Electric birefringence of recombinant spectrin segments 14, 14-15, 14-16, and 14-17 from Drosophila alpha-spectrin

Members of the spectrin protein family can be found in many different cells and organisms. In all cases studied, the major functional role of these proteins is believed to be structural rather than enzymatic. All spectrin proteins are highly elongated and consist mainly of homologous repeats that constitute rigid segments connected in tandem. It is commonly believed that the details of the spectrin function depend critically on the flexibility of the links between the segments. Here we report on a work addressing this question by studying the transient electric birefringence of recombinant spectrin fragments consisting of segments 14, 14-15, 14-16, and 14-17, respectively, from Drosophila alpha-spectrin. Transient electric birefringence depends sharply on both molecular length and flexibility. We found that the birefringence relaxation time of segment 14 measured at 4 degrees C, but scaled to what is expected at 20 degrees C, equals 16 ns (+/-15%) at pH 7.5 and ionic strength 6 mM. This is consistent with this single segment being rigid, 5 nm long and having an axial ratio equal to about two. Under the same conditions, segments 14-15, 14-16 and 14-17 show relaxation times of 45, 39 and 164 ns (all +/-20%), respectively, scaled to what is expected at 20 degrees C. When the temperature is increased to 37 degrees C the main relaxation time for each of these multisegment fragments, scaled to what is expected at 20 degrees C, increased to 46, 80, and 229 ns (all +/-20%), respectively. When the ionic strength and the Debye shielding is low, the dynamics of these short fragments even at physiological temperature is nearly the same as for fully extended weakly bending rods with the same lengths and axial ratios. When the ionic strength is increased to 85 mM, the main relaxation time for each of these multisegment fragments is reduced 20-50% which suggests that at physiological salt and temperature conditions the links in 2-4-segment-long fragments exhibit significant thermally induced flexing. Provided that the recombinant spectrin fragments can serve as a model for native spectrin, this implies that, at physiological conditions, the overall conformational dynamics of a native spectrin protein containing 20-40 segments equals that of a flexible polymer.     

New data on clonal anomalies of chromosome 14 in ataxia telangiectasia: tct (14;14) and inv(14)

Summary From a series of 53 patients with ataxia telangiectasia, two large clones with a t tan or tct(14;14) and two with an inv(14) were observed among phytohaemagglutinin (PHA)-stimulated lymphocytes. Smaller clones with the same inv(14) were observed in two other cases. Similar breakpoints may exist, both for t(14;14) and inv(14): q11.1/q11.2 and q32, and it is postulated that the rearrangements are related to a recombination of the -chain of the T-cell receptor and IgH clusters of genes.     

Assimilative Pathway of Methanol in Candida sp. Incorporation of 14C-Methanol, 14C-Formaldehyde, 14C-Formate and 14C-Bicarbonate into Cell Constituents

1. The incorporation of ¹⁴C-methanol, ¹⁴C-formaldehyde, ¹⁴C-formate and ¹⁴C-bicarbonate into a methanol-utilizing yeast, Candida N–16, was examined by paper-chromato-graphy and radioautography.

2. At the earliest time period examined, the highest percentage of radioactivity fixed from ¹⁴C-methanol or ¹⁴C-formaIdehyde into methanol-grown cells was found in fructose phosphate. The percentage distribution of radioactivity in fructose phosphate decreased as time elapsed. The radioactivity fixed from these compounds into glucose-grown cells was negligible compared with that fixed into methanol-grown cells.

3. The incorporation of ¹⁴C-formate into methanol-grown cells was extremely low. The highest percentage of radioactivity fixed for short time incubation was found in serine. The incorporation pattern of glucose-grown cells was similar to that of methanol-grown cells.

4. At the earliest time period, over 70% of radioactivity fixed from ¹⁴C-bicarbonate into methanol- or glucose-grown cells was found in aspartate.

5. These results suggest that in Candida N–16 methanol is specifically assimilated by a route with hexose phosphate as a primary stable intermediate.

     

Mammalian Pex14p: membrane topology and characterisation of the Pex14p-Pex14p interaction

Peroxisomal biogenesis is a complex process requiring the action of numerous peroxins. One central component of this machinery is Pex14p, an intrinsic peroxisomal membrane protein probably involved in the docking of Pex5p, the receptor for PTS1-containing proteins (peroxisomal targeting signal 1-containing proteins). In this work the membrane topology of mammalian Pex14p was studied. Using a combination of protease protection assays and CNBr cleavage, we show that the first 130 amino acid residues of Pex14p are highly protected from exogenously added proteases by the peroxisomal membrane itself. Data indicating that this domain is responsible for the strong interaction of Pex14p with the organelle membrane are presented. All the other Pex14p amino acid residues are exposed to the cytosol. The properties of recombinant human Pex14p were also characterised. Heterologous expressed Pex14p was found to be a homopolymer of variable stoichiometry. Finally, in vitro binding assays indicate that homopolymerisation of Pex14p involves a domain comprising amino acid residues 147-278 of this peroxin.     

Metabolism of 14C-Chlorobenzilate and 14C-Chloropropylate by Rhodotorula gracilis

Rhodotorula gracilis metabolizes Chlorobenzilate (ethyl 4,4'-dichlorobenzilate) and Chloropropylate (isopropyl 4,4'-dichlorobenzilate) to several metabolites in a basal medium supplemented by sucrose and by several intermediates of the citric acid cycle. Three identified metabolites resulting from the degradation of either acaricide, were 4,4'-dichlorobenzilic acid, 4,4'-dichlorobenzophenone, and carbon dioxide. Chlorobenzilate, i.e., ethyl ester of 4,4'-dichlorobenzilic acid, was more easily hydrolyzed than Chloropropylate, i.e., isopropyl ester of this acid, so that larger amounts of carbon dioxide and 4,4'-dichlorobenzophenone were obtained from Chlorobenzilate degradation. Regardless of acaricides used, longer incubation caused a higher accumulation of 4,4'-dichlorobenzophenone. The probable steps of the degradation pathway are: Chlorobenzilate (or Chloropropylate) --> 4,4'-dichlorobenzilic acid --> 4,4'-dichlorobenzophenone plus carbon dioxide. It appears that the decarboxylation of 4,4'-dichlorobenzilic acid to 4,4'-dichlorobenzophenone was hindered by alpha-ketoglutarate and enhanced by succinate.     

Mouse Chromosome 14

Chair of Committee for Mouse Chromosome 14     

Ring-14 and trisomy 14q in the same child

The case of a male child with three cell lines is described: one cell line with ring chromosome 14, another trisomic for 14q, due to a derived metacentric 14q;14q, and a third one with a normal male karyotype. The clinical findings are compatible with those of the r(14) syndrome.