Generation of small mutation in large genomic fragments by homologous recombination: description of the technique and examples of its use

We have developed a technique of homologous recombination in bacteria which allows the mutagenesis of large genomic fragments cloned in cosmids. The desired mutation is first introduced into a plasmid clone and is then transferred to the appropriate cosmid clone by the means of double antibiotic selection coupled with phenotypic selection. We describe three different types of construct made by this technique.     

Constancy of synaptic ribbon numbers in the retina of the arctic chary,Salvelinus alpinus

Synopsis At high latitudes, such as in Iceland, the daily photoperiod varies from almost continuous darkness in winter to virtually constant light in summer. Previous studies of detailed retinal structure in vertebrates have shown significant daily and annual effects of photoperiod. We sampled arctic charr in Iceland during the summer, including fish that were both light- and dark-adapted, during both day and night. We observed retinomotor responses characteristic of light- and dark-adaptation, but found no difference in the number of synaptic ribbons in the retina. The morpho-physiological changes, appearing as retinomotor responses, are thus not expressed at the synaptic level.     

Studies on new,centrally active and reversible acetylcholinesterase inhibitors

We have synthesized the tertiary amines of pyridostigmine and neostigmine, 3-pyridinol dimethylcarbamate (norpyridostigmine) and 3-dimethylaminophenol dimethylcarbamate (norneostigmine) respectively, and we have tested their abilities to cross the blood-brain barrier and inhibit mouse brainAChE activity. The in vivo inhibition of AChE activity by norpyridostigmine reaches 72% at 10 minutes which is comparable to that seen with physostigmine (73% at 10 minutes). Inhibition by norneostigmine is less effective (50% at 10 minutes) and approaches that obtained with tetrahydroaminoacridine (57% at 10 minutes). These data show that both norpyridostigmine and norneostigmine cross the blood-brain barrier and that they are effective inhibitors of mouse brain AChE activity. These drugs could be useful in the treatment of memory, impairment associated with Alzheimer's disease, and other memory disorders.     

Octanoylation of the lipoyl domains of the pyruvate dehydrogenase complex in a lipoyl-deficient strain of Escherichia coli

The overexpression of a subgene encoding a hybrid lipoyl domain of the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex of Escherichia coli has previously been shown to result in the formation of lipoylated and unlipoylated products. Overexpression of the same subgene in a lipoic acid biosynthesis mutant growing under lipoate-deficient conditions has now been shown to produce domains modified by octanoylation as well as unmodified domains. It was concluded from the mass of a lipoyl-binding-site peptide that the modification involves N6-octanoylation of the lysine residue (Lys244) that is normally lipoylated, and this was confirmed by the trypsin-insensitivity of the corresponding Lys244-Ala-245 bond, and the absence of modification in a mutant domain in which Lys244 is replaced by Gln. This novel protein modification raises interesting questions concerning the pathway of lipoic acid biosynthesis and the mechanism of enzyme lipoylation.     

Intrinsic polymorphism of variable number tandem repeat loci in the human genome.

In the human genome, short tandem repetitive (STR) DNA sequences often show restriction fragment length polymorphisms (RFLPs) due to variation in the number of copies of the repeat unit. For a subset of these sequences known as minisatellites or variable number tandem repeat loci (VNTR), it has been proposed that a homologous "core" sequence of 10-12 nucleotides is involved in the mechanism(s) generating the polymorphism. In our present study we have prepared oligonucleotide probes complementary to one or two repeat units of several VNTR loci. Under stringent hybridization and wash conditions these probes hybridize locus specifically thus allowing the evaluation of the intrinsic polymorphism of individual loci. Our results indicate that not all of the loci having STR DNA sequences are polymorphic despite the fact that they share the "core" sequence. This suggests that more than the DNA sequence of the locus is involved in the mechanism(s) generating the polymorphism.     

Glucose utilization and growth response to protein hydrolysates byFusobacterium species

Glucose uptake was measured in the supernatants of 18 strains ofFusobacterium species cultured in BM medium. Some species, such asF. nucleatum andF. necrophorum, used between 25% and 48% of the glucose in the medium, but the terminal pH remained near neutral. By contrast, strains ofF. mortiferum andF. necrogenes used on average over 90% of the available glucose in the medium and produced a predictably low acidic pH. Strains ofF. varium used between 86% and 91% of the glucose present but produced a near neutral pH of between 5.8 and 5.9. The metabolic fate of glucose inF. varium was, therefore, examined in more detail. Glucose stimulated the growth of this species, and [¹⁴C]glucose was incorporated into the metabolic end products and various cellular components. Protein hydrolysates, tested for their growth-promoting effects onFusobacterium species, produced two general growth response patterns. Most species grew prolifically on trypticase, proteose peptone, and yeast extract, but poorly in casamino acids and vitamin-free casamino acids. Growth in bactocasitone was poor, but for three species,F. necrophorum, F. varium, andF. nucleatum, there was an approximately linear growth response up to 0.5%. These results suggest a major role for nitrogen metabolism but do not preclude glucose as an energy source in at least some species ofFusobacterium.     

Haemostasis and oral contraceptives

Selected hemostasis parameters were assayed in 131 women ingesting different kinds of oral contraceptives and 36 control women. No differences were noted in the plasma levels of fibrinogen, antithrombin III, prekallikrein, alpha 2 antiplasmin, fibrinopeptide A, platelet factor 4 and beta-thromboglobulin. Protein C and plasminogen levels were significantly higher in pill users (p less than 0.001, p less than 0.01) and fibronectin levels were lower (p less than 0.05). Canonical correlation, performed between clinical parameters and hemostasis data, revealed a negative correlation between antithrombin III levels and family history for thromboembolic diseases. A positive correlation was noted between fibrinogen and fibronectin levels in obese OC users. The data suggest that women taking OCs are not in a state of "hypercoagulability."