A class of amphipathic proteins associated with lipid storage bodies in plants. Possible similarities with animal serum apolipoproteins

The lipid-storing tissues of plants contain many small (0.2-1 microns) lipid (normally triacylglycerol) droplets which are surrounded and stabilized by a mixed phospholipid and protein annulus. The proteinaceous components of the lipid storage bodies are termed oleosins and are not associated with any other cellular structures. The major oleosins of rapeseed and radish have been isolated by preparative SDS-PAGE and are respectively classes of 19 kDa and 20 kDa proteins. Both protein classes were N-terminally blocked for direct sequencing, but were partially sequenced following limited proteolytic digestion. The major rapeseed oleosin was made up of at least two 19 kDa polypeptides, termed nap-I and nap-II, which have closely related but different amino acid sequences. A single 20 kDa oleosin, termed rad-I, was found in radish. A near full length cDNA clone for a major rapeseed oleosin was sequenced and found to correspond almost exactly to the sequence of nap-II. The sequences of nap-I and rad-I show very close similarity to one another, as do the sequences of nap-II and the previously determined sequence for the major oleosin from maize. All four oleosins have a large central hydrophobic domain flanked by polar N- and C-terminal domains. Secondary structure predictions for the four oleosins are similar and a novel model is proposed based on a central hydrophobic beta-strand region flanked by an N-terminal polar alpha-helix and a C-terminal amphipathic alpha-helix. The possibility that oleosins exhibit structural and functional similarities with some animal apolipoproteins is discussed.     

COL5a1: fine genetic mapping and exclusion as candidate gene in families with nail-patella syndrome, tuberous sclerosis 1, hereditary hemorrhagic telangiectasia, and Ehlers—Danlos syndrome type II

COL5A1, the gene for the α1 chain of type V collagen, has been considered a candidate gene for certain diseases based on chromosomal location and/or disease phenotype. We have employed 3′-untranslated region RFLPs to exclude COL5A1 as a candidate gene in families with tuberous sclerosis 1, Ehlers—Danlos syndrome type II, and nail-patella syndrome. In addition, we describe a polymorphic simple sequence repeat (SSR) within a COL5A1 intron. This SSR is used to exclude COL5A1 as a candidate gene in hereditary hemorrhagic telangiectasia (Osler—Rendu—Weber disease) and to add COL5A1 to the existing map of “index” markers of chromosome 9 by evaluation of the COL5A1 locus on the CEPH 40-family reference pedigree set. This genetic mapping places COL5A1 between markers D9S66 and D9S67.     

Synthesis of dl-4,5,6-trinor-3,7-inter-m-phenylene-3oxaprostaglandins including one which inhibits platelet aggregation.

The synthesis of dl-4,5,6-trinor-3,7-inter-m-phenylene-3-oxaprostaglandins of the E1 and F1alpha series from 6-endo-(1-heptenyl)-bicyclo[3:1:0]hexan-3-one (III), is described. Preliminary biological screening data for gerbil colon smooth muscle stimulation, rat blood pressure and substrate specificity toward 15-hydroxyprostaglandin dehydrogenase is presented. Platelet function studies, both in vitro and in vivo of dl-4,5,6-trinor-3,7-inter-m-phenylene-3-oxa-PGE1, methyl ester (VIII) are presented.     

Synthesis of d1-4,5,6-trinor-3,7-inter-m-phenylene-3-oxaprostaglandins including one which inhibits platelet aggregation

The synthesis of $\text{d1}$-4,5,6-trinor-3,7-inter-$\text{m}$-phenylene-3-oxaprostaglandins oxaprostaglandins of the E₁ and F₁α series⁷ from 6-endo-(1-heptenyl)-bicyclo[3:1:0]hexan-3-one (III), is described. Preliminary biological screening data for gerbil colon smooth muscle stimulation, rat blood pressure and substrate specificity toward 15-hydroxyprostaglandin dehydrogenase is presented. Platelet function studies, both $\text{in vitro}$ and $\text{in vivo}$ of $\text{d1}$-4,5,6-trinor-3,7-inter-$\text{m}$-phenylene-3-oxa-PGE₁, methyl ester (VIII) are presented.     

The effect of ethylnitrosourea on chromosome aberrations in vitro and in vivo.

The effect of ENU on (A) human chromosomes from blood lymphocyte cultures in vitro, and on (B) rat and mouse bone marrow chromosomes in vivo, was investigated. Doses of 25, 50, 100 and 200 mug/ml were tested in vitro and cells with chromosome breakage were found to be dose dependent. Chromosome damage was also dependent on time; maximum damage was seen when cells were treated 2--6 hrs before harvest. Two doses of 100 and 200 mg/kg were studied in rat and mouse in vivo and a dose effect could be shown in both species. The highest number of abnormal cells was found 6 hrs after treatment; there was a sharp decrease at 18 hrs and thereafer. Types of aberrations were also analyzed, in both in vitro and in vivo studies.     

Germ-line mosaicism in tuberous sclerosis: how common?

Two-thirds of cases of tuberous sclerosis complex (TSC) are sporadic and usually are attributed to new mutations, but unaffected parents sometimes have more than one affected child. We sought to determine how many of these cases represent germ-line mosaicism, as has been reported for other genetic diseases. In our sample of 120 families with TSC, 7 families had two affected children and clinically unaffected parents. These families were tested for mutations in the TSC1 and TSC2 genes, by Southern blotting and by single-strand conformational analysis. Unique variants were detected in six families. Each variant was present and identical in both affected children of a family but was absent in both parents and the unaffected siblings. Sequencing of the variants yielded two frameshift mutations, one missense mutation, and two nonsense mutations in TSC2 and one nonsense mutation in TSC1. To determine which parent contributed the affected gametes, the families were analyzed for linkage to TSC1 and TSC2, by construction of haplotypes with markers flanking the two genes. Linkage analysis and loss-of-heterozygosity studies indicated maternal origin in three families, paternal origin in one family, and either being possible in two families. To evaluate the possibility of low-level somatic mosaicism for TSC, DNA from lymphocytes of members of the six families were tested by allele-specific PCR. In all the families, the mutant allele was detected only in the known affected individuals. We conclude that germ-line mosaicism was present in five families with mutations in the TSC2 gene and in one family with the causative mutation in the TSC1 gene. The results have implications for genetic counseling of families with seemingly sporadic TSC.     

Site-directed mutagenesis of lysine 58 in a putative ATP-binding domain of the calmodulin-sensitive adenylate cyclase from Bordetella pertussis abolishes catalytic activity

A 2.7-kb cya A gene fragment encoding the amino-terminal end of the calmodulin-sensitive adenylate cyclase from Bordetella pertussis has been placed under the control of the lac promoter for expression in Escherichia coli. Following induction with isopropyl beta-D-thiogalactoside, calmodulin-sensitive adenylate cyclase activity was detected in a cell extract from E. coli. The expression vector directed the synthesis of a 90-kDa polypeptide that was recognized by rabbit polyclonal antibodies raised against the catalytic subunit of B. pertussis adenylate cyclase. Inspection of the deduced amino acid sequence of the cya A gene product revealed a sequence with homology to consensus sequences for an ATP-binding domain found in many ATP-binding proteins. On the basis of the analysis of nucleotide binding proteins, a conserved lysine residue has been implicated in the binding of ATP. A putative ATP-binding domain in the B. pertussis adenylate cyclase possesses an analogous lysine residue at position 58. To test whether lysine 58 of the B. pertussis adenylate cyclase is a crucial residue for enzyme activity, it was replaced with methionine by oligonucleotide-directed mutagenesis. E. coli cells were transformed with the mutant cya A gene, and the expressed gene product was characterized. The mutant protein exhibited neither basal nor calmodulin-stimulated enzyme activity, indicating that lysine 58 plays a critical role in enzyme catalysis.