Endothelial cell junctions

In the course of a freeze-cleave study on intercellular junctions in the regenerating rat liver, we observed an unusual array of intramembranous particles located in regions of contact between endothelial cells lining the hepatic sinusoids. These arrays were characterized by an accumulation of particles which resembled a zonula occludens in their linear deployment but differed in that the contact regions were composed of individual particles which remained separated from each other by regular particle-free intervals.     

Super Suppressors in NEUROSPORA CRASSA I. Induction, Genetic Localization and Relationship to a Missense Suppressor

Genetic analyses have been made to test the feasibility of using coincident reversions to prototrophy of multiple mutants to select super suppressors (ssu) in Neurospora crassa. Of five double-mutant strains examined, only those mutant combinations in which both members had the properties of nonsense mutations did revert coincidently. Forty-eight genetically purified coincident revertants were crossed to the wild type, and each was shown to contain a suppressor mutation. Five super suppressors were examined more thoroughly. Tetrad and random spore analysis was used to demonstrate that each behaved as a single gene in crosses. Two super suppressors, ssu-1 and ssu-4 were localized respectively on the right and left arm of linkage group 7. Two others, ssu-2 and ssu-3, appear to map on the right arm of linkage group 1. The fifth super suppressor mapped, ssu-7, lies between ad-8 and ylo-1 on linkage group 6. One super suppressor, ssu-1, was interesting because it mapped near the location reported for the suppressor of the missense mutant tryp-3(td201) (Yourno and Suskind 1964a). However, no overlap was found in action spectrum of the two suppressors. Tetrad analysis showed the two suppressors were located about 10 map units apart, the missense suppressor being the more distal to the centromere.     

Solid-phase synthesis of a range of O-phosphorylated peptides by post-assembly phosphitylation and oxidation.

A completely general method for the O-phosphorylation of peptides of any given composition using solid-phase methodology is described. Peptides were assembled using Fmoc amino acid active esters, with base used for Fmoc deprotection. Unprotected amino acid side chain hydroxyl groups were phosphitylated and oxidised at the end of the assembly using bis(benzyloxy)(diisopropylamino)phosphine and tert.-butylhydroperoxide respectively. TFA was used for final deprotection of the amino acid side chains and for simultaneous cleavage from the resin. The synthesis of O-phosphopeptides of up to 15 residues in length is described.     

Histone deacetylation is required for the maturation of newly replicated chromatin

The effects of inhibiting histone deacetylation on the maturation of newly replicated chromatin have been examined. HeLa cells were labeled with [3H]thymidine in the presence or absence of sodium butyrate; control experiments demonstrated that butyrate did not significantly inhibit DNA replication for at least 70 min. Like normal nascent chromatin, chromatin labeled for brief periods (0.5-1 min) in the presence of butyrate was more sensitive to digestion with DNase I and micrococcal nuclease than control bulk chromatin. However, chromatin replicated in butyrate did not mature as in normal replication, but instead retained approximately 50% of its heightened sensitivity to DNase I. Incubation of mature chromatin in butyrate for 1 h did not induce DNase I sensitivity: therefore, the presence of sodium butyrate was required during replication to preserve the increased digestibility of nascent chromatin DNA. In contrast, sodium butyrate did not inhibit or retard the maturation of newly replicated chromatin when assayed by micrococcal nuclease digestion, as determined by the following criteria: 1) digestion to acid solubility, 2) rate of conversion to mononucleosomes, 3) repeat length, and 4) presence of non-nucleosomal DNA. Consistent with the properties of chromatin replicated in butyrate, micrococcal nuclease also did not preferentially attack the internucleosomal linkers of chromatin regions acetylated in vivo. The observation of a novel chromatin replication intermediate, which is highly sensitive to DNase I but possesses normal resistance to micrococcal nuclease, suggests that nucleosome assembly and histone deacetylation are not obligatorily coordinated. Thus, while deacetylation is required for chromatin maturation, histone acetylation apparently affects chromatin organization at a level distinct from that of core particle or linker, possibly by altering higher order structure.