Biochemical analysis of mouse FKBP60, a novel member of the FKPB family.

We have identified mouse and human FKBP60, a new member of the FKBP gene family. FKBP60 shares strongest homology with FKBP65 and SMAP. FKBP60 contains a hydrophobic signal peptide at the N-terminus, 4 peptidyl-prolyl cis/trans isomerase (PPIase) domains and an endoplasmic reticulum retention motif (HDEL) at the C-terminus. Immunodetection of HA-tagged FKBP60 in NIH-3T3 cells suggests that FKBP60 is segregated to the endoplasmic reticulum. Northern blot analysis shows that FKBP60 is predominantly expressed in heart, skeletal muscle, lung, liver and kidney. With N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a substrate, recombinant GST-FKBP60 is shown to accelerate effectively the isomerization of the peptidyl-prolyl bond. This isomerization activity is inhibited by FK506. mFKBP60 binds Ca2+ in vitro, presumably by its C-terminal EF-hand Ca2+ binding motif, and is phosphorylated in vivo. hFKBP60 has been mapped to 7p12 and/or 7p14 by fluorescence in situ hybridization (FISH).     

Heterozygote advantage in Tay-Sachs carriers?

Chi-square analyses of new data as well as data previously reported by Myrianthopoulos have shown that grandparents of Tay-Sachs carriers die from proportionally the same causes as grandparents of noncarriers. It is unlikely that there is any advantage to being a Tay-Sachs carrier insofar as resistance to tuberculosis is concerned. Our results are further evidence to support Fraikor's claim that the high carrier frequency of the allele in Ashkenazi Jews is probably caused by a combination of founder effect, genetic drift, and differential immigration patterns.     

In situ hybridization of semithin Epon sections with BrdU labelled oligonucleotide probes

Summary We recently described a nonradioactive method for in situ hybridization with 5-bromo-2-deoxyuridine (BrdU) labelled oligonucleotide probes. An antibody to BrdU and immunocytochemistry were used in order to detect the hybridization signal. We have now applied this method to semithin Epon sections, in order to hybridize consecutive sections through single cells with different probes and to stain them with antibodies to neuropeptides. It could be shown that Epon embedding preserves mRNA well. In the present study we used a BrdU labelled synthetic oligonucleotide probe complementary to a fragment of the vasopressin precursor and an antibody to Arg-vasopressin. Vasopressin mRNA was demonstrable in a fraction of the vasopressin immunoreactive neurons in the magnocellular nuclei. In addition some of the magnocellular neurons showed either hybridization or vasopressin immunostaining only, perhaps indicating different stages of synthetic and secretory activity. The method described seems to be a valuable tool for studying synthetic activity in peptidergic neurons on a single cell level. The method might also have potential for in situ hybridization on the electronmicroscopical level.     

Synaptonemal complexes of normal and mutant yeast chromosomes (Saccharomyces cerevisiae)

Synaptonemal complex (SC) analysis of six laboratory yeast strains showed the SC karyotypes to be repeatable within strains. Chromosomal differences were found between strains. In five of the strains, two SCs insert into the nucleolus. This represents a single bivalent with a nucleolar organizer in a medial position as is suggested by genetic data or two bivalents each with a terminal nucleolar organizer. In the first interpretation, n=16; in the second, n=17. Strain Tris has a single nucleolar SC and n=17. In strains DCx374, DCx416 and x 8366a the genetically determined rearrangements of linkage group III could not be identified. Presumably the short SC (0.33 m) associated with linkage group III cannot accommodate an inversion loop or a translocation configuration. The strains however were found to harbour a reciprocal translocation involving the nucleolar chromosome. Trisomy for one of the longer chromosomes was observed in Tris and spo10. It is concluded that rearrangements of the medium and long but not short yeast chromosomes can be detected cytologically. — Measurements of nuclear volumes show SC length to vary with artifactually induced swelling of the nucleus. Linear regression of SC length over nuclear radius indicates that actual SC length is only about one-half the observed length. As a result the DNA packing per SC unit length is higher then previously estimated.     

A ‘Problematic fossil’ revealed:Pycnoporidium ? eomesozoicum Flügel, 1972 (Late Triassic,Tethys)—not an enigmatic alga but a strophomenid brachiopod (Gosaukammerella n.g.)

Summary The microproblematicumPycnoporidium ? eomesozoicum Flügel, 1972, from Upper Triassic reefs of the Alpine-Mediterranean region, Turkey Oman and Iran (originally interpreted as possible alga) represents the type species of a new strophomenid brachiopod genus (Gosaukammerella n.g.). The genus is characterized by a very small, millimeter-sized plano-convex shell, whose ventral valve is attached to the substratum (mainly sponges) by symmetrically arranged outgrowths developing from a pseudopunctate, lamellose foliated shell wall and composed of densely spaced subparallel ‘tubes’ comparable with productide spines secreted by papillose extensions of the mantle.Gosaukammerella seems to be the only reliable candidate for the existence of post-Paleozoic strophomenid (productid ?) brachiopods. Gosaukammerella eomesozoica is restricted to possibly cryptic, shaded reef environments inhabited predominantly by sponges serving as substrates for micromorphic brachiopods.     

Cloning, sequencing, and phenotypic analysis of laf1, encoding the flagellin of the lateral flagella of Azospirillum brasilense Sp7.

Azospirillum brasilense can display a single polar flagellum and several lateral flagella. The A. brasilense Sp7 gene laf1, encoding the flagellin of the lateral flagella, was isolated and sequenced. The derived protein sequence is extensively similar to those of the flagellins of Rhizobium meliloti, Agrobacterium tumefaciens, Bartonella bacilliformis, and Caulobacter crescentus. An amino acid alignment shows that the flagellins of these bacteria are clustered and are clearly different from other known flagellins. A laf1 mutant, FAJ0201, was constructed by replacing an internal part of the laf1 gene by a kanamycin resistance-encoding gene cassette. The mutant is devoid of lateral flagella but still forms the polar flagellum. This phenotype is further characterized by the abolishment of the capacities to swarm on a semisolid surface and to spread from a stab inoculation in a semisolid medium. FAJ0201 shows a normal wheat root colonization pattern in the initial stage of plant root interaction.     

Intercellular junctions in the uterine epithelium of Salamandra salamandra (L.) (Amphibia,Urodela)

Summary Intercellular junctions in the uterine epithelium of the ovoviviparous urodele Salamandra salamandra were studied in pregnant and non-pregnant females by freeze-fracture technique. Junctional complexes consist of zonulae occludentes (tight junctions) and numerous maculae adhaerentes (desmosomes); z. adhaerentes and nexuses (gap junctions) could not be identified. Tight junctions are of the flexible type exhibiting loosely interconnected fibrils. The fibrillary network appears stretched more often in pregnant females possibly due to the mechanical stress of pregnancy. The structure and occurrence of the junctions identified, especially that of the tight junctions, is discussed with regard to the functions of the uterus during pregnancy.Zusammenfassung Mit Hilfe der Gefrierbruchtechnik wurden im Uterus-epithel trächtiger und nichtträchtiger Feuersalamanderweibchen (Salamandra salamandra) Zonulae occludentes und Maculae adhaerentes, jedoch keine Z. adhaerentes sowie Nexus identifiziert. Die Z. occludentes sind flexibel. Ihr fibrilläres Netzwerk ist bei trächtigen Weibchen öfter gestreckt; das ist möglicherweise auf die stärkere Ausdehnung des Uterusgewebes während der Trächtigkeit zurückzuführen. Das Vorkommen der verschiedenen Kontakt-strukturen, namentlich das der Z. occludentes, wird im Hinblick auf die Funktionen des Uterus während der Trächtigkeit diskutiert.We are indebted to Mrs. K. Ott for excellent technical assistance and to Miss Dr. U. Beigel for linguistic help