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Photoperiod controls several responses throughout the plant life cycle, like germination, flowering, tuber formation, onset of bud dormancy, leaf abscission, and cambium activity. From these processes, flowering has been most extensively studied, especially in Arabidopsis thaliana. Photoperiod sensing by the function of photoreceptors and the circadian clock appears to regulate flowering time via Arabidopsis CONSTANS (AtCO), a putative transcription factor that accelerates flowering in response to long days. The genetic factors controlling plant photoperiodic responses other than flowering are little known. However, interspecific grafting experiments demonstrated that the flower-inducing (florigen) and tuber- inducing (tuberigen) signals are functionally exchangeable. Here we show that constitutive overexpression in potato of the Arabidopsis flowering-time gene AtCO impairs tuberization under short-day inductive conditions; AtCO overexpressing lines require prolonged exposure to short days to form tubers. Grafting experiments using these lines indicated that AtCO exerts its inhibitory effect on tuber formation by acting in the leaves. We propose that a conserved photoperiodic functional module may be involved in controlling distinct photoperiod-regulated evocation responses in different species. This module would involve the action of CONSTANS in the production of the elusive and long-distance acting florigen-tuberigen signal(s) in the leaves.

Solanum tuberosum ssp. andigena plants require a short-day (SD) photoperiod for tuber formation, a process that is also affected by gibberellins (GAs). Grafting experiments have confirmed that the photoperiod is perceived in the leaves. Tuber formation, however, usually takes place in the underground stolons. In this review, photoperiod-dependent tuberization has been divided into five chronological events: SD photoperiod perception, short-term adaptive responses to SD conditions, generation and transport of tuber-inducing signal(s), tuber formation, and long-term adaptive responses to tuber growth. Within this frame of study, the interaction of GAs and photoperiod is revised. Similar to the flowering process in Arabidopsis, we suggest the existence of two independent pathways that control tuber formation: a photoperiod-dependent pathway and a GA-dependent pathway. Nevertheless, photoperiod-dependent tuber formation requires the action of GAs at specific stages to orchestrate this complex process of development.

Background: Spinal tuberculosis can produce kyphosis with neurologicaldeficit, despite antibiotic treatment. When there is no response to medical treatment, the recommended procedure is debridementand interbody fusion with bone autograft. The biological characteristicsof Mycobacterium tuberculosis do not prevent osteosynthesisof the infected bone from being performed., Patients and Method: Five patients with spinal tuberculosis and neurological deficitunderwent debridement, interbody fusion and anterior osteosynthesisin addition to medical treatment. In order to ensure stability, posterior fusion was also performed in three patients. All 5 patientsdisplayed weakness and paralysis of their lower extremities, two patients suffered an L4 radiculopathy, one developed paraparesiaand one was excluded due to a short follow-up. Mean value of vertebralkyphosis was 22,8 degrees and mean follow-up was 3,1 years., Results: No patient had septic loosening or progression of the disease. Correction of kyphosis was 104,5% postoperatively and 80,5% atthe end of follow-up. All patients, apart from one with an L4radiculopathy, exhibited neurological recovery., Conclusions: Anterior instrumentation allows spinal decompression, septic focusdebridement, deformity correction and autologous bone grafting. In severe kyphotic flattening, it is advisable to associate alimited posterior arthrodesis. When pathological fractures appearor there is no response to antibiotic treatment, the combinationof medical and surgical treatment improves patients' outcome.

S. tuberosum ssp. andigena potato plants require short days (SD) for tuberization. We have isolated PHOR1 (photoperiod-responsive 1), which shows upregulated expression in induced leaves (SD). PHOR1 encodes an arm repeat protein with homology to the Drosophila segment polarity protein armadillo. Antisense inhibition of PHOR1 produces a semidwarf phenotype similar to that of GA-deficient plants, and the antisense lines show reduced GA responsiveness combined with a higher endogenous GA content than wild-type plants. Feedback regulation of GA biosynthetic genes is also altered in these lines. Conversely, transgenic lines overexpressing PHOR1 show an enhanced response to GA. GA application induces rapid migration of PHOR1-GFP protein to the nucleus. Thus, PHOR1 appears to be a general component of GA signaling pathways that relocalizes to the nucleus in the presence of GA.

Polymerase chain reaction (PCR) methods are very useful techniques for the detection and quantification of genetically modified organisms (GMOs) in food samples. These methods rely on the amplification of transgenic sequences and quantification of the transgenic DNA by comparison to an amplified reference gene. Reported here is the development of specific primers for the rapeseed (Brassica napus) BnACCg8 gene and PCR cycling conditions suitable for the use of this sequence as an endogenous reference gene in both qualitative and quantitative PCR assays. Both methods were assayed with 20 different rapeseed varieties, and identical amplification products were obtained with all of them. No amplification products were observed when DNA samples from other Brassica species, Arabidopsis thaliana, maize, and soybean were used as templates, which demonstrates that this system is specific for rapeseed. In real-time quantitative PCR analysis, the detection limit was as low as 1.25 pg of DNA, which indicates that this method is suitable for use in processed food samples which contain very low copies of target DNA.

Since 1994 an extensive epidemic of infections with Salmonella enteritidis (S. enteritidis) has affected Chile. In order to understand the diversity of infective sources, the possible origin of the epidemic, and the epidemiological relationships between clinical, food, and poultry isolates, we carried out phage typing of three groups of samples: 1) 310 S. enteritidis clinical samples collected between 1975 and 1996, 2) 47 food isolates obtained during S. enteritidis outbreaks, and 3) 27 strains isolated in surveillance studies of poultry-raising establishments. With the clinical samples, a total of 13 phage types were identified, 2 isolates could not be typed, and 1 was considered atypical. The phage types that were identified most frequently were 1 (56.8%) and 4 (31.3%), trailed by type 8 (4.8%) and type 28 (1.9%). Over time and in different regions of the country there were major changes in the distribution of the phage types. In the first years of collection the only phage types registered were 8 and 28, which disappeared around 1980 and then began reappearing sporadically in 1996. With the gradual S. enteritidis expansion that started in 1988, in the central and southern areas of the country phage type 4 began to appear; that type had not been found before in Chile. In 1991 in the northern area of the country phage type 1 began to predominate; it was another type that had not been reported before in Chile. In the food isolates the only phage types identified were 1 and 4, which were also the most common in the poultry isolates. Phage typing of S. enteritidis has proved to be useful in guiding the epidemiological analysis of the infections caused by this pathogen.

Background: Salmonella enteritidis (SE) is a frequent cause of diarrhea, and is transmitted mainly by SE contaminated eggs or poultry meat. The frequency of SE contaminated eggs or chicken meat and the risk for acquiring this pathogen is unknown in Chile., Aim: To measure SE contamination in eggs poultry meat and entrails offered in retail markets in the Metropolitan Area during two consecutive years (1998-1999)., Material and Methods: Samples were placed in sterile bags and transported to the laboratory before 4 hours at 4 degrees C. Microbiologic detection was done using a standard procedure and an immunodetection assay., Results: SE was found in one of 1081 egg samples (0.09%). The contaminated sample was offered in a supermarket under their own commercial name. Six percent of 1154 poultry meat samples were contaminated by SE and 2.3% by other Salmonella serotypes. Entrails had even higher rates with 10.2% of 370 samples harboring SE and 2.7% other serotypes. Total Salmonella sp. isolates and SE isolates declined during 1999. Nine SE phagotypes were identified, predominating types 4 and 7., Conclusions: Eggs and other avian products are contaminated by different SE phagotypes and other Salmonella serotypes, implicating a risk for the consumers (Rev Méd Chile 2000; 128: 1075-83).

Up to three gibberellin (GA) 20-oxidase genes have now been cloned from several species including Arabidopsis, bean (Phaseolus vulgaris), and potato (Solanum tuberosum). In each case the GA 20-oxidase genes exhibit different patterns of tissue expression. We have performed extensive northern analysis on one of the potato GA 20-oxidase genes (StGA20ox1), which is the only one that shows significant transcript levels in leaves. We show that levels of StGA20ox1 transcript are elevated in transgenic antisense plants that have reduced levels of phytochrome B (PHYB) compared with wild-type plants, implicating PHYB in the control of GA biosynthesis. We show that StGA20ox1 transcript levels vary in leaves of different age throughout the plant and cycle throughout the day, furthermore they are up-regulated by light and down-regulated in the dark. The degree of the response to the light-on signal is similar in potato plants deficient in phytochrome A or PHYB and wild-type plants. The induction of StGA20ox1 by blue light raises the possibility that a blue light receptor may be involved in the control of this gene by light.

Gene StGA20ox1 encoding potato GA 20-oxidase is expressed to relatively high levels in leaves and regulated by daylength. To investigate whether this gene is involved in photoperiodic regulation of tuber formation, we have obtained transgenic potato plants expressing sense and antisense copies of the StGA20ox1 cDNA. Over-expression of this cDNA resulted in taller plants that required a longer duration of a short day photoperiod (SD) to tuberize. Tubers from these plants had a decreased time of dormancy and developed sprouts with elongated internodes. Plants expressing antisense copies of the StGA20ox1 cDNA had shorter stems, a decreased length of the internodes and tuberized earlier than control plants, showing increased tuber yields. Antisense inhibition of this gene had no visible effect on the time of dormancy of the tubers, although at the end of dormancy these formed sprouts with shortened internodes. Decreased levels of endogenous GA20 and GA1 were detected in the apex and first leaves of the antisense lines. These results demonstrate the involvement of the GA 20-oxidase activity encoded by StGA20ox1 in the control of stem elongation and in tuber induction but not in tuber dormancy, indicating that the latter may be regulated by another member of the gene family.

Comparative analysis by differential RNA display (DDRT-PCR) of the expression patterns of potato plants induced and non-induced to tuberize, led to the isolation of a cDNA clone, C40.4, that is strongly upregulated in the leaves of tuberizing plants. Leaf expression of this transcript was shown to be light-dependent, with increased levels of mRNA and protein being detected during the light hours. Sequence analysis revealed near complete identity to potato CDSP34, a thylakoid protein induced by drought stress, and strong homology to the carotenoid-associated proteins fibrillin, ChrB and PAP from pepper fruit, and CHRC from cucumber flowers. By using an antibody against pepper fibrillin, we were able to demonstrate a chloroplastic location of the C40.4 protein in association with the thylakoid membranes. Two-dimensional analysis of thylakoid pigment-protein complexes showed a specific association of the protein with the photosystem II (PSII) multi-subunit complex. Antisense plants with reduced levels of accumulation of C40.4 showed a stunted growth and decreased tuber yield and exhibited reduced values of non-photochemical quenching of chlorophyll a fluorescence. Altogether, these results indicate a preferential association of the C40.4 protein with the light harvesting complex of photosystem II (LHCII) antenna pigment complexes, and suggest a functional role of this protein in photosynthesis, by modulating photosynthetic efficiency and dissipation of excess absorbed light energy within the antenna complex.

Tuber formation in potato (Solanum tuberosum) is promoted by short photoperiods and is inhibited by gibberellins (GAs). Endogenous levels of GA1 were shown to decrease in stolons and leaves of potato plants induced to tuberize, which suggests that photoperiodic regulation of GA biosynthesis may play a role in tuber induction. We report the isolation of three potato cDNA clones (StGA20ox1-3) encoding GA 20-oxidase, a key regulatory enzyme in the GA-biosynthetic pathway. Using northern analysis, we detected a differential pattern of tissue-specific expression of the mRNAs corresponding to these clones. StGA20ox mRNAs were also very abundant in leaves of the potato ga1 mutant, which is blocked in the 13-hydroxylation step, and were strongly down-regulated by gibberellic acid, suggesting a feedback regulation of these genes. In plants grown in short-day (inductive) conditions, levels of the StGA20ox transcripts in leaves fluctuated during a 24-h period, with a peak of accumulation observed about 4 h after the lights were turned off. Interruption of the night with a 30-min "night break" of light (noninductive conditions) did not have a marked effect on the levels of accumulation of the three GA 20-oxidase mRNAs during the day, but it induced a second peak of expression of StGA20ox1 and StGA20ox3 transcripts late in the night. This observation, together with the finding that StGA20ox1 mRNA is expressed at high levels in leaves, suggests that night-break induction of this gene might play a role in the control of tuberization by regulating endogenous levels of GAs in response to daylength conditions.

A partial cDNA clone for the potato wound-inducible metallocarboxypeptidase inhibitor (PCI) was isolated from a cDNA library constructed from mRNA of abscisic acid (ABA)-treated potato leaves. The full 5' region of the cDNA was obtained through a RACE-PCR protocol. PCI mRNA encodes a precursor polypeptide which comprises a 29 residue N-terminal signal peptide, a 27 residue N-terminal pro-region, the 39 residue mature PCI protein, and a 7 residue C-terminal extension. Northern blot analysis demonstrates that the PCI gene is transcriptionally activated by wounding, and wound signaling can be induced by ABA and jasmonic acid. Subcellular localization of the protein was investigated by immunocytochemistry and electron microscopy, showing that PCI accumulates within the vacuole. A partial PCI precursor form, comprising the mature protein and the C-terminal extension, has been expressed in Escherichia coli and characterized. Its inability to inhibit carboxypeptidases, and stability to carboxypeptidase digestion, suggest that the C-terminal pro-domain may have, besides a probable vacuolar sorting function, a role in modulation of the inhibitory activity of PCI.

We have used the degenerated oligonucleotide primers-PCR (DOP-PCR) technique to determine the presence of Ty1/copia-related retrotransposons in the wild species of tomato, Lycopersicon chilense. Using degenerated oligonucleotides corresponding to highly conserved domains in the Ty1/copia retrotransposons, fragments of roughly 300 bp were obtained by PCR amplification. These were cloned in a plasmid vector and the nucleotide sequence determined for 20 clones, 19 of which showed sequence homology to retrotransposon-related sequences. Comparison of the deduced amino-acid sequence of these clones with those reported for other retrotransposons has allowed their classification into four distinct families: TLC1-TLC4. The level of amino-acid sequence similarity between these elements extends from 66.7% (between TLC1 and TLC2) to 42.6% (between TLC2 and TLC3). Altogether, the four families comprise about 0.17% of the L. chilense genome. RT-PCR analysis shows that the four TLC families are transcriptionally active, suggesting a mechanism for the generation of the observed diversity between the L. chilense retrotransposons.

Abscisic acid (ABA) is an essential component in the wound signalling cascade. Increased levels of endogenous ABA were observed after wounding and shown to be a requisite for wound-induced expression of the proteinase inhibitor II genes. We have taken advantage of the dominant character of the Arabidopsis abi1-1 mutation, to investigate whether ABl1 has a function in ABA signalling in response to wounding. Transgenic tomato plants carrying copies of either the wild-type ABI1 or the mutant abi1-1 alleles were obtained and assayed for wound-induction of the pin2 or LAP genes. While normal levels of gene induction were observed in the transgenic ABI1 plants, the abi1-1 transformants displayed a severe wilty phenotype and reduced seed dormancy. Expression of the abi1-1 dominant mutation blocked accumulation of the drought-induced TAS14 and LE25 mRNAs in response to ABA, as well as ABA- and wound-induced expression of the defense-associated pin2 and LAP transcripts. MeJA-induction of the pin2 and LAP mRNAs, on the contrary, was not affected in the abi1-1 transformants. These results indicate that abi1-1 inhibits wound-induced expression of the pin2 and LAP transcripts by blocking ABA-induction of these genes. This implicates ABI1 in wound-signalling and suggests that a common early ABA signalling pathway may function in the responses to wounding and water stress.

Grafting experiments between phytochrome B antisense and wild-type potato (Solanum tuberosum L. subsp. andigena [line 7540]) plants provide evidence that phytochrome B is involved in the production of a graft-transmissible inhibitor of tuberization, the level of which is reduced in the antisense plants, allowing them to tuberize in noninducing photoperiods.

Tomato and potato leucine aminopeptidase (LAP) mRNAs are induced in response to mechanical wounding and the wound signal molecules, ABA and jasmonic acid. Here, we report the isolation of two LAP genes, LAP17.1A and LAP17.2, from tomato. Functional analysis in transgenic tomato and potato plants show that fusions of the corresponding 5' non-coding regions to the gusA gene are constitutively expressed in flowers and induced in leaves upon wounding or by treatment with methyl jasmonate (MeJA). Comparison of the 5' non-coding regions of the two genes revealed a region from -317 to -3 relative to the ATG, which is strongly conserved in both promoters. This 0.3 kb proximal promoter fragment is sufficient to direct flower-specific and MeJA-inducible GUS activity in transgenic potato plants, and thus contains a MeJA-responsive element that mediates induction by MeJA. Dimeric TGACG motifs or G-box elements similar to those found in other MeJA-inducible genes are not observed in this region, which suggests that a different DNA sequence is involved in MeJA induction of the LAP genes.

A cDNA clone (Ban17), encoding a protein homologous to pectate lyase, has been isolated from a cDNA library from climacteric banana fruit by means of differential screening. Northern analysis showed that Ban17 mRNA is first detected in early climacteric fruit, reaches a steady-state maximum at the climacteric peak, and declines thereafter in overripe fruit. Accumulation of the Ban17 transcript can be induced in green banana fruit by exogenous application of ethylene. The demonstrates that expression of this gene is under hormonal control, its induction being regulated by the rapid increase in ethylene production at the onset of ripening. The deduced amino acid sequence derived from the Ban17 cDNA shares significant identity with pectate lyases from pollen and plant pathogenic bacteria of the genus Erwinia. Similarity to bacterial pectate lyases that were proven to break down the pectic substances of the plant cell wall suggest that Ban17 might play a role in the loss of mesocarp firmness during fruit ripening.

Background: Salmonella enteritidis infections have increased worldwide in the last years. Isolation of this pathogen was remarkably rare in Chile until 1994, when the still ongoing outbreak emerged., Aim: To assess the main epidemiological characteristics of the Salmonella enteritidis epidemic in Chile., Materials and Methods: Cases of clinical infections by Salmonella enteritidis were recorded from bacteriological and demographic notifications obtained at The National Reference Laboratory for Enterobacteria. Infection rates were calculated using the total Chilean population and the population of the different Health Services along the country., Results: Until 1993, 13.67 Salmonella enteritidis strains per year were received at the Reference Laboratory. The figures increased to 478 and 432 in 1994 and 1995, respectively. National rates were 3.41 and 3.04 notifications/100,000 inhabitants in 1994 and 1995 respectively. Northern regions were the most affected and 90% of observed cases during 1994 came from Arica and Antofagasta. At the present time, 20% of cases are observed in Santiago, located in the mid-portion of the country. The outbreak has mainly affected children and young adults (70% of cases). Strains have been isolated from stool cultures, suggesting gastrointestinal infections as the main clinical presentation. More than 98% of strains are susceptible to chloramphenicol, ampicillin, tetracycline, sulfa-trimetroprim, cefotaxime or ciprofloxacin., Conclusions: The obtained data clearly indicate the existence of an epidemic outbreak of Salmonella enteritidis infections, with a geographic progression from North to South.

Differential screening of a potato leaf cDNA library with cDNA probes made from tuberizing and non-tuberizing Solanum demissum plants led to the identification of a clone that is upregulated in leaves and other tissues upon tuberization. This clone was also shown to have a high level of expression in green tomato fruit, its expression falling off as the fruit turns red. No sucrose or hormonal regulation of the expression of this clone was observed and it did not respond to wounding or heat stress. Clone 32B is 532 bp long and contains an open reading frame encoding a small protein of 98 amino acids. The deduced protein sequence has a putative signal peptide for ER transport and a 10 amino acid domain in the C-terminal region of the protein, both of which are also found in the cotton LEA5, Arabidopsis Di21 and the mungbean Arg2 proteins.

From 1977 to 1986, Chile experienced an important typhoid fever epidemic, despite statistics that indicated apparently improving levels of sanitation of drinking water and sewage disposal. The lack of antibiotic resistance among the Salmonella typhi strains isolated during this period, the mild clinical presentation of the disease, and the initially low level of efficacy of the S. typhi Ty21a vaccine in the population exposed to the epidemic suggested that this epidemic might have resulted from the dissemination of S. typhi strains with unique characteristics. To investigate this hypothesis, we used conventional methods (bacteriophage typing and biotyping) and molecular methods (restriction fragment length polymorphism analysis, ribotyping, IS200 typing, and PCR amplification of the fliC-d gene) to study a population of 149 S. typhi isolates during 1977, 1981, and 1990, the years that included periods with low (when the disease was endemic) and high (when the disease was epidemic) morbidities. Our results indicate that these S. typhi isolates in Chile represent a number of highly diverse variants of the clone of S. typhi with a worldwide distribution described by Selander et al. (R. K. Selander, P. Beltran, N.H. Smith, R. Helmuth, F.A. Rubin, D.J. Kopecko, K. Ferris, B.D. Tall, A. Cravioto, and J.M. Musser, Infect. Immun. 58:2262-2275, 1990). For example, we detected 26 PstI and 10 ClaI ribotypes among 47 and 16 S. typhi strains belonging to this clone, respectively. These results suggest that the Chilean epidemic was probably produced by multiple sources of infection because of deficient sanitary conditions. These findings illustrate the usefulness of molecular methods for characterizing the potential causes of the typhoid epidemics and the possible routes of transmission of S. typhi strains in typhoid epidemics.

A cDNA clone encoding a novel Myb-related protein, designated MybSt1, was isolated from a potato cDNA expression library by South Western screening using the CaMV 35S promoter domain A as a probe. Sequence comparison shows a small region with some homology to the highly conserved DNA binding domain of the c-myb proto-oncogene consisting of three imperfect repeats. The Myb motif of the MybSt1 protein is distinct from the plant Myb DNA binding domain described so far. In contrast to the known plant Myb proteins, with two repeats required for the DNA binding activity, the clone mybSt1 contains only one such repeat. Nevertheless, the Myb-related protein MybSt1 is able to bind to DNA in a sequence-specific manner. In addition to the Myb-like region, the protein MybSt1 contains an acidic segment in its central region as well as a proline-rich region near the C-terminus. Applying the random binding site selection technique, high-affinity DNA binding sites for MybSt1 were identified, sharing the core motif GGATA. In transient expression assays using plant protoplasts, clear evidence was obtained for this myb clone functioning as a transcriptional activator.

A DNA clone encoding a cathepsin D inhibitor CathInh was isolated from a potato genomic library using a CathInh cDNA as hybridization probe. The amino acid sequence of the coding region is nearly identical with a CathInh cDNA and CathInh proteins previously isolated from a tuber-specific cDNA library and from tubers, respectively. Analysis of GUS activity resulting from expression of chimeric CathInh promoter-GUS genes in transgenic potato plants revealed expression exclusively confined to potato tubers. No GUS activity could be detected in any other organ of the transgenic plants either constitutively or after wounding or treatment with abscisic and jasmonic acid (JA). Interestingly, part of the promoter region of the CathInh gene, essential for GUS activity in tubers, shows striking similarity to promoter regions of tuber-specific class I patatin genes.

TGA1a and PG13 constitute a family of tobacco basic leucine zipper (bZIP) proteins that bind to activating sequence-1 (as-1), which is one of the multiple regulatory cis elements of the cauliflower mosaic virus (CaMV) 35S promoter. After truncation of the CaMV 35S promoter down to position -90 (CaMV 35S [-90] promoter), transcription stringently depends on the presence of as-1, which is recognized by nuclear DNA binding proteins called ASF-1. The role of the TGA1a/PG13 bZIP family in the formation of ASF-1 and in transcriptional activation of the CaMV 35S (-90) promoter has not yet been demonstrated in vivo. We constructed transgenic tobacco plants expressing a mutant of potato PG13, which lacks its wild-type DNA binding domain. This mutant acts as a trans-dominant inhibitor of ASF-1 formation and of expression from the CaMV 35S (-90) promoter, showing that PG13 can specifically interact with proteins necessary for these processes. Although we did not observe any other obvious phenotypic changes, these transgenic plants are a potentially valuable tool in identifying whether TGA1a and PG13 are involved in controlling promoters encoded in the plant genome.

A protein encoded by a potato cDNA homologous to a leucine aminopeptidase (LAP) from bovine lens (Hildmann et al. 1992) was expressed in Escherichia coli cells and biochemically characterized by hydrolysis of leucine p-nitroanilide. Activity was highest under alkaline conditions with an optimum at about pH 10. Maximal activities were measured at 65 degrees C. Apart from leucine p-nitroanilide the enzyme could also efficiently hydrolyze the p-nitroanilides of arginine and methionine. Complete inhibition of the enzyme was achieved by incubating bacterial extracts with bestatin and EDTA, which classifies the enzyme as a metalloprotease belonging to the same group as the homohexameric LAPs from mammals. Protein blots showed low constitutive expression of the LAP in all organs of potato plants: buds, flowers, tubers, roots and leaves. An increase in steady-state protein that was paralleled by an increase in total LAP activity was observed in leaf extracts after supplying jasmonic acid via the petioles. Plants containing the cDNA in antisense orientation behind the constitutive Cauliflower Mosaic Virus 35S promoter showed nearly complete reduction of the corresponding mRNA in leaves. However, in these plants LAP activities were only decreased by about 20% as compared to non-transgenic potato plants, while after feeding with jasmonic acid the activity of transgenic plants was reduced to about 5% of that of non-transgenic plants also induced by jasmonic acid. There was no phenotypic difference between wild-type and LAP antisense plants.

Exogenous application of abscisic acid (ABA) has been shown to induce a systemic pattern of proteinase inhibitor II (pin2) mRNA accumulation identical to that induced by mechanical wounding. Evidence is presented that the ABA-specific response is not restricted to pin2 genes but appears to be part of a general reaction to wound stress. Four other wound-induced, ABA-responsive genes that encode two additional proteinase inhibitors, the proteolytic enzyme leucine aminopeptidase, and the biosynthetic enzyme threonine deaminase were isolated from potato plants. Wounding or treatment with ABA resulted in a pattern of accumulation of these mRNAs very similar to that of pin2. ABA-deficient plants did not accumulate any of the mRNAs upon wounding, although they showed normal levels of expression upon ABA treatment. Also, application of methyl jasmonate (MeJA) induced a strong accumulation of these transcripts, both in wild-type and in ABA-deficient plants, thus supporting a role for jasmonic acid as an intermediate in the signaling pathway that leads from ABA accumulation in response to wounding to the transcriptional activation of the genes.

After a period of time of 5 years, we made a review of all the patients registered at our Center. Eight cases of closed traumatism at the scapulohumeral belt, with vascular injury associated (0.2% of all closed traumatisms at this level registered in our center during this time) were reported. Six patients presented also an osteoarticular injury associated. Three different vascular injuries were showed: three cases of complete arterial section, two arterial contusions and a partial arterial section. From the two cases without osteoarticular injuries, one of them showed a complete section and the other presented an arterial contusion. Polytraumatic patients showed multiple osteoarticular injuries. We registered one fracture-luxation at the shoulder, 3 diafisaric humeral fractures, one humeral neck fracture, one clavicle fracture, one fracture-luxation at the cervical column and one simple luxation of the shoulder.

A 16 base-pair palindrome upstream of the Agrobacterium tumefaciens octopine synthase (ocs) gene functions as a positive regulatory element in plant cells (Ellis et al. (1987) EMBO J. 6, 3203-3208; Fromm et al. (1989) Plant Cell 1, 977-984). We have converted it into a negative element by locating two copies flanking the TATA-box of the constitutively expressed CaMV 35S promoter. The reduced promoter activity is very likely due to sterical hindrance of the ocs binding protein (OCSTF) x ocs complex with the transcription initiation complex. We propose that this type of constructs can be used for the identification of recognition sites for DNA-binding proteins which are labile in vitro as well as for determining the DNA-binding activity of a trans-acting factor in vivo.

Authors present their experience (32 patients affected by a traumatic femoropopliteal injury) with vascular traumatisms at the lower limb. Causes, treatment and successful results with the new technics and the early clinical management to the patients are commented.

The 5'-upstream region of the class I patatin gene B33 directs strong expression of the beta-glucuronidase (GUS) reporter gene in potato tubers and in leaves treated with sucrose. Cis-acting elements affecting specificity and level of expression were identified by deletion analysis in transgenic potato plants. A putative tuber-specific element is located downstream from position -195. Nuclear proteins present in leaf and tuber extracts bind specifically to a conserved AT rich motif within this region. A DNA fragment between -183 and -143, including the binding site is, however, not able to enhance the expression of a truncated 35S promoter from cauliflower mosaic virus. Independent positive elements contributing to a 100-fold increase relative to the basic tuber-specific element are located between -228 and -195; -736 and -509, -930 and -736 and -1512 and -951. Sucrose inducibility is controlled by sequences downstream of position -228, indicating that the tuber-specific and sucrose-inducible elements are in close proximity.

Potato nuclear proteins specifically bind to a DNA sequence at the most 5' distal region of the promoter of a potato proteinase inhibitor II gene. Binding studies using the electrophoretic mobility-shift assay showed the appearance of two protein-DNA complexes in the presence of both tuber and leaf nuclear protein extracts. Mechanical wounding of the leaves had no effect on the amount of specific protein-DNA complexes formed. DNase I protection analysis and binding to synthetic oligonucleotides identified the sequence 5'-GAGGGTATTTTCGTAA-3' as the target for the noncooperative binding of two potato nuclear proteins to the upstream element. Methylation interference experiments showed that guanine nucleotides separated by one turn of the DNA helix were in close contact with the proteins. The binding ability of a series of mutated synthetic oligonucleotides further defined the sequence requirements for protein binding, which appeared to contact one side of the DNA helix.

Potato tubers are modified stems that have differentiated into storage organs. Factors such as day-length, nitrogen supply, and levels of the phytohormones cytokinin and gibberellic acid, are known to control tuberization. Morphological changes during tuber initiation are accompanied by the accumulation of a characteristic set of proteins, thought to be involved in N-storage (i.e. patatin) or defense against microbial or insect attack (i.e. proteinase inhibitor II). Additionally, deposition of large amounts of starch occurs during tuber formation, which is paralleled by an increase in sucrose synthase and other enzymes involved in starch biosynthesis (i.e. ADP-glucose pyrophosphorylase, starch synthases, and branching enzyme). Potential controlling mechanisms for genes expressed during tuberization are discussed.

A case of aseptic necrosis of the medial segment of the clavicle with X-ray images of fragmentation and few clinical symptoms is presented. This condition develops spontaneously and leads to early sternoclavicular osteoarthritis. The benign nature of the disease and the absence of treatment make it important to establish the correct diagnosis, differentiating it from other disorders in this area.

Plants respond to wounding or pathogen attack by a variety of biochemical reactions, involving in some instances gene activation in tissues far apart from the actual site of wounding or pathogen invasion. One of the best analyzed examples for such a systemic reaction is the wound-induced expression of proteinase inhibitor genes in tomato and potato leaves. Local wounding of potato or tomato plants results in the accumulation of proteinase inhibitors I and II throughout the aerial part of the plant. In contrast to wild-type plants, abscisic acid-deficient mutants of potato (droopy) and tomato (sit) show a drastically reduced induction of these genes in response to plant wounding. High levels of proteinase inhibitor II gene expression are obtained in mutant and wild-type plants upon exogenous application of abscisic acid. Measurements of the endogenous abscisic acid levels in wild-type plants show that wounding results in increased levels of this phytohormone in wounded and nonwounded systemically induced leaves. Thus these results show that the plant hormone abscisic acid is involved in the wound-induced activation of the proteinase inhibitor II gene. Furthermore, they are compatible with a model assuming this hormone to be the actual mediator of the systemic wound response.

A collection of cDNA clones, corresponding to a group of maize endosperm proteins classified in the glutelin-2 (or reduced soluble proteins) and in the zein-2 subfractions, has been identified and characterized. The nucleotide sequence of three of these clones has been obtained and the amino acid sequence deduced. They appear to correspond to a small family of genes that are specifically expressed in immature endosperm simultaneously to zeins, the best characterized proteins from this tissue. Unlike zeins, the proteins of the glutelin-2 and zein-2 family contain sequences homologous to storage proteins from other cereals such as gliadins or hordeins. The cDNA clones encoding for the two types of proteins have been compared, and a high degree of homology has been observed for both the nucleotide and amino acid sequences. The differences existing in both the coding and non-coding regions allow the definition of multiple types of variability in their sequence. An hypothesis is proposed on how sequence diversity may have been generated in this particular class of plant proteins.

Proteins present in tobacco nuclear extracts bind to a truncated cauliflower mosaic virus (CaMV) 35S promoter fragment (from -90 to +2 relative to the transcription start site) in a sequence specific manner. Gel mobility shift assays show the presence of two protein-DNA complexes that are not competed by a -47/+2 promoter fragment. DNAse I protection and DNA methylation interference reveal two protected sites in the slower migrating complex; both include the pentamer TGACG, separated by a stretch of eight nucleotides where G methylation does not prevent the binding of the proteins. The faster complex is the prevalent form at low protein concentrations. As the protein concentration increases a non-linear rise in the amount of the slower migrating complex relative to the faster one is seen suggesting that cooperative effects are involved in the binding to the second site.

The cDNA coding for a glutelin-2 protein from maize endosperm has been cloned and the complete amino acid sequence of the protein derived for the first time. An immature maize endosperm cDNA bank was screened for the expression of a beta-lactamase:glutelin-2 (G2) fusion polypeptide by using antibodies against the purified 28 kd G2 protein. A clone corresponding to the 28 kd G2 protein was sequenced and the primary structure of this protein was derived. Five regions can be defined in the protein sequence: an 11 residue N-terminal part, a repeated region formed by eight units of the sequence Pro-Pro-Pro-Val-His-Leu, an alternating Pro-X stretch 21 residues long, a Cys rich domain and a C-terminal part rich in Gln. The protein sequence is preceded by 19 residues which have the characteristics of the signal peptide found in secreted proteins. Unlike zeins, the main maize storage proteins, 28 kd glutelin-2 has several homologous sequences in common with other cereal storage proteins.

To evaluate the effect of hepatitis delta virus on the level of replication of hepatitis B virus and to assess the clinical significance that such an effect might have on the final outcome of the infection, the serological profile of hepatitis B virus DNA was investigated in 153 patients with acute or chronic hepatitis B virus infection with or without associated delta infection. Serum hepatitis B virus DNA was detected in 57% of patients with acute hepatitis B, 67% of those with acute hepatitis B virus-hepatitis delta virus coinfection and 25% of HBsAg carriers with hepatitis delta virus superinfection during the first week after the onset of symptoms. Patients with acute hepatitis B and those with acute hepatitis B virus-hepatitis delta virus coinfection did not differ significantly with respect to the serological profile of hepatitis B virus DNA and final clinical outcome. Within the group of HBsAg carriers with hepatitis delta virus superinfection, all patients who were initially negative for hepatitis B virus DNA developed chronic hepatitis delta virus infection, whereas 3 of the 4 patients with active hepatitis B virus infection at the time of superinfection showed transient inhibition of hepatitis B virus replication followed by termination of hepatitis delta virus infection in two patients. Therefore, although delta virus may inhibit the replication of hepatitis B virus among chronic HBsAg carriers, this effect is not readily apparent among patients with hepatitis B virus-hepatitis delta virus coinfection.