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Objective: The objective of this study is to present the characteristics of urological cancers in the Hérault department based on data of this department's cancer registry., Methodology: We analysed the course of all urological data over a 14-year period, from 1987 to 2000, based on the Hérault cancer registry database. Incidence and mortality chronological trends, the risk of cancer before the age of 75 years, and regional estimations were calculated for each cancer site. Data for each cancer site were also compared to French, European and international data., Results: 9,556 urological cancers were reported over a period of fourteen years: 8,779 in males and 777 in females. Urological cancers represented 30.1% of all male cancers and 3.6% of all female cancers. The probability of developing a genital cancer before the age of 75 years for a man was 12.6% and the probability of a urinary tract cancer was 3.2% in males and 0.8% in females. The incidence of prostate cancer has more than tripled over this 14-year period, while the mortality has remained stable. The incidence of renal cancers in both sexes is also increasing, while that of bladder cancers is decreasing. The incidence of cancers of the penis, testis and upper urinary tract has remained stable over time.

Background: Despite the fact that new drugs have emerged from clinical research in urothelial cancer during the last decade, the prognosis of patients with advanced disease remains poor with a median survival of 12 to 14 months. We designed a feasibility study of gemcitabine and oxaliplatin (GO) in patients with advanced urothelial cancer., Patients and Methods: Twenty patients received bimonthly cycles of gemcitabine 1500 mg/m2 and oxaliplatin 85 mg/m2. The cycles were given at 2-week intervals without G-CSF support. Thirteen patients were treated with the GO combination as first-line chemotherapy because of a poor performance status or a creatinine clearance < 1 ml/s., Results: The median number of cycles of GO was 5 (1-7). The median number of days between cycles was 14 throughout the treatment. Seven (8%) out of 87 cycles had to be delayed because of neutropenia or asthenia. A 25% dose reduction in the doses of cytotoxic drugs was necessary in 2 patients. Chemotherapy was stopped before the sixth cycle because of an early death related to a myocardial infarction in 1 patient, a grade 3 neuropathy in 1 patient and a progressive disease in 9 patients., Conclusion: Using these doses and schedules, the GO regimen appears a safe therapy for patients with advanced urothelial cancer. Phase II studies are required to assess the possible role of this combination in advanced urothelial cancer.

Ageing is linked to the accumulation of replicatively senescent cells. The best model system to date for studying human cellular ageing is the progeroid Werner's syndrome (WS), caused by a defect in WRN, a recQ-like helicase that also possesses exonuclease activity. In this paper, we characterise the interaction between WRN and an essential replication factor, PCNA. We show that wild-type WRN protein physically associates with PCNA at physiological protein concentrations in normal cells, while no association is seen in cells from patients with WS. We demonstrate co-localisation of WRN and PCNA at replication factories, show that PCNA binds to two distinct functional sites on WRN, and suggest a mechanism by which association between WRN and PCNA may be regulated in cells on DNA damage and during DNA replication.

One of the ultimate goals of biological research is to understand mechanisms of cell function within living organisms. With this in mind, many sophisticated technologies that allow us to inspect macromolecular structure in exquisite detail have been developed. Although knowledge of structure derived from techniques such as X-ray crystallography and nuclear magnetic resonance is of vital importance, these approaches cannot reveal the remarkable complexity of molecular interactions that exists in vivo. With this in mind, this review focuses on the use of microscopy techniques to analyze cell structure and function. We describe the different basic microscopic methodologies and how the routine techniques are best applied to particular biological problems. We also emphasize the specific capabilities and uses of light and electron microscopy and highlight their individual advantages and disadvantages. For completion, we also comment on the alternative possibilities provided by a variety of advanced imaging technologies. We hope that this brief analysis of the undoubted power of microscopy techniques will be enough to stimulate a wider participation in this rapidly developing area of biological discovery.

Human aging is associated with accumulation of cells that have undergone replicative senescence. The rare premature aging Werner's syndrome (WS) provides a phenocopy of normal human aging and WS patient cells recapitulate the aging phenotype in culture as they rapidly lose the ability to proliferate or replicate their DNA. WS is associated with loss of functional WRN protein. Although the biochemical properties of WRN protein, which possesses both helicase and exonuclease activities, suggest an involvement in DNA metabolism, its action in cells is not clear. Here, we provide experimental evidence for a role of the WRN protein in DNA replication in normally proliferating cells. Most importantly, we demonstrate that in the absence of functional WRN protein, replication forks from origins of bidirectional replication fail to progress normally, resulting in marked asymmetry of bidirectional forks. We propose that WRN acts in normal DNA replication to prevent collapse of replication forks or to resolve DNA junctions at stalled replication forks, and that loss of this capacity may be a contributory factor in premature aging.

It is widely assumed that the vital processes of transcription and translation are spatially separated in eukaryotes and that no translation occurs in nuclei. We localized translation sites by incubating permeabilized mammalian cells with [3H]lysine or lysyl-transfer RNA tagged with biotin or BODIPY; although most nascent polypeptides were cytoplasmic, some were found in discrete nuclear sites known as transcription "factories." Some of this nuclear translation also depends on concurrent transcription by RNA polymerase II. This coupling is simply explained if nuclear ribosomes translate nascent transcripts as those transcripts emerge from still-engaged RNA polymerases, much as they do in bacteria.

Epidemiological and experimental studies suggest the involvement of lipid peroxidation (LPO) in retinal diseases. Clinicians usually prescribe antioxidants to help in the treatment of proliferative diabetic vitreoretinopathy and age-related macular degeneration. In spite of this, these processes inexorably induce visual impairment and may progress towards blindness. In addition to other pathogenic mechanisms not fully understood, it may be that peroxidic aldehydes from LPO occurring in the eyes, acting as cytotoxic chemicals, mediate in these chronic disorders. To test the mechanisms of removing peroxidic aldehydes from retinal cells and in an attempt to understand long-lasting changes induced by LPO, the distribution and activity of aldehyde dehydrogenases (ALDH) in the rat retina were studied and compared with the LPO sites induced by iron/nicotine adenine dinucleotide phosphate. Histochemical and immunocytochemical assays revealed the colocalization of LPO and ALDH, mainly in the photoreceptors and inner retinal layers. This suggests the involvement of ALDH in detoxifying peroxidic aldehydes from the retina. Any change in ALDH retinal expression and distribution might be of crucial importance in assessing the paths of LPO-mediated vitreoretinopathies. Further research is needed to evaluate these findings and their application to new ophthalmic therapy., (Copyright 2000 S. Karger AG, Basel)

The control of RNA synthesis from protein-coding genes is fundamental in determining the various cell types of higher eukaryotes. The activation of these genes is driven by promoter complexes, and RNA synthesis is performed by an enzyme mega-complex-the RNA polymerase II holoenzyme. These two complexes are the fundamental components required to initiate gene expression and generate the primary transcripts that, after processing, yield mRNAs that pass to the cytoplasm where protein synthesis occurs. But although this gene expression pathway has been studied intensively, aspects of RNA metabolism remain difficult to comprehend. In particular, it is unclear why >95% of RNA polymerized by polymerase II remains in the nucleus, where it is recycled. To explain this apparent paradox, this review presents a detailed description of nuclear RNA (nRNA) metabolism in mammalian cells. We evaluate the number of active transcription units, discuss the distribution of polymerases on active genes, and assess the efficiency with which the products mature and pass to the cytoplasm. Differences between the behavior of mRNAs on this productive pathway and primary transcripts that never leave the nucleus lead us to propose that these represent distinct populations. We discuss possible roles for nonproductive RNAs and present a model to describe the metabolism of these RNAs in the nuclei of mammalian cells.-Jackson, D. A., Pombo, A., Iborra, F. The balance sheet for transcription: an analysis of nuclear RNA metabolism in mammalian cells.

Recent evidence suggests that active RNA polymerases are concentrated in discrete 'factories' where they work together on many different templates. The evidence that such factories specialize in the transcription of particular groups of genes is reviewed.

The path that RNA takes through nuclear pores was mapped using two high-resolution techniques. Unexpectedly, no RNA in HL60 cells was detected by immunogold labelling in the central axis of the pore complex on its way to the transporter at the nuclear membrane; instead, it was distributed around the sides, apparently entering just before the membrane. In rat liver nuclei, poly(A)(+) RNA, hnRNPs A1 and C, mrnp 41, ASF, and a phosphorylated subset of SR proteins were also distributed like mRNA, as were various transport factors and their cargoes (NTF2, Ran, RCC1, karyopherin (beta), Rch1, transportin (alpha), m(2,2,7)-trimethylG). Many pores were associated with particular transport factors/cargoes to the exclusion of others; some were associated with poly(A)(+) RNA or phosphorylated SR proteins (but not NTF2), others with NTF2 (but not poly(A)(+) RNA or the SR proteins). Electron spectroscopic imaging confirmed these results. Some pores contained phosphorus-rich RNA apparently entering from the sides; others lacked any phosphorus, and were surrounded by a ribosome-free zone in the cytoplasm. The results also suggest that pores have different functional zones where SR proteins are dephosphorylated, and where hnRNP C is removed from messages.

Efficient centromeric and multicopy vectors have been constructed for the yeast Kluyveromyces marxianus using homologous ARS and centromere sequences. A homologous promoter of a purine-cytosine permease gene called PCPL3 has been cloned, using an expression system based on GUS. Its strength has been estimated in K. marxianus by putting the homologous beta-glucosidase gene under its control. This promoter is very efficient as activities higher than the ones obtained with the Saccharomyces cerevisiae PGK promoter were obtained. This promoter appears to be constitutive in various conditions tested. Its five transcription start sites have been mapped, and a derivative expression vector for K. marxianus has been constructed.

Some SR proteins are associated with eukaryotic transcripts as they move from synthetic sites (transcription "factories"), through downstream sites, to nuclear pores. Downstream sites can also be isolated as large nuclear ribonucleoprotein particles of approximately 200 S (diameter approximately 50 nm). In ultrathin sections of HeLa nuclei, indirect immunogold labeling with a specific antibody gives many small clusters of approximately 10 gold particles (diameter 50-80 nm). We gauged errors in estimating the diameter of underlying structures marked by immunogold probes (lengths approximately 20 nm). We examined systematically how probe dimensions affected cluster diameter. Probes contained one to three immunoglobulin molecules, sometimes a protein A molecule, and a gold particle of 5-15 nm. We found that (a) immunolabeling particles were tightly packed, (b) reducing particle size by 5 nm reduced cluster diameter by 10 nm, (c) reducing the number of immunoglobulins in the immunolabeling sandwich from three to two reduced cluster diameter by approximately 4 nm, (d) replacing the last immunoglobulin in a sandwich with protein A increased diameter by approximately 7 nm and led to a peripheral concentration of particles, and (e) increasing the number of layers in the sandwich increased sensitivity. Assuming that underlying structures had diameters of 50 nm, we find that errors ranged from -20% to +50%.

Using HeLa cells, we have developed methods to determine 1) the number of RNA polymerases that are active at any moment, 2) the number of transcription sites, and 3) the number of polymerases associated with one transcription unit. To count engaged polymerases, cells were encapsulated in agarose, permeabilized, treated with ribonuclease, and the now-truncated transcripts extended in [32P]uridine triphosphate; then, the number of growing transcripts was calculated from the total number of nucleotides incorporated and the average increment in length of the transcripts. Approximately 15, 000 transcripts were elongated by polymerase I, and approximately 75,000 were elongated by polymerases II and III. Transcription sites were detected after the cells were grown in bromouridine for <2.5 min, after which the resulting bromo-RNA was labeled with gold particles; electron microscopy showed that most extranucleolar transcripts were concentrated in approximately 2400 sites with diameters of approximately 80 nm. The number of polymerases associated with a transcription unit was counted after templates were spread over a large area; most extranucleolar units were associated with one elongating complex. These results suggest that many templates are attached in a "cloud" of loops around a site; each site, or transcription "factory," would contain approximately 30 active polymerases and associated transcripts.

Current models for RNA synthesis involve an RNA polymerase that tracks along a static template. However, research on chromatin loops suggests that the template slides past a polymerase immobilized in a large transcription factory. The evidence for immobilized polymerases is reviewed, and a model for transcription by such fixed enzymes is presented. According to the model, gene activation would involve reducing gene-factory distance and increasing the affinity of a promoter for a factory. Locus controlling regions and enhancers would attach to a factory and increase the chances that a promoter could bind to a polymerase; after transcriptional termination, the gene would detach from the factory. As some RNA processing occurs cotranscriptionally, processing sites are also likely to be associated with the factory.

Two cases of cystic tumour are reported. The first consisted of a pseudohaemorrhagic cyst of the right adrenal gland discovered during aetiological assessment of HT refractory to medical treatment. Plasma and urinary assays did not reveal any abnormality suggestive of secreting adrenal tumour. Imaging (ultrasonography. CT and MRI) was in favour of a necrotic malignant tumour and a normal isotope scan (MIBG iodine 131) eliminated phaechromocytoma. Adrenalectomy was easily performed via a subcostal laparotomy and the postoperative course was uneventful. The second case consisted of a right adrenal cyst detected incidentally on ultrasonography. The laboratory assessment demonstrated only a slight elevation of urinary metanephrine and imaging (CT and MRI) was not suggestive of a malignant lesion. Simple annual CT follow-up was decided in this case. The various pathological types of rare cystic lesion of the adrenal gland are described with particular emphasis on their diagnostic and therapeutic modalities.

The authors report 3 cases of fibroepithelial polyps, one of which was an incidental finding and 2 were symptomatic, presenting in the form of macroscopic haematuria and chronic back pain, respectively. These lesions required 2 nephroureterectomies because of their renal repercussions or their multifocal nature, combined with segmental resection of the ureter, including the base of the tumour. In the light of these cases, the authors review the literature and analyse the current management of this rare disease, in particular the indications for endourological techniques which appear to have a major diagnostic contribution as a complement to IVU and retrograde urography, but whose therapeutic value has yet to be defined.

Objectives: To evaluate the morbidity of renal transplant biopsies performed after simple ultrasonographic identification of the transplants, using a Vim-Silverman or Tru-cut needle., Methods: From January 1987 to April 1991, 360 renal transplant biopsies were performed after simple ultrasonographic identification of the transplants, using a Vim-Silverman (n = 204) or Tru-cut (n-156) needle. In 221 transplants, these biopsies were performed because of a rise of serum creatinine (n = 319) or proteinuria (n = 17) or were even performed systematically (n = 24). One to 5 (mean = 1.6) transplant biopsies were performed systematically and the interval between renal transplantation and biopsy varied between 3 days and 11 years., Results: 290 biopsies (80.6%) allowed the analysis of a minimum of 3 glomeruli (mean = 9.3). The yield of the Vim-Silverman needle was significantly greater than that of the Tru-cut model (p = 0.02). 147 biopsies (50.7%) demonstrated acute or chronic rejection, 57 (19.7%) revealed cyclosporin nephrotoxicity, 41 (14.1%) showed acute tubular necrosis and 14 (4.8%) showed glomerulopathy, while 31 (10.7%) were strictly normal. The morbidity of these biopsies was reflected by 37 complications (10.3%), including 30 minor and 7 major complications (2 cases of haemoperitoneum, 4 cases of obstructive anuria and 1 arteriovenous fistula). However, only one case required transplantectomy. These problems were significantly more frequent following inadequate biopsies (< 3 glomeruli, purely medullary, extra-renal)., Conclusion: Despite the considerable risk of iatrogenic lesions, these biopsies were justified by their potential diagnostic and therapeutic benefit. The prophylaxis of complications of this procedure is based on strict respect of blood pressure and haematological criteria and on real-time ultrasound monitoring of the biopsy and miniaturization of the trocars. The treatment of severe complications has been greatly improved by the development of endourology and interventional radiology, but surgery, and especially transplantectomy, is still occasionally required.

The streptokinase gene of the Streptococcus pyogenes strain CIP 56.57 was cloned and sequenced. This sequence coding for a 441 amino acid protein is well conserved among streptococcus species: there are two very conserved domains separated by a more variable region.

From 1972 to 1993, we carried out 803 consecutive renal transplants including 8 third transplants. Exclusively cadaveric, these third renal transplants were implanted by intraperitoneal approach in right iliac position, without previous homolateral transplantectomy in 5 cases. The arterial anastomoses were common (7) or external iliac and hypogastric (1), and the venous anastomoses external (1) and common iliac (3), or inferior vena cava (4). Restoration of urinary continuity was by ureteronecystostomy (Politano-Leadbetter = 4, Grégoir-Lich = 3) or ureteroureteric anastomosis (1). The level of HLA compatibility varied from 2 to 5 identities (mean 3.1) and 4 of the 7 patients explored were hyperimmunized with lymphocytotoxic antibody levels > or = 80%. With the exception of the first of these third transplants, the immunosuppressive protocol associated azathioprine, prednisolone, antilymphocytic serum and cyclosporin. Postoperative sequels were marked by 3 vascular rejections and 1 death from hyperkalemia. Moreover, 1 urinary fistula on ureteroureteric anastomosis settled after percutaneous nephrostomy and placing of an uteric stent endoprosthesis. With a postoperative follow-up of 8-32 months (mean 24), 5 of the transplanted patients (62.5%) have a functional renal transplant with a serum creatinine from 120 to 180 microM/l (mean 140). This brief series, whose failures are exclusively immunological, reveals the remarkable technical reliability for these third renal transplants in right iliac implantation, by median transabdominal approach and above a former transplant site.

Two fragments containing both an autonomous replicating sequence (ARS) and a centromere have been isolated and sequenced from the yeast Kluyveromyces marxianus. The ARS and centromeric core sequences are only 500 bp apart, but ARS activity could be separated from the centromeric sequences. Centromeric sequences are organized in a similar way to those of budding yeasts: two well-conserved elements: CDEI (5' TCACGTG 3') and CDEIII (5' TNTTCCGAAAGTWAAA 3'), are separated by a 165 bp AT-rich (+/- 90%) CDEII element whose length is twice that of Saccharomyces cerevisiae CDEII but almost identical to that of K. lactis. The ARS-core consensus sequence (5' TTTATTGTT 3') is also similar to that of K. lactis. Both ARS and centromeric elements function in this strain, albeit inefficiently, but not in S. cerevisiae. A third ARS-containing fragment with a different organization has been isolated and sequenced.

The endocytic-lysosomal system of proliferating and differentiated astrocytes in primary culture was investigated using a combination of cytochemical, immunocytochemical and biochemical procedures. These included impregnation with osmium tetroxide and potassium iodide, phosphotungstic acid staining, cytochemical demonstration of acid phosphatase and thiamine pyrophosphatase activities and incorporation of cationized ferritin. The acid phosphatase activity was also analyzed using biochemical techniques. Our results indicate that while all astrocytes in primary culture have a developed endocytic-lysosomal system, this system is different in proliferating cells from that in differentiated astrocytes. Whereas in proliferating astrocytes it appears to be composed mainly of a variety of vacuoles and vesicles displaying a heterogeneous osmium tetroxide staining pattern, differentiated cells are characterized by the presence of small size vesicles showing an intense reaction. Both types of astrocyte showed abundant lysosomes, including multivesicular bodies, which presented an intense phosphatase acid activity. Biochemical analyses demonstrated that this activity increase during the proliferation period, reaching a maximum at 15 days of culture. Incorporation of cationized ferritin revealed that lysosomes and endosomes constitute separate systems. Finally, we have also found that the activity of thiamine pyrophosphatase, a marker for the Golgi complex, increases throughout the culture period. These results indicate that astrocytes could play an important role in regulating the macromolecular composition of the extracellular space.

Kluyveromyces marxianus can be transformed with an efficiency of 10(5) transformants/microgram of DNA by a replicative plasmid using electroporation. In order to obtain this efficiency, we isolated ura- mutants cells which can be complemented by the URA3 gene from Saccharomyces cerevisiae. The URA3 gene and KARS2, a replicative origin from Kluyveromyces lactis which functions in K. marxianus, were ligated together in a plasmid which can be used as a vector to transform this strain.

The authors report a case of leiomyosarcoma of the infrarenal inferior vena cava presenting as an abdominal mass. CT and MR imaging and immunotyping led to the diagnosis of retroperitoneal leiomyosarcoma whose origin in the inferior vena cava was only confirmed by histological examination of the resection specimen. Surgical resection was followed by radiotherapy (4,500 rads) with a favourable course after 12 months. The progress in medical imaging and histology has considerably simplified the diagnosis of these rare retroperitoneal tumours. In the absence of any truly effective adjuvant therapy, treatment essentially consists of surgery and the clinical course is characterised by a high incidence of local recurrences.

This study has investigated the effect of prenatal alcohol exposure on the qualitative and quantitative ultrastructure of proliferating and differentiated astrocytes in primary cultures as well as on the cytochemical activity of several subcellular phosphatase markers, including acid phosphatase, uridine diphosphatase, thiamine pyrophosphatase, 5'-nucleotidase and glucose-6-phosphatase. The astrocytes were obtained from 21-day-fetuses of both control and alcohol-fed rats. Our results show that several cell components, such as mitochondria, rough endoplasmic reticulum and lysosomes, exhibit qualitative and/or quantitative ultrastructural changes during the process of astrocyte maturation. In some cases these morphological changes are accompanied by variations in the cytochemical activity of enzymes located in these and other cell components, suggesting that these enzymes, and therefore the functional state of these organelles, are modulated during astrocyte development. When prenatally exposed to ethanol, both proliferating and differentiated astrocytes showed striking ultrastructural alterations compared with controls, including an increment of lysosomes as well as a decrease in the values of stereological parameters relative to mitochondria, rough endoplasmic reticulum and Golgi apparatus. Cytochemical analysis of these cells indicates that prenatal exposure to ethanol decreased the activities of all the enzymes tested, except for acid phosphatase, which was increased in both groups of treated astrocytes. These results suggest that prenatal exposure to ethanol could affect astrocytes during development in two different but probably complementary ways: a) by causing a delay in astrocyte maturation and, b) by inducing a direct toxic effect on these cells.

We report about two cases of cancer of the testis occurring 2 and 7 years after Hodgkin's disease treated with combined chemotherapy and radiation therapy. From an etiopathogenic point of view, this association may, of course, be accidental, but it may also have an iatrogenic origin or be caused by contiguous carcinogenesis, or even by an immune deficiency. In addition to orchidectomy, the therapeutic history of these patients requires an adaptation of the treatment of their tumors of the testes.

From January 1984 to January 1989, 139 kidneys were retrieved from 74 brain dead donors in our institution. The transplantation was performed either locally (79), or in an other French institution (40). The five year actuarial survival rate, for the 139 kidneys retrieved in Montpellier, was 65 percent. Many factors about the donor, the retrieval and the recipient, which may affect the graft survival, were entered in a Cox multivariate analysis. The minimal follow up duration was 18 months. The risk factors studied included: donor parameters (age, sex, cause of death, haemodynamic parameters and renal function); retrieval parameters (kidney alone or multiorgan harvesting, discoloration and renal perfusion quality); organ characteristics (multiple arteries and cold ischemia time); recipients parameters (age, sex, prior transplantation, local transplantation or not, and HLA matching). A first multivariate analysis included only pretransplant risk factors. The risk factors for graft loss, as identified by the Cox model, were in the order: donor's age (P = 0.03), arterial pressure (P = 0.01), prior transplantation of the recipient (P = 0.01) and kidney discoloration quality during the retrieval (P = 0.008). Early post transplant parameters were included within this Cox model (poor early renal function, need for dialysis, serum creatinine level at one week). The need for dialysis therefore was identified as the main predictive value (P = 0.002). The 4 other risk factors, selectioned in the first model, always remained significant.

This paper reports the DNA sequence of a segment of 9.8 kb of the chromosome III. The sequenced DNA contains the MAT alpha locus. The new sequence of the MAT alpha locus differs from the previously reported sequence by six modifications in the W segment. We have found the same modifications in the HML locus. The corrected sequence contains, in HML, an open reading frame (ORF) of 190 codons which ends at the border between the W segment and the flanking DNA. In the MAT locus, this ORF extends in the flanking DNA up to 538 codons. This ORF corresponds to a gene independently identified as BUD5 (Chant et al., 1991). This gene presents homologies with the exchange factors SDC25 and CDC25. A large ORF of 1399 codons is found on the opposite side of MAT alpha (toward the telomere). This ORF corresponds to a new gene YCR724. Next to this gene is a small ORF, YCR725, of 127 codons. The localization of this fragment on chromosome III, originally supposed to be distal from the MAT locus based on genetic distance, illustrates variation in recombination frequency along the chromosome and suggests the existence of hot spots of recombination between MAT and the THR4 locus.

In order to study the regulation of expression of the iso2-cytochrome c gene, we have constructed a fused gene between the 5'flanking region of the gene coding for the yeast iso2-cytochrome c and the coding region of the E. coli beta-galactosidase lacZ gene. When introduced in yeast cells this hybrid gene is expressed and regulated like the production of iso2-cytochrome c: it is under the control of the general catabolic repression and of the unlinked trans-acting CYP1 gene whose CYP1-18 allele causes an overproduction of iso2-cytochrome c. The expression of hybrid genes whose upstream region has been progressively shortened or altered by internal deletions was studied either in wild-type CYP1+ cells or in cells carrying the CYP1-18 allele grown either on glucose or on glycerol. It appears that the expression and the regulation of the iso2-cytochrome c gene is controlled by an upstream regulatory site composed of a positive and a negative element. This site is the target of regulation by the CYP1 gene product and, directly or through this gene, of the control by the general catabolic repression.

The relationship between the promoter length of the Kluyveromyces fragilis beta-glucosidase gene and the level of its expression in Saccharomyces cerevisiae was studied by gene fusion between deleted promoter fragments of various lengths and the promoterless beta-galactosidase gene of Escherichia coli. The removal of a region from position -425 to -232 led to a tenfold increase in the expression of the gene. The same results were obtained for the reconstructed beta-glucosidase gene with the same promoter length. It is likely that the deletion of this part of the promoter removes negative regulatory elements which are functional in Saccharomyces cerevisiae. This increase in activity is the main event which may explain the high increase in gene expression (60-fold) previously observed for an upstream deletion obtained during subcloning experiments of the beta-glucosidase gene. It is also shown that the expression of the gene greatly depends upon the nature of the recipient strain, the growth phase of the cell and that of the vector carrying it.

Two forms of yeast RNA polymerase A are resolved by phosphocellulose chromatography. One of these, called RNA polymerase A, is lacking two polypeptide chains of 48,000 and 37,000 daltons. The properties of the two enzymes are compared in the present paper. RNA polymerase A transcribes d(A-T)n with a similar efficiency as the complete enzyme, but it is comparatively much less active with native DNA. The two enzymes can also be differentiated on the basis of their ionic strength and divalent cation requirements. RNA polymerase A has a particularly low activity at high salt and low Mg2+ concentrations. Thermal inactivation curves of the two enzymes are different when residual activity is assayed with native DNA. In contrast with d(A-T)n as template the apparent inactivation curves of the two enzymes are identical. The data suggest that the two dissociable polypeptide chains play an important role in transcription. The template specificity of yeast RNA polymerase B was further investigated using SV40 DNA-FI as template. RNA polymerase B is able to retain [3H]SV40 DNA-FI on nitrocellulose filters but the enzyme-DNA complex is very unstable. The observation that RNA polymerase B can transcribe to some extent a supercoiled DNA but not a linear double stranded template supports the hypothesis that the enzyme needs some unpaired DNA structure to initiate transcription.