CYP77A19 and CYP77A20 characterized from Solanum tuberosum oxidize fatty acids in vitro and partially restore the wild phenotype in an Arabidopsis thaliana cutin mutant.

Cutin and suberin represent lipophilic polymers forming plant/environment interfaces in leaves and roots. Despite recent progress in Arabidopsis, there is still a lack on information concerning cutin and suberin synthesis, especially in crops. Based on sequence homology, we isolated two cDNA clones of new cytochrome P450s, CYP77A19 and CYP77A20 from potato tubers (Solanum tuberosum). Both enzymes hydroxylated lauric acid (C12:0) on position ω-1 to ω-5. They oxidized fatty acids with chain length ranging from C12 to C18 and catalysed hydroxylation of 16-hydroxypalmitic acid leading to dihydroxypalmitic (DHP) acids, the major C16 cutin and suberin monomers. CYP77A19 also produced epoxides from linoleic acid (C18:2). Exploration of expression pattern in potato by RT-qPCR revealed the presence of transcripts in all tissues tested with the highest expression in the seed compared with leaves. Water stress enhanced their expression level in roots but not in leaves. Application of methyl jasmonate specifically induced CYP77A19 expression. Expression of either gene in the Arabidopsis null mutant cyp77a6-1 defective in flower cutin restored petal cuticular impermeability. Nanoridges were also observed in CYP77A20-expressing lines. However, only very low levels of the major flower cutin monomer 10,16-dihydroxypalmitate and no C18 epoxy monomers were found in the cutin of the complemented lines.

Inhibition of proliferation of primary avian fibroblasts through expression of histone H5 depends on the degree of phosphorylation of the protein.

To obtain stable and constitutive expression of histone H5 at levels comparable to those observed in normal chicken erythrocytes, an avian self-inactivating retroviral vector was used to transfer the H5 gene into cells which do not express this protein. The vector, pDAH5, was obtained by removing the CAAT and TATA boxes of the 3'LTR of the avian leukosis virus RAV-2 and inserting the H5 sequence. Infection of QT6 quail cells with the recombinant virus (DAH5) led to the stable integration of the foreign H5 gene at low copy number, to the formation of correctly initiated mRNA transcripts and to the production of H5 protein. The amount of H5 expressed was equivalent to that of a mature chicken erythrocyte. Expression of histone H5 in DAH5 transformed cells, such as QT6 or AEV-ES4, transformed chicken embryo fibroblasts had only slight effects on the growth rate and did not inhibit cell replication. Conversely, the effect of H5 expression on normal quail and chicken fibroblasts was dramatic: cells acquired the aspect of quiescent fibroblasts, grew very slowly, and nuclei looked compacted, often extruded from the cell. The H5 histone produced in QT6-transformed cells was found to be phosphorylated while in normal chicken fibroblasts the protein lacked this posttranslational modification. It is proposed that the chromatin-condensing role of histone H5 is inhibited by its phosphorylation.

Synthesis and translation site of light-induced mRNAs in etiolated Euglena gracilis.

Poly(a)-mRNA synthesis has been studied in etiolated Euglena gracilis exposed to 1 or 2 h of illumination. 1. Labeling kinetics of mRNAs containing poly(A) sequences, during illumination or after return to darkness, reach a plateau in 10 or 20 min according to nutritional conditions. When the cultures are returned to darkness, the mRNA synthesis decreases rapidly. Thus, the synthesis of these mRNAs (light-induced mRNAs) is dependent on light and their half-life can be evaluated. 2. Cycloheximide induces accumulation of label in poly(A)-containing mRNA; such an accumulation is not observed after addition of lincomycin. Labeling during illumination of mRNA in a chloroplast mutant is similar to that in the wild type strain. These data suggest that the poly(A)-mRNAs synthesized in the two first hours of illumination are translated on cytoplasmic polyribosomes.

Molecular cloning and sequencing of a cDNA encoding a beta-thyroid hormone receptor in muscovy duckling.

A cDNA clone encoding a beta-thyroid hormone receptor (TRbeta) from muscovy duckling liver was isolated and sequenced. Comparison with the chicken TRbeta sequence showed a high degree of homology. This cDNA was used as a probe to characterize the TRbeta mRNA transcripts expressed in muscovy duckling liver.

Evolution of the primate beta-globin gene region. High rate of variation in CpG dinucleotides and in short repeated sequences between man and chimpanzee.

A 5500 base-pair fragment including the beta-globin gene downstream from codon 122 and about 4000 base-pairs of its 5' flanking sequence was cloned from chimpanzee DNA and thoroughly sequenced before being compared with the corresponding human sequence: 88 point differences (83 substitutions and 5 deletions or insertions of 1 base-pair) were detected as well as seven more important deletion/insertion events. These changes occur preferentially in two kinds of structure. First, 40% of the CpG dinucleotides present in either human or chimpanzee sequences are affected by nucleotide variations. This corresponds to a divergence level considerably higher than that expected. Second, most short repeated sequences found in the 5' extragenic sequence are involved in mutational events (amplification or contraction of the number of basic motifs as well as point substitutions or deletions/insertions of 1 base-pair). Considering the very low level of nucleotide sequence divergence between these two closely related species, our data provide direct evidence for CpG and tandem array instability.

In situ expression of helper-free avian leukosis virus (ALV)-based retrovirus vectors in early chick embryos.

Defective avian leukosis-based vectors expressing the bacterial lacZ gene were used as helper-free preparations to infect early stage Brown-Leghorn embryos. Both in toto X-gal staining and DNA analysis using Southern blot technique were applied to detect virus integration and expression. Our results demonstrate a low efficiency of in vitro infection in early stages of embryonic development. Southern blot analysis reveals that only 1% of embryonic cells integrate the vector genome after infection using 2 to 12 virus particle per embryonic cell. In situ expression of the lacZ marker gene was detected in only 0.06% of embryonic cells. These results lead us to conclude that only 6% of infected cells express efficiently the lacZ marker gene. This low level of expression could result from avian leukosis virus LTRs inhibition in chicken embryonic cells at an early stage of development. In spite of the low efficiency of infection, no evidence for tissue restrictive expression was observed. However, vector containing LTRs from RAV-2 virus allows preferential expression of provirus vector in neural tube tissue, whereas cardiac localization of the preferential expression was observed using vector containing the RAV-1 LTRs. The chronological analysis of the marker gene expression in terms of location of expression foci and sizes of these foci, lead us to hypothesize the putative regulation of retrovirus expression linked to embryonic development.

CG sites and their nucleotide environment in the human gamma-delta-beta-globin gene cluster: distribution, frequency and possible frame for differential methylation.

Available restriction endonucleases including CG dinucleotides in their target sequences (most of them being unable to cut the DNA when the cytosine of the CG sequence is methylated) have been used to map cloned DNA covering the human gamma-delta-beta globin gene cluster. Since the human DNA fragments were cloned in Escherichia coli, only the internal cytosine in the sequence CCAT GG could be methylated. Thus, any recognized "CG enzyme" site can be detected since they are unmethylated. Results show that frequencies of "CG enzyme" sites regularly decrease from the gamma-globin region to the beta-globin region, the latter being very poor in "CG enzyme"' sites. The array of enzymes used here detects 4 times more CG sites than the classical MspI/HpaII system. Examination of previously sequenced parts of the gamma-delta-beta globin gene cluster shows that CG dinucleotides correspond to an average frequency of 1 out of 104 nucleotides in the gamma-globin region and 1 out of 138 nucleotides in the beta-globin region. In the gamma-globin region, 1 CG out of 4 or 5 may be detected by the enzymes used; the detected frequency is less than 1 out of 10 CG in the beta-region. Analysis of nucleotide environment around CG dinucleotides shows occurrence of local differences, the main sequences being CGG in the 5' side flanking the gamma genes and ACG in the corresponding area of the beta gene. The results presented introduce some new considerations about analysis of cytosine methylation which has been previously proposed as playing a role in the control of the activity of gamma, delta and beta genes respectively.

Intratesticular inoculation of avian leukosis virus (ALV) in chickens--production of neutralizing antibodies and lack of virus shedding into semen.

In order to investigate the possibility of producing transgenic chickens by injection of avian leukosis virus-based vectors into testis, we have analyzed the infection rate of testicular cells following inoculation of Rous-associated virus type 1 (RAV-1) into the gonads of adult and 1-wk-old brown leghorn males. Viroproduction, neutralizing antibody production, and vital DNA presence in testis, blood, muscle, and semen were analyzed at various times after infection. Inoculation of RAV-1 into the gonads of adult males resulted in a low level of viroproduction in testis and blood, followed by the appearance of neutralizing antibody 2 or 3 wk later. Neither viroproduction in semen nor viral DNA presence in sperm were detected even though the infected chickens were found to produce RAV-1 in testis. One week after intratesticular inoculation of 1-wk-old males with RAV-1, a high level of viroproduction was found in blood and testis, and viral DNA was detected in gonadal cells. Further, by 6 wk after inoculation, the production of virus decreased in all tissues, viral DNA could not longer be detected in the testis, and neutralizing antibodies appeared in blood. All together these data show that it is possible to infect testicular cells by direct inoculation of RAV-1 in the testis, and that the immune response of both adult and young chickens seems to reduce this infection. Moreover, no evidence of spermatozoa infection was found; this result suggests that RAV-1 inoculation into testis may not induce genetic transmission of virus, and consequently would not be useful in the production of transgenic chickens.

Identification and structure analysis of endogenous proviral sequences in a Brown Leghorn chicken strain.

In a Brown Leghorn chicken strain, four endogenous proviral loci have been identified. The DNA mapping data show strong homology between their structures and that of the Rous-associated virus O (RAV-O) genome. Two of them seem similar to ev3 and ev6 loci previously described in White Leghorn chickens; the two others are unknown in White Leghorns. Using DNA amplification methods, envelope genes of these endogenous viral structures have been partially sequenced. The results demonstrate that subgroup-specific sequences of the endogenous loci were largely homologous with those of RAV-O.

Structure and expression of endogenous retroviral sequences in the permanent LMH chicken cell line.

From DNA mapping data, four endogenous proviral loci have been observed in the chicken permanent cell line LMH. The locus corresponding to endogenous virus (ev) ev1 is present in duplicate whereas the locus corresponding to ev3 is present in one copy. The other loci are probably ev6 and a solitary long terminal repeat. A RNA Northern blot analysis revealed both ev3 and ev6 transcripts but no ev1 transcript was detected. Using avian leukosis virus (ALV)-based vectors, transcomplementing assays were performed. They demonstrate the correct expression and maturation of endogenous env proteins and the absence of production of functional gag and pol components, indicating that these cells are not competent for viral production.

Characterization of a cDNA encoding an alpha thyroid hormone receptor in muscovy duckling.

A complementary deoxyribonucleic acid (cDNA) clone encoding an alpha thyroid hormone receptor (TR alpha) from muscovy duckling liver was isolated and sequenced. Comparison with the chicken TR alpha sequence showed a high degree of homology. Despite 45 nucleotide substitutions, the deduced peptide sequence was similar. This cDNA was used as a probe to characterize the TR alpha mRNA transcripts expressed in muscovy duckling liver and skeletal muscle.

[Functional outcome of laparoscopically transposed ovaries in the multidisciplinary treatment of cervical cancers. Analysis of risk factors]. / Avenir fonctionnel des ovaires transposés par coelioscopie dans le traitement multidisciplinaire des cancers du col utérin. Analyse des facteurs de risque.

OBJECTIVES: To evaluate the place of ovarian transposition by laparoscopy in the treatment of cervical cancers. METHODS: From March 1992 to November 1994 at Institut Bergonié, 11 patients (mean age: 40 years; 36-44 years) with invasive squamous cell carcinoma of the uterine cervix stages Ib (4 cases) and IIb (7 cases) underwent lateral high ovarian transposition by laparoscopy performed during a staging inter-iliacal lymphadenectomy. There was no complication during surgery but one phlebitis occurred postoperatively. The treatment for the cervical cancer included: brachytherapy (11 cases), external beam radiotherapy (EBRT) (9 cases), surgery (6 cases), chemotherapy (2 cases). Ovarian radiation dosis was calculated and hormonal status assessed. RESULTS: Ovarian preservation was achieved in 30% of the cases. The mean lowest cumulative dosis to the ovaries was 1.78 Gy. Age was the most predictive factor for ovarian preservation. CONCLUSION: With ovarian laparoscopic transposition, ovarian function can be preserved in selected patients requiring first line radiotherapy for cancer of the cervix. After the age of 40 years, transposition should be restricted to small T1 tumors treated by brachytherapy. When EBRT is required for larger lesions, transposition should be reserved to younger patients.

The delta-beta-crossing-over site in the fusion gene of the Lepore-Boston disease might be localized in a preferential recombination region.

The delta-beta-fusion gene of heterozygous Lepore-Boston patient contains a large intervening sequence (IVS2) which could derive entirely from the beta-gene. This conclusion was obtained using a specific beta-IVS2 probe to analyse the physical map of the Lepore-Boston DNA which has shown the presence of a specific beta-AvaII site at the 5' end of the delta-beta-Lepore fusion gene. Thus, the crossing-over between delta- and beta-globin genes to explain the delta-beta-Lepore recombination could be localized between the delta-codon 87 and the 5' end of the beta-IVS2. The second protein-encoding region might represent a preferential recombination position in disorders associated with structurally abnormal globin chains resulting from fusion genes.

Colonic granulometry in the malabsorption syndromes.

After transit through the small intestine barium enters the large intestine and its characteristics in that gut segment can be studied. The pattern of distribution of barium in the colon is always altered in patients with malabsorption syndromes. The physical basis for this alteration is analysed in a manner analagous to that used in soil mechanics. The dispersal of barium granules within the liquid-solid content of the colon is related to certain factors among which is the polarisation and electrical potential of barium particles. A technique using four radiographs which permits evaluation of barium dispersal in the colon - colonic granulometry - is described. Lastly, the authors point out the necessity of classical barium studies in identifying the rare anatomical anomalies that can be the starting point of a typical clinical coeliac syndrome.

S1-nuclease mapping of the genomic Lepore-Boston DNA demonstrates that the entire large intervening sequence of the fusion gene is of beta-type.

Several reports have suggested but not proven that the large intervening sequence of Lepore delta-beta fusion gene was of beta-type (3-5). A method able to detect rearrangements as small as 4 nucleotide pairs directly into genomic DNA (6) has been applied to the total DNA of a heterozygous Lepore-Boston patient in order to identify accurately the origin of the large intervening sequence of the delta-beta fusion-gene. Hybrid duplexes were formed between genomic Lepore DNA and single-stranded DNA used as probes, then submitted to S1-nuclease treatment. Our data demonstrate that the entire large intervening sequence of the Lepore fusion gene is of beta-type. Moreover, no large modification was detected in any delta- and beta parts of the delta-beta fusion gene.

Analysis of conserved and non-conserved amino acids critical for ALSV (Avian leukemia and sarcoma viruses) integrase functions in vitro.

Retroviral integrase (IN) is the viral enzyme responsible for the integration of viral DNA into host cellular DNA. In vitro, recombinant IN protein is able to catalyze the 3'-processing, strand transfer and disintegration activities. In order to analyze the importance of specific residues of ALSV (Avian leukemia and sarcoma viruses) IN protein, we introduced 31 amino acid substitutions either in residues previously shown by others to be involved in IN oligomerization or in selected conserved and non-conserved residues through the IN sequence. We tested, in vitro, the three catalytic activities of these mutants as well as their capacity to bind DNA. We found that (i) 88% of the substitutions occurring on well-conserved residues have an effect on IN activities (ii) two mutants (S85T in the central catalytic domain and N197C in the C-terminal domain) present a reduced efficiency of DNA binding compared to the wild type protein. Moreover, all mutations made on the dimer interface of C-terminal domain present reduced activities, suggesting an important role of this part of the protein. Finally, for some mutations, we observed differences between the ALSV and HIV (Human immunodeficiency virus) IN corresponding residues.

Influence of expression and cis-acting sequences from avian leukosis viruses (ALVs) on stability of (ALV)-based retrovirus vectors.

Defective avian leukosis virus (ALV)-based vectors expressing the neo and LacZ genes were constructed under the control of cis-acting elements originated from 4 avian retroviruses: avian erythroblastosis virus (AEV), Rous associated viruses 1 (RAV-1) and 2 (RAV-2), and the Schmidt Ruppin strain of Rous sarcoma virus subgroup D (SR-RSV-D). We used these vectors to study the long-term stability of beta-galactosidase expression (encoded by the LacZ gene) in a permanent cell line from quail fibroblasts (QT6). Infection of the immortalized QT6 cell line with these vectors resulted in unstable beta-galactosidase expression. We determined whether this instability of provirus expression was correlated with: (1) presence of G418 selection; (2) deletion in the proviral genome; (3) hypermethylation of the proviral genome; (4) position of the neo and LacZ genes in the proviral genome; and (5) the transcriptional activity of the long terminal repeat (LTR) elements of proviral vectors. We observed that G418 selection pressure applied to infected QT6 cells lead to a more stable LacZ gene expression. Moreover, our results suggest a correlation between the stability of proviral gene expression and the level of gene expression driven by the LTR elements and depending on the strain origin of these.