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.     

Separation, isolation and amino acid composition of uremic peptides

Gel filtration and thin layer chromatography were conducted on sera from uremic patients and normal subjects for the isolation of nitrogenous substances unique to uremia. Many ninhydrin-positive substances were found in greater amounts in uremic patients compared to normal subjects. Some of these ninhydrin-positive substances were also detected by staining with chlorine-tolidine. Amino acid analysis of these substances showed considerable qualitative and quantitative differences, perhaps reflecting interference with enzymatic activity by the uremic environment.     

Evidence for a highly asymmetric arrangement of ether- and diacyl-phospholipid subclasses in the plasma membrane of Krebs II ascites cells

(1) Krebs II ascites cells were taken as a model of the neoplastic cells to investigate the transverse distribution of phospholipids in the plasma membrane. The experimental procedure was based on non-lytic degradation of phospholipids in the intact cell by Naja naja phospholipase A₂ and Staphylococcus aureus sphingomyelinase C and on phopholipid analysis of purified plasma membranes. It was shown that the three major phospholipids, i.e., phosphatidylcholine, phosphatidylethanolamine and sphingomyelin, are randomly distributed between the two halves of the membranes, whereas phosphatidylserine remains located in the inner leaflet. (2) The membrane localization of phosphatidylcholine and phosphatidylethanolamine subclasses (diacyl, alkylacyl and alkenylacyl) was also examined, using a new procedure of ether-phospholipid determination. The method involves a selective removal of diacyl species by guinea pig pancreas phospholipase A₁ and of alkenylacyl species by acidolysis. This analysis revealed a 50% increase of ether phospholipids in the plasma membrane as compared to the whole cell (36.5 and 23.1% of total phospholipid, respectively). Furthermore, a strong membrane asymmetry was demonstrated for the three phosphatidylcholine subclasses, since 1-alkyl-2-acyl-sn-glycerol-3-phosphocholine (alkylacyl-GPC) was entirely found in the inner leaflet, whereas both diacyl- and alkenylacyl-GPC displayed an external localization. The same pattern was observed for phosphatidylethanolamine subclasses, except for 1-alkenyl-2-acyl-sn-glycero-3-phosphoethanolamine, which was found randomly distributed. These results are discussed in relation to the process of cell malignant transformation and to the biosynthesis of platelet-activating factor (PAF-acether or 1-alkyl-2-acetyl-GPC).     

Phosphorylation of the androgen receptor by a nuclear cAMP-independent protein kinase

The androgen receptor was purified from rat ventral prostate. The purified receptor migrated as a single band of mol. wt. 87000 on SDS-polyacrylamide gels, had a kd for R-1881 (17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-trien-3-one) binding as 6 nM, and sedimentation coefficient of 4.5 S. Phosphorylation of the purified receptor was studied by incubating it with [gamma-32P]ATP in the presence of several purified protein kinases including cAMP-dependent protein kinase, and four cAMP-independent protein kinases (which were active towards substrates such as phosvitin and casein). Phosphorylation of the 87000 mol. wt. androgen receptor protein occurred only in the presence of a nuclear cAMP-independent protein kinase (of the N2 type). No auto-phosphorylation of the receptor was detected. The results indicate that the androgen receptor is a phosphoprotein. Further, phosphorylation of the androgen receptor by only a specific nuclear cAMP-independent protein kinase may be important in determining the dynamics of its function.     

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     

Effects of polyamines on prostatic chromatin- and non-histone-protein-associated protein kinase reactions.

Studies are presented on the influence of polyamines on prostatic chromatin- and non-histone-protein-associated protein kinase reactions involving both exogenous and endogenous substrates. The activities toward the model acidic protein substrate, dephosphophosvitin, were maximal at 160--200mM-NaCl (or -KCl or -NH4Cl). Under these conditions, spermidine and spermine added in concentrations up to 2mM were essentially without effect. However, without addition of NaCl to the medium, marked stimulation of these reactions was elicited by these polyamines at 1--2mM concentrations. The stimulatory effects were not due to non-specific changes in the ionic strength or to substitution of spermine for Mg2+, as maximal stimulation by 1 mM-spermine was observed only at optimal (2--4mM) Mg2+ concentrations. Qualitatively similar effects of polyamines were observed with enzyme preparations from the prostates of castrated rats, and with chromatin and non-histone-protein preparations from other tissues besides ventral prostate. When phosphorylation of endogenous non-histone proteins of the chromatin was measured, spermine stimulated both the initial rates and the final extent of transphosphorylation, even in the presence of optimal concentration of NaCl. By contrast, spermine or spermidine had no effect on the chromatin- and non-histone-protein-associated protein kinase reactions determined with lysine-rich histones as substrates. Chemically NN-dimethylated dephosphophosvitin was a less active substrate for the chromatin-associated protein kinase, but its phosphorylation was more markedly stimulated by spermine in comparison with unmodified dephosphophosvitin. These observations hint that the polyamine stimulations of the various protein kinase reactions may be due to effects on the conformations of the non-histone protein substrates rather than on the kinases themselves.     

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.