Effect of Radopholus similis, Pratylenchus araucensis, Meloidogyne spp. and their interaction on Musa AAB 'Dominico Hartón' seedlings.

The effect of Radopholus similis, Pratylenchus araucensis, Meloidogyne spp., and their interaction was evaluated in seedlings of Musa AAB 'Dominico Hartón'. The study was conducted in a nursery in Palestina, Caldas department, Colombia. Forty-day-old plantain seedlings were infected separately with 750, 1,500, 2,250 and 3,000 of each species of nematodes/plant. Two experiments were conducted to evaluate the damage of R. similis, P. araucensis, Meloidogyne spp. and the mixture of 750 R. similis + 750 P. araucensis + 750 Meloidogyne spp. compared with the mixture of different proportions (1,500, 2,250 and 3,000 of each species of nematodes). Noninfected plants were included as a control treatment, for a total of 17 treatments in a randomized complete block design with ten replications. Twelve weeks after inoculation, all nematodes, both alone and in combination, reduced (p < 0.05) plantain dry root and shoot weight. In two experiments, R. similis, P. araucensis, and Meloidogyne spp. alone, each with a population density of 3,000, reduced (p < 0.05) root dry weight by 32.5%, 9.5% and 49%, respectively, and decreased (p < 0.05) shoot dry weight by 21.5%, 23%, and 31.5%, respectively, compared to the control. The interaction of nematodes with the lowest population decreased root (33%) and shoot (21%) weight. We conclude that the growth of 'Dominico Hartón' seedlings was affected by plant-parasitic nematodes, but the greatest damage occurred with concomitant nematode infection.

Dp71ab/DAPs complex composition changes during the differentiation process in PC12 cells.

PC12 cells express different Dp71 isoforms originated from alternative splicing; one of them, Dp71ab lacks exons 71 and 78. To gain insight into the function of Dp71 isoforms we identified dystrophin associated proteins (DAPs) that associate in vivo with Dp71ab during nerve growth factor (NGF) induced differentiation of PC12 cells. DAPs expression was analyzed by RT-PCR, Western blot and indirect immunofluorescence, showing the presence of each mRNA and protein corresponding to alpha-, beta-, gamma-, delta-, and epsilon-sarcoglycans as well as zeta-sarcoglycan mRNA. Western blot analysis also revealed the expression of beta-dystroglycan, alpha1-syntrophin, alpha1-, and beta-dystrobrevins. We have established that Dp71ab forms a complex with beta-dystroglycan, alpha1-syntrophin, beta-dystrobrevin, and alpha-, beta- and gamma-sarcoglycans in undifferentiated PC12 cells. In differentiated PC12 cells, the complex composition changes since Dp71ab associates only with beta-dystroglycan, alpha1-syntrophin, beta-dystrobrevin, and delta-sarcoglycan. Interestingly, neuronal nitric oxide synthase associates with the Dp71ab/DAPs complex during NGF treatment, raising the possibility that Dp71ab may be involved in signal transduction events during neuronal differentiation.

Differential expression and subcellular distribution of dystrophin Dp71 isoforms during differentiation process.

Dp71 is the major product of the Duchenne muscular dystrophy gene in the brain. In order to study the function of Dp71 in the nervous system we examined the expression of Dp71 isoforms in PC12 rat pheochromocytoma cell line, a well-established system to study neuronal differentiation. We show by reverse transcriptase-polymerase chain reaction and Western blot assays that PC12 cells express two Dp71 isoforms. One isoform lacks exon 71 and the other isoform lacks exons 71 and 78 (Dp71d and Dp71f isoforms respectively). Nerve growth factor-induced neuronal differentiation of PC12 cells results in differential regulation of the expression and subcellular localization of Dp71 isoforms: a) the amount of Dp71f protein increases nine-fold in total extracts while Dp71d increases up to seven-fold in nuclear extracts; b) Dp71f relocates from the cytoplasm to neuritic processes, being prominent at varicosities and the growth cone; c) Dp71d relocates almost entirely to the nucleus and is detected to a lower extent in the cytoplasm and neuritic processes. Dp71f co-localizes with beta-dystroglycan and synaptophysin while Dp71d co-localizes with beta-dystroglycan in the nucleus. Dp71d accumulates at cell-cell contacts where Dp71f is absent. These results suggest that Dp71d and Dp71f associate with different subcellular complexes and therefore may have distinct functions in PC12 cells.

Myotonic dystrophy protein kinase (DMPK) gene expression in lymphocytes of patients with myotonic dystrophy.

BACKGROUND: Myotonic dystrophy (DM) is an autosomal dominant neuromuscular disorder with defects in many tissues, including skeletal muscle myotonia, progressive myopathy, and abnormalities in heart, brain, and endocrine systems. It is associated with a trinucleotide repeat occurring in the 3' (UTR) untranslated region of the myotonic dystrophy protein kinase (DMPK) gene. Several studies have been carried out to determine DMPK gene expression in muscle and non-muscle tissues. METHODS: DMPK gene expression was determined in lymphocytes of adult-onset patients with DM and normal controls. To quantitate total locus expression as well as allele-specific mRNA levels, semiquantitative RT-PCR assay was used. Allele-specific expression was analyzed using a Bpm1 polymorphism located at exon 10 of the DMPK gene. RESULTS: In heterozygous patients with DM, we observed a fourfold difference between mRNA levels produced by the Bpm1-undigested allele (187 bp) compared to the Bpm1-digested allele (136 bp). By using (CTG) trinucleotide (with cytosine, thymine, and guanine) expansion polymorphism, it was shown that the down-regulated allele corresponds to the mutant allele. Interestingly, the reduction in the mutant allele-transcript levels is compensated by an increase of the wild-type allele, yielding no significant differences in total locus mRNA amount between patients and normal individuals. CONCLUSIONS: These results suggest that the expression of the two alleles at the DMPK locus in lymphocytes is coordinated. The reduction in mutant-allele transcript levels is compensated by an increase in wild-type allele mRNA levels.

Alternative splicing regulates the nuclear or cytoplasmic localization of dystrophin Dp71.

The subcellular distribution of Dp71 isoforms alternatively spliced for exon 71 and/or 78 was examined. The cDNA sequence of each variant was fused to the C-terminus of the green fluorescent protein and the constructs were transfected transiently in the cell lines HeLa, C2C12 and N1E-115. The subcellular distribution of the fused proteins was determined by confocal microscope analysis. The Dp71 isoform lacking the amino acids encoded by exons 71 and 78 was found exclusively in the cytoplasm whereas the variants containing the amino acids encoded by exon 71 and/or exon 78 show a predominant nuclear localization. The nuclear localization of Dp71 provides a new clue towards the establishment of its cellular function.

Expression and localization of utrophin in differentiating PC12 cells.

To ascertain the role of utrophin in cultured neuronal cells, we investigated its expression and distribution along the NGF-induced differentiation of PC12 cells grown on different substrata. Utrophin mRNA was measured by RT-PCR assay and utrophin protein was quantified by immunoblot analysis. The distribution of utrophin and beta-dystroglycan was analyzed by confocal microscopy. We demonstrate that utrophin protein was increased 4-fold during differentiation of cells grown laminin. Concomitant with this up-regulation, utrophin was enriched at the growth cones in differentiating cells, where it co-localizes with beta-dystroglycan. These data suggest the presence of a utrophin-beta-dystroglycan complex in PC12 cells that participates in the formation and/or stabilization of the growth cone-extracellular matrix adhesion.

A 24-kDa cloned zinc metalloprotease from Actinobacillus pleuropneumoniae is common to all serotypes and cleaves actin in vitro.

Actinobacillus pleuropneumoniae causes pleuropneumonia in swine. This bacterium secretes proteases that degrade porcine hemoglobin and IgA in vitro. To further characterize A. pleuropneumoniae proteases, we constructed a genomic library expressed in Escherichia coli DH5alpha, and selected a clone that showed proteolytic activity. The recombinant plasmid carries an 800-base pair A. pleuropneumoniae gene sequence that.codes for a 24-kDa polypeptide. A 350-base pair PstI fragment from the sequence hybridized at high stringency with DNA from 12 serotypes of A. pleuropneumoniae, but not with DNA from Actinobacillus suis, Haemophilus parasuis, Pasteurella haemolytica, Pasteurella multocida A or D, or E. coli DH5alpha, thus showing specificity for A. pleuropneumoniae. The expressed polypeptide was recognized as an antigen by convalescent-phase pig sera. Furthermore, a polyclonal antiserum developed against the purified polypeptide recognized an A. pleuropneumoniae oligomeric protein in both crude-extract and cell-free culture media. This recombinant polypeptide cleaved azocoll, gelatin, and actin. Inhibition of the proteolytic activity by diethylpyrocarbonate suggests that this polypeptide is a zinc metalloprotease.

Sp1 and AP2 transcription factors are required for the human fragile mental retardation promoter activity in SK-N-SH neuronal cells.

The promoter of the human fragile mental retardation gene (FMR1) was functionally analyzed in order to identify elements responsible for its regulation. Plasmids carrying the wild type or different deleted-promoter sequences driving the chloramphenicol acetyl transferase gene (CAT) were transiently transfected into the SK-N-SH cells and the CAT activity was assessed. Deletion studies suggested that major regulatory elements are present in a DNA region between positions -123 and -51. Gel mobility shift and footprinting assays using a DNA fragment encompassing that promoter region showed that SP1 and AP2 transcription factors could be involved in the functioning of the FMR1 promoter.

Nus A is involved in transcriptional termination on lambda tI.

The transcriptional terminator tI generates the 3'end of the integrase (int) gene transcript that is read from the lambda PI promoter in lambda phage. We have studied the factors that affect transcription termination in vitro and in vivo at the lambda tI terminator. In vitro transcriptional studies showed that tI is about 80% efficient in the presence of purified NusA protein, whereas it is only about 50% efficient in its absence. In vivo studies, where the readthrough transcript of lambda tI was measured by quantitative dot blot analysis, gave about 80% efficiency in wild-type strains, but only 60% in the nusA1 mutant strain at non-permissive temperatures. These results support the idea that termination at lambda tI in vivo involves interaction with the NusA factor.

Linkage analysis of the hph-1 mutation and the GTP cyclohydrolase I structural gene.

Our research establishes genetic linkage between the hph-1 mutation and the GTP-CH I structural gene. Our results indicate that these two loci are within an 8 cM region with 95% confidence. This finding lends additional support for the use of the hph-1 mouse mutant as a bona fide model system for the human disorder GTP-CH I to further our understanding of the molecular mechanisms involved in the disease pathology of GTP-CH I deficiency.

A novel mutation in the KH domain of polynucleotide phosphorylase affects autoregulation and mRNA decay in Escherichia coli.

Polynucleotide phosphorylase (PNPase) is a key 3'-5' exonuclease for mRNA decay in bacteria. Here, we report the isolation of a novel mutant of Escherichia coli PNPase that affects autogenous control and mRNA decay. We show that the inactivation of PNPase by a transposon insertion increases the half-life of galactokinase mRNA encoded by a plasmid. When the bacteriophage lambda int gene retroregulator (sib/tI ) is placed between pgal and galK, it severely diminishes galactokinase expression because of transcription termination. The expression of galK from this construct is increased by a single base mutation, sib1, which causes a partial readthrough of transcription at tI. We have used this plasmid system with sib1 to select E. coli mutants that depress galK expression. Genetic and molecular analysis of one such mutant revealed that it contains a mutation in the pnp gene, which encodes the PNPase catalytic subunit alpha. The mutation responsible (pnp-71 ) has substituted a highly conserved glycine residue in the KH domain of PNPase with aspartate. We show that this G-570D substitution causes a higher accumulation of the alpha-subunit as a result of defective autoregulation, thereby increasing the PNPase activity in the cell. The purified mutant alpha-subunit shows the same electrophoretic mobility in denaturing gels as the wild-type subunit, as expected. However, the mutant protein present in crude extracts displays an altered electrophoretic mobility in non-denaturing gels that is indicative of a novel enzyme complex. We present a model for how the pnp-71 mutation might affect autoregulation and mRNA decay based on the postulated role of the KH domain in RNA-protein and protein-protein interactions.

Dystrophins in developing retina: Dp260 expression correlates with synaptic maturation.

Dystrophin, the protein altered in Duchenne muscular dystrophy (DMD), is necessary for normal retinal function and exists in several isoforms. We examined the expression of dystrophin and utrophin proteins and transcripts in the rat retina at different developmental stages using Western blots and semi-quantitative RT-PCR. Our results revealed the presence of utrophin (DRP1), G-utrophin and/or DRP2 and four dystrophin isoforms (Dp427, Dp260, Dp140, Dp71) in the normal adult rat retina. Only Dp260 showed a marked progressive increase with age at both protein and mRNA levels. This variation is consistent with the establishment of synaptic functions in the developing retina and suggests a key role for this apo-dystrophin in synaptogenesis.

Pattern of deletions of the dystrophin gene in Mexican Duchenne/Becker muscular dystrophy patients: the use of new designed primers for the analysis of the major deletion "hot spot" region.

We have analyzed 59 unrelated Mexican Duchenne/Becker muscular dystrophy patients (DMD/BMD) using PCR analysis of the 2 prone deletion regions in the DMD gene. Thirty one (52%) of the patients had a deletion of one or several of the exons. Most of the alterations (87%) were clustered in exons 44-52, this being the highest percentage reported until now. In order to improve the molecular diagnosis in the Mexican population, we designed a new multiplex assay to PCR amplify exons 44-52. This assay allowed for the identification of a greater number of deletions in this region compared with the 9 and 5-plex assays previously described and to determine most of the deletion end boundaries. This is a reliable alternative for the initial screening of the DMD patients in the Mexican population.

Analysis of the (CAG)n repeat causing Huntington's disease in a Mexican population.

We investigated the allele distribution of the polymorphic (CAG)n repeat in the IT15 gene in 96 normal subjects from the Mexican population and 83 unrelated patients with Huntington's disease. Our results show that the size distributions of normal and affected alleles do not overlap. Normal alleles range from 13 to 32 triplets, with 18 being the most frequent allele, while HD alleles contain 37 to 76 repeats with 42 being the most frequent. One allele in the range of intermediate alleles was found (32 repeats) in a normal subject. The juvenile onset cases in this study are associated with an expansion greater than 49 repeats. In the available parent-offspring pairs, paternal alleles show instability with an expansion of 28 repeats in one case.

Point mutations in a transcription terminator, lambda tI, that affect both transcription termination and RNA stability.

The terminator tI is located approx. 280 nucleotides beyond the int gene of bacteriophage lambda. Besides its role as a transcription terminator, tI may confer stability to the int message by protecting it from 3' exonucleolytic degradation. In order to study the role of the tI sequence in transcription termination and RNA stability, three different point mutations tI1, tI2, and tI3 were isolated and characterized. All the tI mutations map in the G + C-rich region of dyad symmetry in the terminator and decrease the transcriptional termination of tI in vivo from 99% for the wild type terminator to 81-93% as determined by galactokinase activity and in vitro from 80% for the wild type terminator to 8-12% using the E. coli RNA polymerase. Additionally, the tI mutations cause upstream transcript instability in vivo. This instability defect caused by tI mutations is compensated by the host mutant deficient in polynucleotide phosphorylase resulting in increased steady state levels of these mutant transcripts. The results show that the intact hairpin of tI is essential for efficient transcription termination and for maintaining mRNA stability by blocking the 3' to 5' exonucleolytic activity of polynucleotide phosphorylase.

Expression of dystrophin Dp71 during PC12 cell differentiation.

The expression of dystrophin-protein 71 (Dp71) was investigated during nerve growth factor (NGF) induced differentiation of PC12 cells. A semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay was designed to measure Dp71 mRNA, whereas the Dp71 protein amount was evaluated by immunoblot analysis using an anti-dystrophin monoclonal antibody. Comparison with control cultures showed that Dp71 mRNA and protein levels increased in parallel with NGF treatment peaking with increments of 60% and 1.4 times, respectively. The upregulation of Dp71 expression during PC12 cells differentiation point at PC12 cells as a suitable model for studying the function of Dp71 in neuronal cells.

Carrier detection in Duchenne and Becker muscular dystrophy using dinucleotide repeat polymorphisms. A study in Mexican families.

In order to improve carrier detection of Duchenne and Becker muscular dystrophy, dinucleotide sequences repeats (CA) of introns 44, 45, 49 and 50 were used as well as two markers located at the 5' and 3' ends of the dystrophin gene. Haplotypes of the unaffected and affected persons of ten DMD/ BMD Mexican families were determined. Fifty eight females were studied, 30 of whom were at-risk STR haplotypes. Furthermore, it was possible to identify a recombination event in the dystrophin gene in one family, and a gonadal mosaicism was found in another family.

Comparative analysis of plasmids from nosocomial Klebsiella pneumoniae strains isolated from two hospitals.

A total of 46 clinical isolates of Klebsiella pneumoniae were studied. Of these, 33 were from "Hospital Infantil de México" (HIM) and 13 from "Hospital General de México" (HGM). The susceptibility of these strains to five antibiotics, as well as the plasmid DNA profiles, were determined for each group. Antibiotic susceptibility profiles were very similar in strains from both hospitals; however, most of the strains analyzed exhibited heterogeneous plasmid DNA profiles. Results showed that strains isolated in the two hospitals did not differ regarding morphology, biochemical profiles, antibiotic susceptibility or plasmid populations, and these characteristics may not be used as markers to differentiate Klebsiella pneumoniae strains from different hospitals.

[Restoration of transcription antitermination of N- bacteriophages in Escherichia coli with a mutant RNA polymerase]. / Restauración de la antiterminación de la transcripción de bacteriófagos N-en Escherichia coli con una RNA polimerasa mutante.

The N protein of bacteriophage lambda modifies Escherichia coli RNA polymerase in such a way that it transcribes through termination signals, in a process called antitermination. In general N-mutants are not able to perform transcription antitermination. In this paper we report the suppression of N7 and Nmar3 mutations by Escherichia coli ron-lon strain. The lon mutation causes the N protein half-life to raise, suggesting that excess of N7 fragment or Nmar3 protein overcome the defect in antitermination. Under these conditions the lambda N-phages produced a titer similar to lambda wild type, although the plaques were smaller. These observations highlight the relevance of N half life in the regulation of transcription antitermination.

Analysis of dystrophin gene deletions in patients from the Mexican population with Duchenne/Becker muscular dystrophy.

Forty unrelated Mexican patients with Duchenne/Becker muscular dystrophy were analyzed for intragenic DMD gene deletions, using the multiplex amplification of 15 deletion-prone exons described by Chamberlain et al. and Beggs et al. The percentage of deletions was 52.5%, and the majority of them (86.3%) were located at the hot spot deletion region which encompasses exons 44-55. This frequency is higher than that found in American and European populations. There were no correlations between deletion size, location and clinical severity.