Studies on protein kinases involved in regulation of nucleocytoplasmic mRNA transport.

The rate of energy-dependent nucleoside triphosphatase (NTPase)-mediated nucleocytoplasmic translocation of poly(A)-containing mRNA [poly(A)+mRNA] across the nuclear envelope is thought to be regulated by poly(A)-sensitive phosphorylation and dephosphorylation of nuclear-envelope protein. Studying the phosphorylation-related inhibition of the NTPase, we found that phosphorylation of one polypeptide of rat liver envelopes by endogenous NI- and NII-like protein kinase was particularly sensitive to poly(A). This polypeptide (106 kDa) was also phosphorylated by nuclear-envelope-bound Ca2+-activated and phospholipid-dependent protein kinase (protein kinase C). Activation of kinase C by tumour-promoting phorbol esters resulted in inhibition of nuclear-envelope NTPase activity and in a concomitant decrease of mRNA (actin) efflux rate from isolated rat liver nuclei. Protein kinase C, but not nuclear envelope NI-like or NII-like protein kinase, was found to be solubilized from the envelope by Triton X-100, whereas the presumable poly(A)-binding site [the 106 kDa polypeptide, representing the putative carrier for poly(A)+mRNA transport] remained bound to this structure. RNA efflux from detergent-treated nuclei lost its susceptibility to phorbol esters. Addition of purified protein kinase C to these nuclei restored the effect of the tumour promoters. Protein kinase C was found to bind also to isolated rat liver nuclear matrices in the absence but not in the presence of ATP. The NII-like nuclear-envelope protein kinase co-purified together with the 106 kDa polypeptide which specifically binds to poly(A) in an ATP-labile linkage.     

Proteins from rat liver cytosol which stimulate mRNA transport. Purification and interactions with the nuclear envelope mRNA translocation system

Two polysome-associated proteins with particular affinities for poly(A) have been purified from rat liver. These proteins stimulate the efflux of mRNA from isolated nuclei in conditions under which such efflux closely stimulates mRNA transport in vivo, and they are therefore considered as mRNA-transport-stimulatory proteins. Their interaction with the mRNA-translocation system in isolated nuclear envelopes has been studied. The results are generally consistent with the most recently proposed kinetic model of mRNA translocation. One protein, P58, has not been described previously. It inhibits the protein kinase that down-regulates the NTPase, it enhances the NTPase activity in both the presence and the absence of poly(A) and it seems to increase poly(A) binding in unphosphorylated, but not in phosphorylated, envelopes. The other protein, P31, which probably corresponds to the 35,000-Mr factor described by Webb and his colleagues, enhances the binding of poly(A) to the mRNA-binding site in the envelope, thus stimulating the phosphoprotein phosphatase and, in consequence, the NTPase. The possible physiological significance of these two proteins is discussed.     

Growth hormone and insulin-like growth factor I concentrations in bulls of various growth hormone genotypes

A leucine/valine substitution at amino acid position 127 was identified by the polymerase chain reaction and restriction fragment length polymorphism in the bovine growth hormone gene. Genotyping was performed in 84 AI bulls of three different breeds, in which plasma concentrations of growth hormone (GH) and insulin-like growth factor I (IGF-1) were also measured. Gene frequencies of variants L (leucine) und V (valine) were 0.80/0.20 (Black and White), 0.90/0.10 (Brown), 0.71/0.29 (Simmental). Hormone concentrations were measured during different physiological conditions (normal feeding, fasting, realimentation) in the majority of animals. Generally, genotype LL was associated with higher concentrations of GH than LV. This difference was significant in Black and White bulls (P < 0.05). In contrast, IGF-1 concentrations were higher in LV than in LL animals. This was most pronounced in mature, realimented Simmental bulls. We conclude that the various GH alleles influence the circulating concentrations of GH and IGF-1.     

Isolation of U3 snoRNP from CHO cells: a novel 55 kDa protein binds to the central part of U3 snoRNA.

U3 snoRNP, the most abundant of the small nucleolar ribonucleoprotein particles (snoRNPs), has previously been demonstrated to participate in pre-rRNA maturation. Here we report the purification of U3 snoRNP from CHO cells using anti-m3G-immunoaffinity and mono Q anion-exchange chromatography. Isolated U3 snoRNPs contain three novel proteins, of 15, 50 and 55 kDa respectively. These proteins may represent core U3 snoRNP proteins whose binding mediates the association of other proteins, such as fibrillarin, that are lost during purification. Using a rabbit antiserum raised against the 55 kDa protein, and an in vitro reconstitution assay, we have localised the 55 kDa protein binding site on the U3 snoRNA. Stable binding of the 55 kDa protein requires sequences located between nucleotides 97 and 204 of the human U3 snoRNA, including the evolutionarily conserved B and C sequence motifs.     

Effect of organic matter on growth and cell yield of ammonia-oxidizing bacteria

The effect of various organic compounds on the growth of ammonia-oxidizing bacteria was examined.Nitrosococcus oceanus, a strongly halophilic bacterium, had a very low tolerance to organic matter compared with other organisms tested. Organic compounds scarcely affected the growth of theNitrosomonas strains whereas nitrite formation by bothNitrosococcus mobilis strains was inhibited by nearly all of the substances tested. The growth ofNitrosospira strain Nsp₁ was enhanced more than 30% by acetate and formate, but not growth was detectable in the presence of pyruvate. On the contrary,Nitrosospira strain Nsp₅ was stimulated only by pyruvate. Nitrite formation by the twoNitrosovibrio tenuis strains tested was similar. The growth of both strains was enhanced considerably by formate and glucose; acetate and, to a greater extent, pyruvate inhibited these bacteria.In batch culture, the energy efficiency of autotrophically grown ammonia-oxidizing bacteria varied from strain to strain. The cell yield of mixotrophically grown cultures, per unit of ammonia oxidized, was increased in comparison with autotrophic ones. No heterotrophic growth was detected.     

Balbiani ring RNA in the cytoplasm of Chironomus thummi salivary gland cells

In this paper experimental results on the size, transport and stability of cytoplasmic Balbiani ring RNA and on its appearance in polysomes are presented. Cytoplasmic RNA of salivary gland cells from Chironomus thummi contains two large RNA fractions of about 20×10⁶ dalton and 10×10⁶ dalton in size. These RNA fractions correspond both to Balbiani ring BR 1 RNA and BR 2 RNA and are apparently transported from nucleus into cytoplasm without a significant size reduction. Chase experiments illustrate a great stability of giant cytoplasmic Balbiani ring RNA molecules and exclude the possibility of a precursor-product relationship between these and smaller BR RNA molecules also found in cytoplasm. A part of giant cytoplasmic Balbiani ring RNA molecules is bound to poly(U)-sepharose columns and should, therefore, contain poly(A)-sequences. — Polysomes of salivary gland cells extracted by a gentle lysis procedure and centrifuged through sucrose gradients are characterized by a rather broad sedimentation profile. Polysome sizes up to about 800 S have been detected, but in no case a distinct polysome fraction corresponding in size to Balbiani ring RNA has been observed. Hybridization of polysomal RNA with salivary gland chromosomes in situ resulted in labelling of both Balbiani rings BR 1 and BR 2.