Genomic organization of the canrep repetitive DNA in Brassica juncea

Canrep is a heterogeneous, tandemly repeated, 176 bp nucleotide sequence that contains a single Hind III site and is present in high copy numbers in the genomes of many Brassica species. Complete clusters of repeats of this DNA were cloned from the nuclear DNA of Brassica juncea. Restriction-fragment dimers and higher multimers of the 176 bp sequence have arisen by mutations within the Hind III recognition sequence. Adjacent repeats from within the same cluster usually have different nucleotide sequences with features indicating that diversity is generated by a mechanism that causes site-specific base substitutions. While most of the units of canrep DNA are clustered in long arrays of tandem repeats, some are dispersed throughout the genome as isolated copies or in small clusters. Regardless of the size of the arrays, each cluster begins and ends with a variable-length, truncated repeat and is flanked by inverted copies of the sequence 5-ATCTCAT3-,which is not part of the basic sequence of the canrep family of DNAs. Furthermore, some clusters are located close to nucleotide sequences related to those of known plant transposons. Thus, canrep elements may be dispersed by transposition. There are two distinct subfamilies of canrep sequences in B. juncea, and one of these is closely related to one of the two subfamilies of this type of DNA from B. napus, indicating that it originated from B. campestris, the common diploid ancestor of both amphidiploid species. Neither the repetitive DNA nor nucleotide sequences flanking canrep clusters are transcribed in seedlings, suggesting that even small arrays of repeats are located in heterochromatic regions and might be involved in chromatin condensation and/or chromosome segregation.     

The biosynthetic pathway of the S-alk(en)yl-L-cysteine sulphoxides (flavour precursors) in species of Allium

Pulse labelling experiments with ³⁵SO₄ ^2- fed for 24h to intact plants (shooted onion sets)of Allium cepa (onion) showed that >70% of the label appeared in the S-alkenyl-L-cysteine sulphoxides within 18h, reached a maximum at 48h and thereafter decreased. The amount of label detected in the -glutamyl peptide fractions was below 20% of the total label at any time. It is concluded that in intact plants (at the growth stage used) the -glutamyl peptides are not the immediate precursors of the S-alkenyl-L-cysteine sulphoxides. The major S-alkenyl-L-cysteine sulphoxide in onion was found to be compartmentalized mainly within the endoplasmatic reticulum.Abbreviations AllCysSO (+)-S-2-propenyl-L-cysteine sulphoxide - MeCysSO (+)-S-methyl-L-cysteine sulphoxide - PrenCysSO trans-(+)-S-1-propenyl-L-cysteine sulphoxide - ProCysSO (+)-S-propyl-L-cysteine sulphoxide     

A short purine oligonucleotide forms a highly stable triple helix with the promoter of the murine c-pim-1 proto-oncogene.

A homopurine-homopyrimidine region of murine c-pim-1 proto-oncogene was chosen as a target for triple-helix-forming oligonucleotide. Oligonucleotide 5'-GGG-GAGGGGGAGG-3' was shown to bind to its target sequence in the presence of 50 mM Na+ or K+, 10 mM MgCl2 and 20 mM Tris-acetate, pH 7.5. This oligonucleotide is bound in an antiparallel orientation with respect to the homopurine sequence. As was shown by co-migration assay the triplex is stable up to 65 degrees C. At 37 degrees C it was practically irreversible: after 24 hours of co-migration assay there was no traces of triplex dissociation. The rate of triplex formation was highly accelerated with increase of temperature and Mg2+ concentration. This rate was higher for superhelical DNA when compared to the linear and circular ones and the preference was dependent from temperature and Mg2+ concentration. The precision of this interaction is extremely high: sequences in c-pim-1 promoter region with only one substitution when compared to the target gave negligible triplex formation under investigated conditions. These data suppose that natural triplex structures could play an important role in eukaryotic gene regulation and/or chromatin structure formation.     

Recombinant protein expression in aDrosophila cell line: comparison with the baculovirus system

In this report, we compare two different expression systems: baculovirus/Sf9 and stable recombinantDrosophila Schneider 2 (S2) cell lines. The construction of a recombinant S2 cell line is simple and quick, and in batch fermentations the cells have a doubling time of 20 hours until reaching a plateau density of 20 million cells/ml. Protein expression is driven by theDrosophila Metallothionein promoter which is tightly regulated. When expressed in S2 cells, the extracellular domain of human VCAM, an adhesion molecule, is indistinguishable from the same protein produced by baculovirus-infected Sf9 cells. Additionally, we present data on the expression of a seven trans-membrane protein, the dopamine D4 receptor, which has been successfully expressed in both systems. The receptor integrates correctly in the S2 membrane, binds [³H]spiperone with high affinity and exhibits pharmacological characteristics identical to that of the receptor expressed in Sf9 and mammalian cells. The general implications for large scale production of recombinant proteins are discussed.     

Mitochondrial ribosome assembly in Neurospora crassa: mutants with defects in mitochondrial ribosome assembly.

Summary We have examined mitochondrial (mt) ribosome assembly and-function in five nuclear and six extranuclear mutants of Neurospora crassa which had previously been characterized as deficient in cytochromes b and aa ₃. All six extranuclear mutants showed phenotypes similar to that previously described for the extranuclear [poky] mutant: small subunit-deficient with 19 S rRNA rapidly degraded. The nuclear mutants have the following phenotypes: 297-24 is mt small subunit deficient with 19 S RNA rapidly degraded. 289-56 is mt small subunit deficient but contains normal ratios of 19 S to 25 S RNA in whole mitochondria. 289-67 and 299-9 show defects in the processing of 25 S RNA leading to accumulation of a large precursor RNA. 289-4 is deficient in large subunits although a substantial, but less than normal, amount of 25 S RNA is present in the mitochondria.The present work provides new insight into the phenotypes of mt small subunit-deficient mutants. Previous studies using chloramphenicol suggest that some defects in the assembly of mt small subunits may arise secondarily as a result of inhibition of mt protein synthesis (LaPolla and Lambowitz, 1977; Lambowitz et al., 1979). Three mutants (289-56, 289-67 and 299-9) appear to show such defects. These strains contain incomplete mt small subunits which sediment more slowly than normal and are deficient in at least two proteins, S-5 and S-9. Correlation of mutant phenotypes with rates of mt protein synthesis in the different strains suggests that mt protein synthesis must be decreased to less than one half of the wild-type rate before secondary defects in mt small subunit assembly are observed. This threshold value is much lower than that which leads to gross deficiencies of cytochromes b and aa ₃. Although several mutants have phenotypes suggestive of alterations in mt ribosomal proteins, no such alterations could be identified by two dimensional gel electrophoresis.     

Cation-induced asymmetry of choline flux across presynaptic plasma membranes

Highly cholinergic synaptosomes from the optic lobes of Sepia officinalis retain their ability to concentrate K⁺ and extrude Na⁺ and to synthesise acetylcholien in vitro. Choline uptake is hemicholinium-3 and Na⁺ sensitive but is not obligatorily coupled to choline metabolism, or an energy supply as shown by the action of metabolic and ion pump inhibitors. The influx and efflux and/or steady-state distributions of choline in the presence of Na⁺, Li⁺, Rb⁺, Cs⁺ and mannitol were studied. The influx studies at different cis-choline concentrations revealed two systems for choline influx with different monovalent cation sensitivity and suggested a 1 : 1 interaction of choline with both mechanisms. Choline efflux was stimulated by trans-choline. Calculations of the internal/external concentration ratio expected if choline transport were coupled to the Na⁺ gradient gave a maximal value of about 10². A secondary active transport of choline, where Na⁺ is the driver solute provides an explanation for the cation sensitivity of the mechanism as well as for the method of coupling of choline transport to the varying demands of the nervous system for acetylcholine.     

Le Paléocène supérieur a Ranikothalia bermudezi et l'Éocène inférieur (Ilerdien basal) a Nummulites fraasi et N. Deserti du Tadémait-E et Tinrhert-W

The Upper Paleocene of E-Tademaït and W-Tinrhertis characterized by a new macrofauna and the appearance of Ranikothalia bermudezi. This is followed by Lower Eocene (Lowermost Ilerdian) Nummulites fraasi and N. deserti, in E-Tademaït.We note in relation with the uplifting of Saharo-Nubianshelf a regressive evolution towards the North, where successively later Nummulites species (deserti, globulus, rollandi, gizehensis) appear during the Lower Eocene.