The rat albumin promoter: cooperation with upstream elements is required when binding of APF/HNF1 to the proximal element is partially impaired by mutation or bacterial methylation.

We have characterized in the accompanying paper (P. Herbomel, A. Rollier, F. Tronche, M.-O. Ott, M. Yaniv, and M. C. Weiss, Mol. Cell. Biol. 9:4750-4758, 1989) six different elements in the albumin promoter. One of them, the proximal element (PE), is the binding site for a strictly liver specific factor, APF/HNF1. This binding site contains a bacterial DAM DNA methylase methylation target sequence which, when methylated, decreases the affinity of the protein for this element. When the different albumin promoter constructions were prepared in an Escherichia coli deoxyadenosine methylase-negative strain, the respective contributions of the elements to the overall promoter activity were strikingly different. An intact proximal element plus the TATA box gave almost full transcriptional activity in transient transfection experiments and only in differentiated hepatoma cells of line H4II, whereas the distal elements (distal element III [DEIII], the NF1-binding site DEII, and the E/CBP-binding site DEI) had become essentially dispensable. Mutations affecting the CCAAT box showed only a two- to threefold decrease. When PE was methylated, mutated, or replaced by the homologous element from the alpha-fetoprotein gene, activity in the context of the short promoter (PE plus the TATA box) was abolished. However, activity was restored in the presence of the upstream elements, showing that cooperation with factors binding to the CCAAT box and distal elements favors the functional interaction of the liver-specific APF/HNF1 factor with lower-affinity binding sites.

The rat albumin promoter is composed of six distinct positive elements within 130 nucleotides.

No fewer than six different positive regulatory elements concentrated within 130 base pairs constitute the rat albumin promoter, which drives highly tissue specific transcription in rat hepatoma cells in culture. Inactivation of each element led to a decrease in transcriptional efficiency: from upstream to downstream, 3- to 4-fold for distal elements III and II, 15-fold for distal element I, and 50-fold for the CCAAT box and the proximal element (PE). Three of these elements, distal elements III and II and, more crucially, the PE, were found to be involved in the tissue-specific character of transcription, with an additional negative regulation possibly superimposed at the level of the PE. Finally, our mapping of these regulatory elements in vivo entirely coincided with footprint data obtained in vitro, thereby allowing the tentative assignment of specific factors to the effects observed in vivo.

Determinants of rat albumin promoter tissue specificity analyzed by an improved transient expression system.

The 150-base-pairs region located upstream of the transcriptional start site of the rat albumin gene contains all of the critical sequences necessary for this gene's tissue-specific expression in rat hepatoma cells. In transient expression assays using an improved CAT system or direct mRNA analysis we were able to detect a faithful transcription from the albumin promoter in albumin-negative dedifferentiated H5 hepatoma cells which was 250-fold weaker than in differentiated H4II hepatoma cells producing albumin. This strong tissue specificity could be completely overcome through the cis action of a non-tissue-specific enhancer. Two upstream regions from nucleotides -151 to -119 and from -118 to -94, were required for efficient transcription in H4II cells. Each region contained a sequence motif highly conserved among different species. The effect of the -151/-119 region was strictly tissue specific, while the -118/-94 region was also involved in the low level of transcription observed in H5 cells. Finally, sequences between the CCAAT box and the TATA box also contributed to the overall tissue specificity of rat albumin gene transcription.

Regulation of albumin gene expression in hepatoma cells of fetal phenotype: dominant inhibition of HNF1 function and role of ubiquitous transcription factors.

Two widely used hepatoma cell lines, mouse BW1J and human HepG2, express gene products characteristic of fetal hepatocytes, including serum albumin, whereas reporter genes driven by the albumin promoter are expressed at very low levels compared with highly differentiated hepatoma cells. We have investigated the low albumin promoter activity in BW1J cells to understand differences in liver gene regulation between fetal and adult cells. Addition of the albumin upstream enhancer, or any other fragment of the albumin gene, failed to modify expression of the transfected promoter in BW1J cells. Analysis of cis elements of the albumin promoter showed that, in contrast to highly differentiated H4II cells, in BW1J cells the activity largely depends on ubiquitous transcription factors. Both BW1J and HepG2 cells produce the liver-enriched transcription factor HNF1; dimerization and DNA binding properties are identical to those of liver HNF1, yet the protein fails to show the anticipated transcriptional stimulatory activity. A transfected HNF1 expression vector strongly trans-activates the albumin promoter in HepG2 but only weakly in BW1J cells, and in hybrids (BW1J x Fao), inefficient HNF1 function is dominant. We conclude that hepatoma cells of the fetal phenotype are deficient in the use of HNF1 to drive transcription of the albumin gene and that they harbor a dominant modulator of HNF1 function.

NFY or a related CCAAT binding factor can be replaced by other transcriptional activators for co-operation with HNF1 in driving the rat albumin promoter in vivo.

Like many eukaryotic genes, the rat albumin promoter contains a CCAAT consensus motif at position -80. In transfected H4II hepatoma cells the strength of this promoter depends to a large extent on the integrity of a hepatic nuclear factor 1 (HNF1) binding site located at position -60 and to a lesser extent on the CCAAT element. However, if the affinity for HNF1 is reduced, the CCAAT-box becomes essential for high, and tissue specific, promoter activity. We wished to determine which, among the different CCAAT binding factors co-existing in eukaryotic cells, was responsible for this co-operativity with HNF1. To this end we prepared a series of mutants of the CCAAT sequence and compared their effects on albumin promoter activity in vivo and on the binding of different CCAAT binding factors in vitro. Our results strongly suggest that a ubiquitous factor NFY (also designated CBF, ACF, CP1) interacts with this CCAAT element in vivo. We propose that during development NFY could facilitate transcription of the albumin gene in hepatocytes when the concentration of HNF1 is limiting. This co-operativity in transcriptional activation is not due to strict co-operativity in DNA binding between the two proteins and is not limited to NFY or a closely related factor, as the CCAAT-box can be replaced by AP1, SP1 or E2 target sites without significantly affecting the final activity.

Human genome dispersal and evolution of 4q35 duplications and interspersed LSau repeats.

We have investigated the evolutionary history of the 4q35 paralogous region, and of a sub-family of interspersed LSau repeats. In HSA, 4q35 duplications were localized at 1q12, 3p12.3, 4q35, 10q26, 20cen, whereas duplicons and interspersed LSau repeats simultaneously labeled the p arm of acrocentric chromosomes. A multi-site localization of 4q35-like sequences was also observed in PTR, GGO, PPY, HLA (Hominoidea) and PAN (Old World monkey), thus indicating that duplications of this region have occurred extensively in the two clades, which diverged at least 25 million years ago. In HSA, PTR and PAN, 4q35-derived duplicons co-localized with rDNA, whereas in GGO and PPY this association was partially lacking. In PAN, the single- and multi-site distribution of rDNA and paralogous sequences, respectively, indicates a different timing of sequence dispersal. The sub-family of interspersed LSau repeats showed a lesser dispersal than 4q35 duplications both in man and great apes. This finding suggests that duplications and repeated sequences have undergone different expansion/contraction events during evolution. The mechanisms underlying the dispersal of paralogous regions may be further derived through studies comparing the detailed structural organization of these genomic regions in man and primates.

An international overview of sterilization.

PIP: Sterilization, both male and female, for family planning purposes developed very rapidly, beginning in Puerto Rico and Japan between 1940 and 1950 and continuing in India and Pakistan on a large scale after the mid-1950s. The legal status of voluntary sterilization around the world is confused, but recent developments show a trend toward liberalization of the laws. Certain countries still consider sterilization punishable under the criminal law, but there is a gap between the law and the practice. Certain countries have made the issue of consent relevant under the law. Certain other governments are studying the question of sterilization officially. Countries with heavy population pressures are employing incentives to encourage sterilization and others, specifically European countries, have legally authorized sterilization. The procedure is becoming increasingly accepted.^ieng

Common hepatic artery aneurysm: diagnosis with duplex sonography and color Doppler.

Hepatic artery aneurysms are uncommon lesions, often with a nonspecific clinical presentation and difficult to diagnose before rupture. The authors report a case which was correctly diagnosed with non-invasive procedures (duplex sonography and color Doppler).

[The problem of abortion in the countries of the European Community]. / Le probleme de l'avortement dans les pays de la Communaute europeenne.

PIP: The abortion laws in effect and being considered in several European countries are summarized briefly (Scandinavian and Eastern European countries, Switzerland, Austria); others are more comprehensively reviewed, including dates, content and individual sponsors of proposed laws (Denmark, England, France, Belgium, Luxembourg, Holland, West Germany, Italy). Denmark and England have liberal abortion laws; Denmark permits abortion on demand up to 12 weeks but to residents only, but England performs about 25% (24,000/year) of its abortions on foreigners. Illegal abortions are estimated at 500,000-1,000,000/year in Germany, 250,000-800,000/year in France, and 30,000-50,000/year in Belgium. All of these countries have repressive abortion laws, usually prescribing 5 years in prison and/or a fine for the woman, and 5 years in prison with suspension of license for the practitioner. Convictions are rare in comparison with illegal abortions not prosecuted. A controversial conviction was that of Dr. Willy Peers, Professor of Gynecology of Brussels, who was imprisoned for performing an abortion on a mentally retarded girl raped by her father. Germany, France, Holland, and Belgium each have proposed liberal legislation during 1971-1973. A condemnation of abortion by Pope Paul has prompted 2 proposed bills in the Italian assembly which stand, however, little chance of being ennacted.^ieng

[Voluntary female sterilization in Europe and the world]. / La stérilisation volontaire féminine en Europe et dans le monde.

Voluntary sterilization is worldwide used about 138 millions women in reproductive ages, not only in the developing world. In the North American continent this method is very popular, and most European countries having legalized contraceptives and abortion consider sterilization as a necessary complementary measure for family planning purposes.

[Contraception: yes, but...]. / Contraception: oui, mais...

PIP: Since the Neuwirth law of 1967 legalizing contraception in France, the apparent fear of the legislature that the birth rate will fall further or the population will become promiscuous has prompted many restrictions. The law has not affected the birth rate, which has stabilized after a falling trend since 1920. The young people are sexually active at increasingly earlier ages, despite restrictions on contraception for minors. The 1967 law permits sale of contraceptives exclusively in pharmacies; not even family planning clinics may dispsense them. Prescriptions must be accompanied by a form "carnet a souche" putting the name and age of the user on file in the health department. Prescriptions may be renewed only for 1 year. Minors under 18 must have written parental consent, as must women 18-21 who want oral contraceptives. These restrictions do not apply to the territories of Martinique, Guadeloupe and Reunion. Restrictions on IUDs cover type of establishment, facilities, physicians' licenses, forms filed with the health department. Information on contraception is spread primarily by private nonprofit associations such as the French Movement for Family Planning, but with small financial resources and no government assistance. In 1972, 2 types of family planning counseling centers were established by the National Assembly: centers for information, consultation or family counseling, and centers of planning or family education. The required staff (gynecologist, psychiatrist, midwife, social worker and family counselor) and facilities of these centers have been set by law.^ieng

The legal battle for reproductive rights in Europe.

PIP: Thanks largely to medical progress, restrictive family planning laws have been increasingly viewed as an intolerable limitation of individual freedom and as intolerable state interference in private life. Most European countries have reformed their legislation to take both medical improvements, e.g. vacuum suction abortion, and the needs of society into account. In France, Belgium, Luxembourg, and the Netherlands, Napoleonic Code sanctioned the segregation of sexes in conjugal and family relations. Due to this legal background and the Catholic culture, very strong taboos developed concerning sexual life. It has taken a long time and a tough fight to establish modern legislation. In France, the family planning debate began in 1965. In December, 1974, a new law made contraceptives widely available, even to minors, and reimbursable by the Social Security system. Abortion was legalized in January, 1975, after the election of Giscard d'Estaing. Sterilization is not illegal. In 1975 the Committee of Ministers of the Council of Europe recommended that member governments make voluntary sterilization available. Scandinavian countries are in the forefront of fertility control. Both Denmark and Sweden have liberal abortion laws allowing the operation up to the 12th week. Poland, Bulgaria, Hungary, Romania, Czechoslovakia, and the German Democratic Republic adopted liberal abortion laws from 1950 to 1960. In most Eastern European countries voluntary sterilization is not practiced.^ieng

Isolation and functional analysis of the promoter of the bovine serum albumin gene.

The bovine serum albumin (bSA) promoter has been cloned from bovine genomic DNA using the polymerase chain reaction. In common with other albumin promoters, this promoter functions efficiently in the differentiated rat hepatoma cell line H4II and not in the its dedifferentiated derivative, H5. Analysis of 5' deletions of the bSA promoter after transient transfection into H4II has revealed that a short construct containing the HNF1 binding site and TATA box functions efficiently but requires the presence of the more upstream sequences to achieve full activity Footprint analysis of the promoter reveals seven sites of DNA protein interaction extending from -31 to -213. One of these sites, extending from -170 to -236, whose deletion results in a four fold increase in promoter activity. This site has not previously been reported in other albumin promoters and is bound by the C/EBP-like family of proteins.

Mutants of Bacillus subtilis temperature sensitive in the outgrowth phase of spore germination.

Thirteen thermosensitive mutants of Bacillus subtilis defective in the outgrowth phase of spore germination were isolated. The spores of the mutants grow into vegetative cells at 35 C but not at 47 C, whereas the vegetative cells grow equally well at both temperatures. At 47 C all the mutant spores are able to initiate germination, but the process stops at an early phase of outgrowth in one strain and in a late phase in the other 12 strains. The spore of the latter gives rise to a swollen cell unable to divide. In all mutants, the normal phenotype is restored when the spores are grown in the presence of 20% sucrose or 2% NaCl. The synthesis of deoxyribonucleic acid and proteins does not seem to be altered in the mutants giving swollen cells. The mutants were grouped into three distinct genetic classes by transformation.

[Vasectomy: legal recognition in Italy, opposition to change in France]. / La vasectomie: liberalisation en Italie, immobilisme en France.

PIP: Voluntary sterilization exclusively for contraceptive purposes is now legal in many countries of Asia, North America, and Europe. Since 1972 most European countries have taken action through statutes, codes of medical ethics, decrees, or court decisions to establish the legality of contraceptive sterilization. France and Italy, neighboring countries with Catholic majorities and a tradition of Roman law, have had great difficulties in recognizing the legality of vasectomy. Italy was 1 of the few countries of the world with a law specifically prohibiting voluntary sterilization. The 1930 law was abrogated by Parliament in 1978, but in 1982 a physician was prosecuted for performing 50 vasectomies on men who had sought the procedure and consented to it. Prosecution argued that sterilization was not decriminalized despite abrogation of the 1930 law, and that voluntary sterilization was prohibited by the article of the penal code dealing with voluntary assault and battery. The interpretation surprised experts in population law, since the physician had received permission from the regional government of Tuscany to perform the vasectomies. The physician won the case, lost it in the Court of Appeals in Florence, and was finally acquitted in the Court of Cassation after a 5-year legal struggle. The court declared explicitly that voluntary sterilization did not constitute a penal infraction, but it recommended legislation to establish a minimum age, assure informed consent, and protect the interests of the spouse. In France, the legality of sterilization for purely contraceptive purposes has not always been explicitly recognized by the courts. Some recent verdicts have held that sterilization without therapeutic motivations is illicit, and some local committees of medical ethics continue to argue that vasectomy is an illicit practice that should only be considered in the presence of precise medical indications. The position is contrary to a resolution of the Committee of Ministers of the Council of Europe which France voted for in 1975, and a resolution of the National Council of the Order of Physicians in 1983. The resolution of the Order relaxed its earlier opposition to sterilization on the grounds that operative techniques had improved and that the chances for successful reversal had improved. It argued that sterilization should only be performed for very serious nonmedical reasons and required informed consent, spousal notification, and a waiting period of 2 months. The resolution has however been largely ignored. Although vasectomies are performed in numerous hospitals in France, their legal status remains ambiguous.^ieng

[Abortion].

PIP: The historical and current (1969) abortion laws in France as well as those in other Western countries are analyzed. France has had a series of punitive abortion codes since the Napoleonic Code of 1810 prescribing solitary confinement for the woman. The reforms of 1920 and 1923 made provocation of abortion or contraceptional propaganda a "crime" (felony), later a "delit" (misdemeanor), called for trial before magistr ate instead of jury, but resulted in only about 200 convictions a year. The decree of 1939 extended the misdemeanor to women who aborted even if they were not pregnant, and provided for professional licenses such as that of surgeon or pharmacist to be suspended. The law of 1942 made abortion a social crime and increased the maximum penalty to capital punishment, which was exercised in 2 cases. About 4000 per year were convicted from 1942-1944. Now the law still applies to all who intend to abort, whether or not pregnant or successful, but punishemnt is limited to 1-5 years imprisonment, and 72,000 francs fine, or suspension of medical practice for 5 years. About 500 have been convicted per year. Since 1955 legal abortion has been available (to about 130 women over 4 years) if it is the only means to save the woman's life. Although pregnancy tests are controlled, the population desregards the law by resorting to clandestine abortion. The wealthy travel to Switzerland (where 68% of legal abortions are done on French women) or to England. Numbers are estimated by the French government at 250,000-300,000 per year, or 1 for every 2 live births, but by hospital statistics at 400,000-1,000,000 per year. The rest of the review covers abortion laws in Scandinavian, Central European, and individual US states as of 1969.^ieng

[Presidential address at the 20th Journees sur la Fertilite et l'Orthogenie]. / Adresse de la Presidente des 20es Journees sur la Fertilite et l'Orthogenie.

PIP: Recollections of the author's early experiences in French family planning and reflections on the ethical issues and legal status of new fertility technologies are the major topics of this address. The group that was to become le Mouvement francais pour le Planning Familial (MFPF) was founded in 1956 by the author and others and opened its first family planning center in 1961, before contraception became legal. Despite opposition from physicians and Catholic clergy, the MFPF operated 100 centers serving over 100,000 clients by 1965. In 1969 the MFPF began a study of abortion. Despite legalization, the controversy concerning the legitimacy of abortion has not subsided. More recently, the practices of artificial insemination with donor sperm, in vitro fertilization, surrogate mothers, and other medically assisted fertility technologies have raised moral, ethical, and legal questions for all societies. The questions range from the legitimacy of gamete donation to the status of unused embryos. The dissociation of different stages of the reproductive process affects concepts of filiation, paternity and maternity. In the absence of appropriate legislation, it has been left largely to the courts to create a new body of law at this frontier. The review of legislation in France, elsewhere in Europe, and in the U.S. concerning contested filiation in cases of artificial insemination, the legitimacy of insemination of a woman after the death of her spouse, the preservation and donation of embryos, surrogate motherhood, the child's rights to information about his biological parents, and other questions indicate that no consensus has yet emerged concerning the admissibility and legitimacy of specific practices. It would be highly desirable for the European countries to adopt a common legislation in these areas.^ieng

Anatomy of the rat albumin promoter.

The sequences preceding the albumin mRNA start site are able to direct efficient transcription only upon introduction into cells expressing the endogenous albumin gene. In transient expression assays, the activity of a reporter gene (CAT) linked to this promoter is 100-fold higher in H4II differentiated hepatoma cells than in H5 dedifferentiated cells which no longer express their albumin gene. This tissue specificity depends on the very proximal promoter region, composed of a CCAAT box, the proximal element and a TATA box. Deletion of the CCAAT box leads to a two- to threefold decrease in activity, deletion of the proximal element (PE) results in loss of activity. The PE is a high-affinity binding site for HNF1/APF, a strictly liver specific trans-acting factor. When the affinity of this factor for PE is decreased by bacterial methylation (PE includes a dam methylase site), by mutation, or by its replacement with the homologous element from the alpha-fetoprotein gene (AFP), the activity of the short promoter (PE plus the TATA box) is abolished. This activity can be rescued in the presence of the more upstream elements: DEII, DEI and the CCAAT box (recognized, respectively, by the NF1/CTF, C/EBP and NFY/ACF factors) which are then absolutely required. Our results suggest that the upstream elements contribute to promoter activity by stabilizing the HNF1-PE complex and not by direct interaction with TFIID or the RNA polymerase. It is probable that these elements, essentially dispensable in already differentiated hepatoma cells, play a crucial role during development or differentiation to activate the promoter in cells that contain a low concentration of HNF1 and/or an HNF1 unable to open inactive chromatin alone.

Gene amplification in fibroblasts from ataxia telangiectasia (AT) patients and in X-ray hypersensitive AT-like Chinese hamster mutants.

In search of functions involved in the regulation of gene amplification, and given the relevance of chromosome breakage in initiating the process, we analyzed the gene amplification ability of cells hypersensitive to inducers of DNA double-strand breaks and defective in cell cycle control: two human fibroblast strains derived from patients affected by ataxia telangiectasia (AT) and two hamster mutant cell lines belonging to complementation group XRCC8 of the rodent X-ray-sensitive mutants. These mutants are considered hamster models of AT cells. To measure gene amplification, the frequency and the rate of occurrence of N-(phosphonacetyl)-L-aspartate resistant cells were determined. In both hamster mutants, these two parameters were increased by about an order of magnitude compared with parental cells, suggesting that amplification ability was increased by the genetic defect. In primary AT fibroblasts, as in normal human fibroblasts, gene amplification was undetectable and a block in the G(1) phase of the cell cycle was induced upon PALA treatment. These results suggest that in AT fibroblasts, where only the ATM gene is mutated, ATM-independent mechanisms prevent gene amplification, while, in the immortalized hamster cell lines, which are already permissive for gene amplification, the AT-like defect increases the probability of gene amplification.