Direct molecular analysis of the fragile X syndrome in a sample of Egyptian and German patients using non-radioactive PCR and Southern blot followed by chemiluminescent detection

Molecular genetic analysis of individuals from 6 Egyptian and 33 German families with fragile X syndrome and 240 further patients with mental retardation was performed applying a completely non-radioactive system. The aim of our study was the development of a non-radioactive detection method and its implementation in molecular diagnosis of the fragile X syndrome. Furthermore, we wanted to assess differences in the mutation sizes between Egyptian and German patients and between Egyptian and German carriers of a premutation. Using non-radioactive polymerase chain reaction (PCR), agarose gel electrophoresis and blotting of the PCR products, followed by hybridisation with a digoxigenin-labelled oligonucleotide probe (CGG)₅ and chemiluminescent detection, we identified the fragile X full mutation (amplification of a CGG repeat in the FMR-1 gene ranging from several hundred to several thousand repeat units) in all patients. We observed no differences in the length of the CGG repeat between the Egyptian and German patients and carriers, respectively. However, in one prenatal diagnosis, we detected only one normal sized allele in a female fetus using the PCR-agarose assay, whereas Southern blot analysis with the digoxigenin labelled probe StB 12.3 revealed presence of a full mutation. Our newly established nonradioactive genomic blotting method is based on the conventional radioactive Southern blot analysis. Labelling of the probe StB 12.3 with digoxigenin via PCR allowed the detection of normal, premutated and fully mutated alleles. For exact sizing of small premutated or large normal alleles, we separated digoxigenin labelled PCR products through denaturing poly-acrylamide gelelectrophoresis (PAGE) and transfered them to a nylon membrane using a gel dryer. The blotted PCR-fragments can easily be detected with alkaline phosphate-labelled anti-digoxigenin antibody. The number of trinucleotide repeat units can be determined by scoring the detected bands against a digoxigenated M13 sequencing ladder. Our newly developed digoxigenin/chemiluminescence approach using PCR and Southern blot analysis provides reliable results for routine detection of full fragile X mutations and premutations.     

16S-23S and 23S-5S intergenic spacer regions of Streptococcus thermophilus and Streptococcus salivarius,primary and secondary structure

The 16S-23S intergenic spacer region (spacer region 1) of Streptococcus salivarius, S. thermophilus, and Lactococcus lactis subsp. cremoris and the 23S-5S intergenic spacer region (spacer region 2) of S. salivarius and L. lactis subsp. cremoriswere sequenced and compared with the spacer regions 1 and 2 of other streptococci. A high degree of intraspecific conservation was observed for S. thermophilus and L. lactis, and very similar sequences were found for S. salivarius and S. thermophilus. Whereas spacer region 1 is highly conserved in the genus Streptococcus sensu-stricto,only the tRNA gene and the rRNA processing stems are highly conserved in the three genera: Streptococcussensu-stricto, Lactococcus, and Enterococcus. The presence of a unique tRNA^Ala gene without the 3 terminal CCA sequence seems to be a general feature of the streptococci spacer region 1. A secondary structure model was built to show the interaction between the spacer regions 1 and 2 of S. thermophilus and S. salivarius. The rapid evolution of spacer region 1 in streptococci is in part due to insertions and deletions of small RNA stem/loop structures.     

Callus formation from Malus x domestica cv. ‘Jonathan’ protoplasts

Protoplasts could be successfully isolated and cultured from callus and suspension cultures of Malus xdomestica cv. Jonathan. Protoplast-derived colonies were recovered when the osmoticum (glucose) was gradually reduced in semi-solid 8p medium or by the use of feeder plates. Formation of embryo-like structures was induced from the protoplast-derived callus on media supplemented with IAA and BA. These structures formed roots but plants failed to develop. Protoplasts could be isolated from leaves, but not from stems or petioles. The leaf protoplasts failed to divide.List of abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - ABA abscisic acid - IAA indole acetic acid     

Genetic and biosynthetic studies of families carrying hemoglobin J α Mexico: Association of α-thalassemia with Hb J

Summary Hemoglobin J Mexico, an chain mutant, was studied in eight unrelated Algerian families. The quantities of the abnormal hemoglobin in 116 subjects are trimodally distributed: 55% in homozygotes, 31% and 38% in heterozygotes. Both hematological data and the / chain biosynthetic ratio are normal in heterozygotes with 31% Hb J and in homozygotes. In contrast, the MCV and MCH as well as the / biosynthetic ratio are slightly reduced in heterozygotes with 38% Hb J and in their relatives carrying Hb A. The elevated expression of ^J chains in heterozygotes with 38% Hb J may be due to an thalassemia gene trans to the ^>J locus.     

The effect of monochromatic radiation in the range 350 to 750 nm on the carotenogenesis in Verticillium agaricinum

An action spectrum for carotenogenesis in V. agaricinum has maxima at 395, 433, 660 and 737 nm. In a previous study it had been shown that a light-minus-dark difference spectrum of a crude extract from V. agaricinum had maxima at 390 and 420 nm, and furthermore a red, far-red interaction suggesting phytochrome involvement has been proposed. All these data suggest that there may be at least two photoreceptor systems operating in the photoinduction process here; one for the near-ultraviolet (UV-A)-mediated carotenogenesis, presumably a novel pigment, and the other for the red, far-red region, most likely phytochrome.     

Produits neutres et alcaloides de Myrtopsis macrocarpa,M. myrtoidea,M. novae-caledoniae et M. sellingii

Stems and leaves of Myrtopsis macrocarpa, M. myrtoidea, M. novae-caledoniae and M. sellingii yielded terpenes, sterols, coumarins, alkaloids (furoquinolines and quinolones) and amides. A new quinolone (8-methoxy flindersine) occurs in Myrtopsis macrocarpa, a new amide (N-benzoyltryptamine) in M. myrtoidea, two new coumarins (myrsellin and myrsellinol) and a new dihydrofuroquinoline (myrtopsine) in M. sellingii. Structures of the new compounds are proposed from chemical and spectroscopic evidence.     

Cowpea Rhizobia Producing Dark Nodules: Use in Competition Studies

During a program of screening rhizobia from West Africa, it was found that some strains produced nodules of unusually dark appearance on cowpeas, but not on peanuts, soybeans, pigeon peas, or mung beans. The dark pigmentation was in the bacteroid zone, was not correlated with nodule effectiveness, and was additional to the leghemoglobin pigment. Only rhizobial strains with a nongummy (“dry”) colony morphology produced dark nodules. Visually distinguishable pink and dark nodules formed on the same root when a mixture of pink and dark strains was applied as inoculum. The dark-nodule phenotype was therefore appraised as a marker and found to be useful for studying nodulation competition with strains of the orthodox pink-nodule type. The competitiveness of 10 pink-nodule strains was examined relative to a black-nodule strain, IRc 256; a range of competitiveness was obtained of less competitive than, equally competitive to, or more competitive than IRc 256. Patterns of primary (early) nodulation were generally the same as patterns of secondary (later) nodulation. Mixed infections by dark and pink strains produced piebald nodules, the frequency of occurrence of which was much greater among primary than among secondary nodules.     

Studies on dehydro-l-ascorbic acid 2-arylhydrazone 3-oximes: Conversion into substituted triazoles and isoxazolines

l-threo-2,3-Hexodiulosono-1,4-lactone 2-(arylhydrazones) (2) were prepared by condensation of dehydro-l-ascorbic acid with various arylhydrazines. Reaction of 2 with hydroxylamine gave the 2-(arylhydrazone) 3-oximes (3). On boiling with acetic anhydride, 3 gave 2-aryl-4-(2,3-di-O-acetyl-l-threo-glycerol-l-yl)-1,2,3-triazole-5-carboxylic acid 5,4¹-lactones (4). On treatment of 4 with liquid ammonia, 2-aryl-4-(l-threo-glycerol-l-yl)-1,2,3-triazole-5-carboxamides (5) were obtained. Acetylation of 5 with acetic anhydride-pyridine gave the triacetates, and vigorous acetylation with boiling acetic anhydride gave the tetraacetyl derivatives. Periodate oxidation of 5 gave the 2-aryl-4-formyl-1,2,3-triazole-5-carboxamides (8), and, on reduction, 8 gave the 2-aryl-4-(hydroxymethyl)-1,2,3-triazole-5-carboxamides, characterized as the monoacetates and diacetates. Controlled reaction of 2 with sodium hydroxide, followed by neutralization, gave 3-(l-threo-glycerol-l-yl)-4,5-isoxazolinedione 4-(arylhydrazones), characterized by their triacetates. Reaction of 2 with HBr-HOAc gave 5-O-acetyl-6-bromo-6-deoxy-l-threo-2,3-hexodiulosono-1,4-lactone 2-(arylhydrazones); these were converted into 4-(2-O-acetyl-3-bromo-3-deoxy-l-threo-glycerol-l-yl)-2-aryl-1,2,3-triazole-5-carboxylic acid 5,4¹-lactones on treatment with acetic anhydride-pyridine.     

The polysaccharides of the brown seaweed Turbinaria murrayana

Polysaccharides of the brown seaweed Turbinaria murrayana were isolated. Laminaran (3.2%) was isolated from the hot-water extract of the algae by using ion-exchange chromatography. Fucans (2.1%) were isolated from the hot-water extract, as well (4.7%) as from the extract of the algae with dilute acid. Acid hydrolsysis of the isolated fucans revealed glucose, amnnose, fucose, glucuronic acid, xylose, and galactose. Alginic acid (22.6%) was separated, and reduced to a neutral polysaccharide. The polysaccharides isolated were analyzed by methylation and Smith degradation.