The duplication in Charcot-Marie-Tooth disease type 1a spans at least 1100 kb on chromosome 17p11.2

Summary Recently, it has been shown that Charcot-Marie-Tooth disease type 1a (CMT1a) is linked with a duplication of a DNA segment that is detected by probe VAW409R3, and that is located on chromosome 17p11.2. Here, we show that this duplication also contains VAW412R3a, but not A10-41 and EW503. Accounting for the duplication in recombination analysis, we found recombinants between CMT1a and EW301 and EW502, but not with A10-41, VAW409R3, and VAW412R3. Using pulsed-field gel electrophoresis analysis, we estimated the minimal size of the duplicated region in CMT1a patients to be 1100 kb.     

Spectrum of phenylketonuria mutations in Western Europe and North Africa,and their relation to polymorphic DNA haplotypes at the phenylalanine hydroxylase locus

Summary A total of 252 chromosomes from 126 patients with phenylalanine hydroxylase (PAH) deficiencies were analyzed for both mutant genotypes and restriction fragment length polymorphism (RFLP) haplotypes at the PAH locus. The mutant genes studied originated either from Western Europe (116 alleles) or from Mediterranean countries (136 alleles). Only 27% of all mutant alleles were found to carry identified mutations, particularly mutations at codon 252 (2.3%), 261 (7.5%), 280 (6.3%), 408 (3.5%) and at the splice donor site of intron 12 (6.3%). The mutant genotypes were associated with RFLP haplotypes 7, 1, 38, 2 and 3 at the PAH locus respectively. Except for the splice mutation of intron 12, these associations were preferential, but not exclusive, since the other four mutations were found on the background of at least two RFLP haplotypes. These results, together with the observation that 85% of PAH deficient patients are heterozygotes for their mutant genotypes, emphasize the great heterogeneity of PAH deficiencies in Mediterranean countries and hamper systematic DNA testing for carrier status in this population.     

Use of pectinases complexed to colloidal gold for the ultrastructural localization of polygalacturonic acids in the cell walls of the fungus Ascocalyx abietina

Summary Three pectinase—gold complexes were used to localize polygalacturonic acids in the fungusAscocalyx abietina (Lagerberg) Schlaepfer-Bernhard. With the pectinesterase and pectin lyase—gold complexes, the labelling was uniformly distributed over the fungus walls and did not seem to be significantly influenced by the tissue preparation. With the polygalacturonase—gold complex, differences in the labelling distribution were noted according to the fixation procedure indicating, therefore, that osmication of the tissues could greatly interfere with the localization of the specific enzyme binding sites. These results demonstrate, for the first time, the possibility of detecting polygalacturonic acids by means of different gold-complexed pectinases.     

Differentiation among populations of Sedum wrightii (Crassulaceae) in response to limited water availability: water relations,CO2 assimilation,growth and survivorship

Summary Sedum wrightii is one of only a few species in the Crassulaceae for which there is evidence for a high degree of variability in the ratio of daytime to nighttime CO₂ assimilation. There are both environmental and genetic components to this variability. S. wrightii grows over a wide altitudinal gradient. The purpose of this study was to compare low, intermediate, and high altitude populations with respect to the degree of CAM expression and the capability to tolerate limited water availability. We utilized clonallyreplicated genotypes of plants from each population in common environment greenhouse experiments. Genetic differences among the populations were found in long-term water use efficiency, in 24 hour CO₂ exchange patterns, in biomass ¹³C values, in carbon allocation, and in water status and ultimately survival during prolonged drought. The differences among the populations appear to be closely related to differences in the native habitats. The low altitude, desert plants had the greatest ability to grow and survive under conditions of limited water availability and appear to have the greatest shift to nighttime CO₂ uptake during periods without water, while the high altitude plants had the poorest performance under these conditions and appear to shut down net carbon uptake when severely water limited.     

Identification of zona binding proteins of rabbit, pig, human, and mouse spermatozoa on nitrocellulose blots

Mammalian fertilization is a multi-step process with different requirements for specificity at each step. In the present report we have examined the binding of spermatozoa to homologous and heterologous zonae pellucidae. The homologous zona binding proteins (ZBP) of ejaculated rabbit, pig and human spermatozoa and epididymal mouse spermatozoa have been identified. The rabbit's ZBPs have relative molecular weights (MW) of 32K, 18K, 16K and 14K; the pig's major ZBP is 16K while human spermatozoa bind human zona protein at 17K and 18K. Mouse sperm ZBPs are 19K, 18K and 16K.     

Binding of penicillin to thiol-penicillin-binding protein 3 of Escherichia coli: identification of its active site

Summary In order to determine the active site of penicillin-binding protein 3 of Escherichia coli (PBP3), the serine residue at position 307 was replaced with alanine, threonine or cysteine by oligonucleotide-directed site-specific mutagenesis. Since a unique BanII site exists at the position corresponding to serine-307, BanII digestion of the plasmid DNA after mutagenesis resulted in significant enrichment of the mutant plasmids. For mutagenesis, the gene coding for PBP3 (ftsI) was inserted into the expression cloning vector pIN-IIB. The hybrid protein produced was able to bind penicillin while mutant PBP3 in which serine-307 was replaced with either alanine or threonine did not lead to any detectable binding. However, contrary to the report of Broome-Smith et al. (1985) thiol-penicillin-binding protein 3, in which serine-307 was replaced with cysteine, was still able to bind penicillin. Replacement of serine-445 with an alanine residue had no effect on penicillin binding to PBP3.     

The common 90-kd protein component of non-transformed ''8S'' steroid receptors is a heat-shock protein.

Non-transformed steroid receptors have an approximately 8S sedimentation coefficient that corresponds to an oligomeric structure of 250-300 kd which includes a non-hormone binding 90-kd protein. A monoclonal antibody BF4 raised against the purified, molybdate-stabilized, 8S progesterone receptor (8S-PR) from chick oviduct, recognizes 8S forms of all steroid hormone receptors. BF4 was found specific for a 90-kd protein present in great abundance in all chicken tissues, including that present in 8S-forms of steroid receptors. Here, using immunological and biochemical techniques, we demonstrate that this ubiquitous BF4-positive 90-kd protein is in fact the chicken 90 kd heat-shock protein (hsp 90): it increased in heat-shocked chick embryo fibroblasts, and displayed identical migration in two-dimensional gel electrophoresis and the same V8 peptide map as the already described hsp 90. We discuss the possibility that the interaction between hsp 90 and steroid hormone-binding subunits may play a role in keeping the receptor in an inactive form.     

Rapid purification of mammalian 70,000-dalton stress proteins: affinity of the proteins for nucleotides.

A new and rapid purification procedure has been developed for the mammalian 70,000-dalton (70-kDa) heat-shock (or stress) proteins. Both the constitutive 73-kDa protein and the stress-induced 72-kDa protein have been purified by a two-step procedure employing DE52 ion-exchange chromatography followed by affinity chromatography on ATP-agarose. The two proteins, present in approximately equal amounts in either the 12,000 X g supernatant or pellet of hypotonically lysed heat-shock-treated HeLa cells, were found to copurify in relatively homogenous form. The purified proteins were covalently labeled with the fluorescent dye tetramethylrhodamine isothiocyanate, and the fluorescently labeled proteins were introduced back into living rat embryo fibroblasts via microinjection. The microinjected cells maintained at 37 degrees C showed only diffuse nuclear and cytoplasmic fluorescence. After heat-shock treatment of the cells, fluorescence was observed throughout the nucleus and more prominently within the nucleolus. This result is consistent with our earlier indirect immunofluorescence studies which showed a nuclear and nucleolar distribution of the endogenous 72-kDa stress protein in heat-shock-treated mammalian cells. The result also indicates that, for at least the 72-kDa protein, (i) the protein has been purified in apparently "native" form and (ii) its nucleolar distribution is stress dependent.