24, which is likely to represent the ancestral position of the OZF gene. This is the first evidence of gene amplification in a mammalian species during evolution.

Structural heterogeneity of hsr(11) in the MDA-MB-134 mammary carcinoma cell line.

The MDA-MB-134 cell line was characterized by the presence of two homogeneously staining region (hsr) carrier chromosomes containing sequences from 8p11-p12, 11q13, and 8q24. Using fluorescence in situ hybridization, a detailed study of the organization of this chromosome has been performed. The hsr carrier chromosomes are shown to be identical and to derive from a chromosome 11, on which large segments of the long arm of chromosome 8 are translocated, forming its short arm. The hsr segment is inserted in the long arm, distally to C1NH gene. It is formed by sequences from both chromosomes 8 and 11. For the genes investigated by both methods the number of copies detected by in situ hybridization is compatible with that expected by quantification of Southern blots. In spite of its complexity, a possible mechanism of formation is proposed.

Excision repair of 8-hydroxyguanine in mammalian cells: the mouse Ogg1 protein as a model.

8-Hydroxyguanine (8-OH-Gua) is a major mutagenic lesion produced on DNA by the oxidative stress induced by either the endogen metabolism or the exposure to external agents. In bacteria and yeast this modified base can be removed by specific DNA glycosylases. Recently a human gene coding for an 8-OH-Gua DNA glycosylase/AP lyase has been identified by its homology to the yeast OGG1. This gene is located in human chromosome 3p25, a region commonly rearranged in various cancers, specially in lung tumor cells. We report here the cloning, by sequence homology to the yeast OGG1, of a mouse cDNA coding for a 8-OH-Gua DNA glycosylase with 84% and 38% identity to the human and yeast relevant proteins, respectively. The Ogg1 gene is localized to the mouse chromosome 6E. The mouse Qgg1 cDNA, when expressed in Eschierichia coli, is capable of suppressing the spontaneous mutator phenotype of a DNA repair deficient fpg mutgamma strain. The mouse Ogg1 protein acts efficiently on duplexes in which the 8-OH-Gua is paired with a cytosine but is inactive on 8-OH-Gua: Ade pair, consistently with its proposed biological role in the avoidance of mutations. A comparison of the mouse enzyme with other eukaryotic Ogg1 enzymes is also presented. The isolation of this gene will allow the development of an animal model to study the effects of oxidative stress on carcinogenesis and degenerative diseases.

Purine metabolism in two human melanoma cell lines: relation to proliferation and differentiation.

Purine nucleotide metabolism was studied in two human cutaneous melanoma cell lines IPC182 and IGR221. IPC182 cells do not differentiate, while IGR221 cells differentiate spontaneously at confluency, with intense melanin production. The activities of 11 enzymes involved in the de novo or salvage synthesis or the catabolic pathway of purine nucleotides were measured at different times (from day 3 to day 18), after subculture, during exponential growth and the stationary phase, with or without differentiation. The results demonstrated remarkable differences in the enzyme activity levels and/or the evolution from exponential growth to the stationary phase for each cell line, as well as between the two cell lines. In the non-differentiating IPC182 cells, the activity of enzymes involved in purine nucleotide synthesis decreased when the growth rate slowed down and remained at a low level with a concomitant increase in catabolic activities. In the differentiating IGR221 cells, the activity of enzymes involved in purine nucleotide salvage synthesis increased during the proliferative phase and was maintained at a high level when the cells reached confluency and differentiated; catabolic activities were always lower than in the IPC182 cells. This suggests that extra purine nucleotides, synthesized preferentially by the salvage pathway, could be required for the differentiation of human melanoma cells. Since the two cell lines were cultured in the absence of any differentiation-inducing agents, these results indicate that various metabolic modifications are associated with the natural processes of cell proliferation and differentiation. This research could help to identify some of the enzymes involved in purine metabolism as the targets for the induction of differentiation.

Patient experiences and preferences: development of practice guidelines in a cancer imaging department.

OBJECTIVE: To improve patient management based on analysis of the results of a survey conducted during their visit to the imaging department of a cancer centre. MATERIALS AND METHODS: A questionnaire comprising 30 single-response questions on a dichotomous scale or a 3- or 4-modality scale was developed by three radiologists specialized in oncology, the head of our quality assurance department, a psycho-oncologist, a psycho-sociologist, a biostatistician and a member of our institute's Patient Committee. Questions concerned reception, information provided about the examinations, examination experiences, the relational qualities and availability of health care professionals, the interview with the radiologist and announcement of the examination results. RESULTS: The questionnaire was given to 190 patients in the waiting room before a standard radiography or ultrasound examination (33%), mammography and breast ultrasound (33%), computed tomography (CT) or magnetic resonance imaging (MRI) (34%). The return rate was 81%. This article analyses the responses to the various questions in terms of either percentages or detailed replies and suggestions. CONCLUSION: Analysis of the patients' experience and their suggestions provided objective elements concerning their real wishes in relation to each step of their management and identified changes and improvements to be made to the organization and daily functioning of the department.

Organization and chromosomal assignment of two human PAG gene loci: PAGA encoding a functional gene and PAGB a processed pseudogene.

A cDNA, designated PAG, was recently isolated by differential cloning between an untransformed and a ras-transformed human mammary cell line. Higher levels of expression were found to be associated with cell proliferation. The absence in the pag protein of known consensus sequence, as well as its close relationship with a gene product involved in the differentiation of a mouse erythroleukemia cell line, has suggested that the PAG gene belongs to a family of genes associated with cell proliferation and differentiation. To further characterize this gene, a physical map has been established from a human genomic cosmid library. The PAG gene spans 13 kb of DNA and contains six exons. The promoter region is GC-rich and contains a TFIID motif located 25 nucleotides upstream of the potential site for initiation of transcription and potential recognition sites for a variety of trans-acting factors. Using fluorescence in situ hybridization, the PAG gene was mapped to human chromosome band 1p34.1. A pseudogene was also isolated, sequenced, and mapped to human chromosome band 9p22.

[The cancerogenic activity of gamma-butyrolactone in mice]. / A propos de l'action cancérigène de la gamma-butyrolactone chez les Souris

Gamma-butyrolactone has been tested for its carcinogenic activity in pure strain XVII/G and C3H Mice. No tumours were observed in mice which received the compound in injections at birth, after skin painting, gavage, or mixed with their food.

Dose-effect studies on estrogen induced mammary cancers in mice.

Castrated male (C3H X RIII) F1 mice were treated with graded doses of estradiol-17-beta and estrone in a life-span experiment. Estradiol-17-beta incorporated into paraffin pellets was implanted under the skin for continuous resorption. These pellets contained 1 microgram, 2.5 microgram, 5 microgram, 10 microgram or 100 microgram of hormone. Estron was given orally, mixed with the food at 3 daily dosages: 0.06 microgram, 0.6 microgram and 6 microgram. Although the smallest dosages of both hormones induced vaginal estrus in castrated females, they did not produce mammary tumors. The mammary tumor incidence reached progressively almost 100% in response to the increase of the hormonal dosages. The absence of effect of low doses of estrogen in mice is compared with the absence of an excess of breast cancers among women using oral contraceptives.

Chromosome anomalies in mammary carcinoma from transgenic WAPRAS mice: compression with human data.

Transgenic WAPRAS mice, obtained by infection of the construct WAP promoter murine gene and the HRAS human protooncogene, develop mammary adenocarcinoma within 1-3 months after pregnancy. A cytogenetic analysis was performed on 17 tumors from 10 mice. Almost all detected anomalies were chromosome gains. The resulting trisomies affected recurrently chromosomes 1, 15, 19, 17, 7, and 12, in decreasing order of involvement. Although in situ hybridization showed that the transgene was integrated in chromosome 1, the duplication of this chromosome did not depend on the presence or absence of the transgene. Comparison with human data indicates that the 3 most frequently duplicated chromosomes in WAPRAS mice correspond to the human chromosome segments most frequently duplicated or amplified in breast cancer, i.e., 1q, 8q, and 11q13. None of the chromosome segments often deleted in human tumors were found to be duplicated in the mouse tumors.

The use of synthetic tandem repeats to isolate new VNTR loci: cloning of a human hypermutable sequence.

Synthetic tandem repeats (STRs) of oligonucleotides have previously been shown to detect polymorphic loci in the human genome. Here, we report results from the use of three such probes to screen a human cosmid library. Nine of the 45 positive clones that were analyzed appear to contain highly polymorphic minisatellite or VNTR loci. The degree of enrichment for minisatellite sequences varied with the choice of STR: one provided a 15- to 20-fold enrichment (4 polymorphic loci among 10 clones), whereas 2 others gave a 3- to 5-fold enrichment (5 polymorphic probes in a total of 35 clones) compared to random screening. The 9 VNTR markers have been localized by linkage analysis in the CEPH panel and/or by in situ hybridization. Eight probes identify new loci, one of which maps to an interstitial region. One of the VNTR loci (identified by probe CEB1) was found to be hypermutable, with 52 mutation events identified among 310 children characterized in 40 CEPH families. The parental origin of the mutation could be identified in all instances, and only one mutation was found to be of maternal origin. The mutation rate in males was estimated to be approximately 15%. Segregation analysis of flanking markers suggests that mutations are not associated with crossing over. As the only previously described hypermutable minisatellite loci in humans have equal rates of male and female mutations, these observations establish that a second type of hypermutable minisatellite exists in the human genome. In neither case does the generation of new alleles appear to be associated with unequal crossing over.

Chromosomal localization and sequence analysis of a human episomal sequence with in vitro differentiating activity.

The genomic fragment carrying the human activator of liver function, previously described as an episome capable of inducing differentiation upon transfection into a dedifferentiated rat hepatoma cell line, was mapped on human chromosome 12q24.2-12q24.3. This chromosomal location was indistinguishable by in situ hybridization from that of the gene coding for the hepatic transcription factor HNF1. The sequence of the integrated form of the episome as well as its flanking sequences show that it is rich in retroposons. It contains a human ribosomal protein L21 processed pseudogene, one truncated L1Hs sequence, and 10 Alu repeats, which belong to different subfamilies.

Evidence for long-range oncogene activation by hepadnavirus insertion.

Insertional mutagenesis of host genes, a common oncogenic strategy of slow transforming retroviruses, has recently been described for a DNA virus of the hepadnavirus group: the woodchuck hepatitis virus. This virus causes insertional activation of myc genes, mainly the intronless N-myc2 oncogene, in > 50% of woodchuck liver tumours. In most remaining tumours, N-myc2 is overexpressed without any apparent genetic alteration. To elucidate the role of the virus in such cases, we have cloned and analysed single integration sites in four woodchuck tumours carrying wild-type myc alleles. All sites were clustered within < 20 kb in a single locus, in which scarce unique sequences showed no detectable transcriptional activity. By fluorescent in situ hybridization, N-myc2 and the new locus (win) were localized to the same region of the long arm of the woodchuck X chromosome, and a 150-180 kb intervening distance was deduced from pulse-field gel analysis. The detection of viral integrations in win in additional tumours that produced abundant N-myc2 transcripts further substantiates the link between these two loci in woodchuck tumorigenesis. We propose that efficient activation of the N-myc2 promoter by the hepadnavirus enhancer acting over a long distance might operate in liver cell transformation.