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.     

Progress in fold recognition

The prediction experiment reveals that fold recognition has become a powerful tool in structural biology. We applied our fold recognition technique to 13 target sequences. In two cases, replication terminating protein and prosequence of subtilisin, the predicted structures are very similar to the experimentally determined folds. For the first time, in a public blind test, the unknown structures of proteins have been predicted ahead of experiment to an accuracy approaching molecular detail. In two other cases the approximate folds have been predicted correctly. According to the assessors there were 12 recognizable folds among the target proteins. In our postprediction analysis we find that in 7 cases our fold recognition technique is successful. In several of the remaining cases the predicted folds have interesting features in common with the experimental results. We present our procedure, discuss the results, and comment on several fundamental and technical problems encountered in fold recognition. © 1995 Wiley-Liss, Inc.     

One of three nuclear localization signals of maize Activator (Ac) transposase overlaps the DNA-binding domain

The nuclear localization sequences (NLSs) of the Ac transposase (TPase) protein have been characterized by indirect immunofluorescence detection of TPase deletion derivatives and TPase/β-glucuronidase (GUS) fusion proteins in transiently transfected Petunia cells. The TPase contains three NLSs near its amino-terminal end, NLS(44–62), NLS(159–178) and NLS(174–206), each of which is sufficient to redirect GUS to the nucleus. Deletion of the N-terminal 102 TPase residues including NLS(44–62) results in strongly reduced nuclear import of the truncated TPase. NLS(44–62) and NLS(159–178) are bipartite NLSs, whereas the structure of NLS(174–206) does not allow a classification into one of the three major NLS categories. NLS(174–206) overlaps with the basic DNA-binding domain of TPase. A substitution of two amino acids in this segment (HiS₁₉₁→Arg and Arg₁₉₃→His) results in a total loss of DNA-binding activity, but retains reduced NLS activity. Accordingly, the two functions can be separated. In addition, we show that a NLS-deficient 71 kDa TPase derivative is co-imported into the nucleus in the presence of wildtype TPase.     

Age-dependent alterations of linoleic, arachidonic and eicosapentaenoic acids in renal cortex and medulla of spontaneously hypertensive rats

The lipid content as well as the fatty acid pattern of triglycerides, free fatty acids (FFA), phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were estimated in renal cortex and medulla of spontaneously hypertensive rats (SHR) and normotensive Wistar rats (WR) at 4,8,26 and 52 weeks of age. In general, the level of triglycerides in renal medulla appeared higher when compared with the cortex. On the other hand, PC and PE, increasing with age, were usually higher in the cortex. A decreased percentage of linoleic acid (LA) in triglycerides, of arachidonic acid (AA) in PC and of eicosapentaenoic acid (EPA) in triglycerides, FFA, PC and PE could be found in the kidneys of SHR at 8 weeks of age, i.e. during the development of hypertension. This was accompanied with a rise of AA in FFA of SHR at 8 weeks of age, which occurred with delay in WR (at 26 weeks of age). From the data presented it can be concluded that systematic alterations in the availability of individual polyunsaturated fatty acids (PUFA) in various renal lipids might be related to the onset of hypertension in SHR which should be elucidated in more detail.     

Transient Responses of Glucose-Limited Cultures of Cytophaga johnsonae to Nutrient Excess and Starvation

Cells from glucose-limited chemostat cultures of Cytophaga johnsonae were subjected to a sudden relaxation of substrate limitation by injecting the cells into fresh batch cultures. Starvation experiments were carried out by injecting glucose-limited cells into batch cultures lacking glucose. Transient responses of biomass, glucose uptake and mineralization, ATP content, and viability on different agar media were monitored during these nutrient-shift experiments. Cells reacted differently depending on growth rate and time spent in the chemostat. Fast-growing cells showed an immediate adaptation to the new growth conditions, despite some initial overshoot reactions in ATP and uptake potential. In contrast, slowly growing cells and long-term-adapted cells showed extensive transient growth responses. Glucose uptake and mineralization potentials changed considerably during the transient growth phase before reaching new levels. During the starvation experiments, all cell types displayed a fast decrease in ATP, but the responses of the substrate uptake and mineralization potentials were strongly dependent upon the previous growth rate. Both potentials decreased rapidly in cells with high growth rates. On the other hand, cells with low growth rates maintained 80% of their uptake and mineralization potentials after 8 h of starvation. Thus, slowly growing cells are much better adapted for starvation than are fast-growing cells.     

Toward a complete linkage map of the human X chromosome: regional assignment of 16 cloned single-copy DNA sequences employing a panel of somatic cell hybrids.

Closely linked restriction fragment length polymorphisms (RFLPs) are potentially useful as diagnostic markers of genetic defects, and, in principle, RFLPs can be employed to construct a complete linkage map of the human genome. On the X chromosome, linkage studies are particularly rewarding because in man more than 120 X-linked genes are known. Thus, it is probable that each X-specific RFLP will be of use as a genetic marker of one or several X-linked disorders. To facilitate the search for closely linked RFLPs, we have regionally assigned 16 cloned DNA sequences to various portions of the human X chromosome, employing a large panel of somatic cell hybrids. These probes have been used to correlate genetic and physical distances on Xp, and it can be extrapolated from these data that the number and distribution of available Xq sequences will also suffice to span the long arm of the X chromosome.     

An improved timm sulphide silver method for light and electron microscopic localization of heavy metals in biological tissues

Summary Modifications of the Timm sulphide silver method for the demonstration of heavy metals are described.To improve the structural preservation of the tissues perfusion with a glutaraldehyde fixative is employed before perfusion with the sodium sulphide solution. For the subsequent staining for light and electron microscopy, procedures for plastic embedding, paraffin embedding and cryostat sectioning are presented. Examples from several tissues are shown, including the pituitary, pancreas, intestine, tongue, kidney, testis and brain. The staining of autolytic, postmortal human brain tissue is demonstrated.     

Evaluation Of Eight Fluorochrome Combinations For Simultaneous Dna-Protein Flow Analyses

Eight fluorescent dye combinations for simultaneous DNA-protein staining have been evaluated spectroscopically and flow microfluoromctrically: propidium iodide (PI) with fluorescein-isothiocyanate (FITC), fluorescamine (FC), and dansylchloride (DANS); diamidinophenylindole (DAPI) with sulphorhodamin (SR101), tetramethylrhodamin isothiocyanate (TRITC), and nitroben-zodiazole (NBD); acriflavine (AF) with stilbene isothiocyanate sulphonic acid (SITS), and DAPI. Three different experimental tumor cell lines have been employed in the investigations. Simultaneous DNA-protein analyses have been carried out with the newly developed HEIFAS instrument. Spectroscopically two groups of dyes were distinguishable according to their excitation maximum below 400 nm and above 450 nm respectively. DANS and NBD were found to be unsatisfactory with respect to their protein distributions obtained by flow analysis. The remaining stains involved in the dye combinations revealed comparable flow distributions of the cellular DNA and protein content. With respect to preparation time and number of centrifugal steps involved in the staining protocols, and in connection with the stability of the dye used, the DAPI-SR101 method proved to be fastest and easiest With this combination DNA and protein flow analysis can be performed simultaneously within 30 min.     

The chromosome fiber: Evidence for an ordered superstructure of nucleosomes

Chromosome fibers isolated from lymphocyte nuclei and prepared for electron microscopy by techniques designed to preserve their native structure have a distinctly knobby appearance, suggesting that DNA and protein are not distributed evenly along the fiber axis. Individual knobs (superbeads) are arranged in tandem and have an average diameter of about 200 Å. Mild nuclease digestion of isolated nuclei releases apparent monomer superbeads that are composed of nucleohistone particles with the properties of nucleosomes. The kinetics of digestion indicate that the superbead is a discrete structural unit containing, on the average, about eight nucleosomes.