Fungal catabolic gene regulation: Molecular genetic analysis of theamdS gene ofAspergillus nidulans

Aspergillus nidulans is an excellent experimental organism for the study of gene regulation. Genetic and molecular analyses oftrans-acting andcis-acting mutations have revealed a complex pattern of regulation involving multiple independent controls. Expression of theamdS gene is regulated by thefacB andamdA genes which encode positively acting regulatory proteins mediating a major and a minor form of acetate induction respectively. The product of theamdR gene mediates omega amino acid induction ofamdS. The binding sites for each of these proteins have been localised throughamdS cis-acting mutations which specifically affect the interaction with the regulatory protein. The global controls of nitrogen metabolite repression and carbon catabolite repression regulate the expression of many catabolic genes, includingamdS. Nitrogen control is exerted through the positively actingareA gene product and carbon control is dependent on thecreA gene product. Each of the characterized regulatory genes encodes a DNA-binding protein which recognises particular sequences in theamdS promoter to activate or repress gene expression. In addition, there is evidence for other genetically uncharacterised proteins, including a CCAAT-binding complex, which interact with the 5 region of theamdS gene.     

Meiotic recombination in an Irish family with beta-thalassaemia

Using the technique of allele-specific priming of the polymerase chain reaction (PCR), the C-T substitution in codon 39 was identified as the cause of -thalassaemia in an Irish family. Analysis of the restriction fragment length polymorphisms (RFLPs) in the -globin gene cluster established linkage of the -thalassaemia mutation to a particular -haplotype but indicated that a recombinational event had occurred in the paternal chromosome in the younger of two affected children. Non-paternity was excluded by DNA fingerprinting analysis with hypervariable minisatellite probes. This is the fourth case of recombination in the -globin gene cluster to be reported. The event has occurred 5 of the polymorphic RsaI site at position-550 bp upstream of the -globin gene mRNA Cap site, within the 9.1-kb region that has been shown to be a hot spot for recombination in the -globin gene cluster.     

Experimental and field evidence of Antarctic foraminiferal tolerance to anoxia and hydrogen sulfide

The common occurrence of foraminifera in strata deposited under anoxic environmental conditions throughout the geologic record raises the question of whether foraminifera actually survive anoxic or reducing conditions. To test this, benthic foraminifera from McMurdo Sound, Antarctica were exposed to anoxic or reducing conditions for 30 days. These Antarctic forms are known to be exposed to anoxic events in their natural environment. An adenosine triphosphate (ATP) assay was used to determine foraminiferal survival, and their ultrastructure was examined using transmission electron microscopy (TEM) to evaluate any possible effect that might have resulted from exposure to anoxic or reducing conditions. The experimental treatments did not have any statistically significant effects upon the foraminifers with regards to survival or average ATP content per individual including the four most common living species. Some ultrastructural evidence for encystment (fibrillar membrane-bound bodies in the cytoplasm of many specimens) was observed. Field data include the observation thatGlobocassidulina cf.G. biora resides in sediments to depths of 7 cm, as evidenced by ultrastructural investigations. A specimen ofGlobocassidulina cf.G. biora collected from sediments containing anoxic pore waters showed numerous bacteria within the confines of the organic lining. The occurrence of cytoplasmic inclusions similar in appearance to car☐ysomes within these bacteria suggest possible affinities to the chemolithotrophicThiobacillus. Bacteria of this type were not observed in specimens collected from oxygenated pore waters. These field and laboratory findings suggest that certain foraminifera may be facultative anaerobes able to withstand reducing conditions.     

Substitution of transferrin by FeCl3 in the development of a low foetal calf serum concentration medium for KB-26.5 hybridoma cell line

KB-26.5, a murine hybridoma cell line producing an IgG₃ monoclonal antibody used in blood type determination, primarily adapted to grow at 5% foetal calf serum (FCS) concentration has been adapted to grow at 0.5% FCS, maintaining its ability to produce antibodies at the same level. In the final step of adaptation, the addition of insulin, transferrin, ethanolamine and selenium to the media formulation was studied, using factorial assay techniques to check the effect of the different compounds and to optimize their required level for satisfactory growth and antibody secretion. KB-26.5 cells required only 20 g/ml of transferrin to adapt to 0.5% FCS medium. Furthermore, transferrin could be substituted by FeCl₃, at a relatively low level of 2 g/ml. Maximum cell density decreased by 31.5% in spinner flask test, but the antibody titer was maintained, thus the specific productivity increased. However, inoculum size had to be increased three-fold with 0.5% FCS medium in order to assure cell growth.     

EVIDENCE OF DARK AVOIDANCE BY PHOTOTROPHIC PERIPHYTIC DIATOMS IN LOTIC SYSTEMS1

Short-term (24–48 h) colonization dynamics of periphytic diatoms on artificial (styrofoam) substrata were examined using fast-flushing, continuous-flow troughs located on the North Thompson River, British Columbia. Two parallel troughs, one exposed to natural light and the other completely darkened, showed significant differences in periphyton biomass, chlorophyll a, and algal taxonomic composition with 24 h. Experiments which commenced at the onset of natural darkness demonstrated that rates of algal immigration during the night were the same in both troughs. Within 2–3 h of sunrise, however, certain diatom species (most notably Hannaea arcus (Ehr.) Pair, and Diatoma tenue Ag.) selectively emigrated from the artificially darkened trough but remained in the trough exposed to natural light. More closely adhering species such as Achnanthes minutissima Kütz, also showed significant emigration from the darkened trough after light deprivation for two photoperiods. Data from adhesion, emigration, and sinking rate experiments indicate that differential egress of cells from the darkened versus the lighted environments is the result of cellular regulation of buoyancy or form resistance.     

Analysis of the effect of potato carboxypeptidase inhibitor pro-sequence on the folding of the mature protein.

Protein folding can be modulated in vivo by many factors. While chaperones act as folding catalysts and show broad substrate specificity, some pro-peptides specifically facilitate the folding of the mature protein to which they are bound. Potato carboxypeptidase inhibitor (PCI), a 39-residue protein carboxypeptidase inhibitor, is synthesized in vivo as a precursor protein that includes a 27-residue N-terminal and a seven-residue C-terminal pro-regions. In this work the disulfide-coupled folding of mature PCI in vitro has been compared with that of the same protein extended with either the N-terminal pro-sequence (ProNtPCI) or both N- and C-terminal pro-sequences (ProPCI), and also with the N-terminal pro-sequence in trans (ProNt + PCI). No significant differences can be observed in the folding kinetics or efficiencies of all these molecules. In addition, in vivo folding studies in Escherichia coli have been performed using wild-type PCI and three PCI mutant forms with and without the N-terminal pro-sequence, the mutations had been previously reported to affect folding of the PCI mature form. The extent to which the 'native-like' form was secreted to the media by each construction was not affected by the presence of the N-terminal pro-sequence. These results indicate that PCI does not depend on the N-terminal pro-sequence for its folding in both, in vitro and in vivo in E. coli. However, structural analysis by spectroscopy, hydrogen exchange and limited proteolysis by mass spectrometry, indicate the capability of such N-terminal pro-sequence to fold within the precursor form.