Studies of the nuclear residual proteins

The nuclear residual proteins of rat liver have been prepared by solubilization with sodium deoxycholate and Sephadex gel filtration. These proteins were examined by analytical ultracentrifugation, DEAE-cellulose chromatography, starch gel electrophoresis, amino acid composition, and alkali-labile phosphorus analysis. The results show that these proteins are heterogeneous, having a high glutamic acid content, and contain phosphoprotein. Partial fractionation was achieved by DEAE-cellulose chromatography and starch gel electrophoresis. Some of these proteins appeared as spheroidal particles with diameters of 100–400 Å. In the presence of sodium deoxycholate, the particles dissociated into smaller units that reassociated upon removal of the detergent.     

Effect of appetitive training on brain lysine level and incorporation into nuclear proteins

The content of free lysine in the brains of mice increased significantly during an appetitive training in which the mice were trained to touch a bar in order to get sweetened milk. The free lysine level reached a maximum at 20–30 min of training, and returned to control levels at 60 min. The specific activity of free lysine was significantly lower in the brains of trained mice than in controls at 20 and 30 min after either subcutaneous or intracerebral administration of the isotopically labeled compound. Subcutaneously injected radioactive lysine disappeared more rapidly from the blood of trained mice than from the blood of control mice during the interval from 20 to 60 min after injection. The specific activities of brain nuclear proteins from trained mice were significantly greater than those of controls after 20 min or more of training. These protein differences were more marked when expressed as relative specific activities that were corrected for changes of specific activity of free lysine that occurred during training.     

Synthesis, transport and incorporation into the nuclear envelope of A-type lamins and inner nuclear membrane proteins

The mammalian NE (nuclear envelope), which separates the nucleus from the cytoplasm, is a complex structure composed of nuclear pore complexes, the outer and inner nuclear membranes, the perinuclear space and the nuclear lamina (A- and B-type lamins). The NE is completely disassembled and reassembled at each cell division. In the present paper, we review recent advances in the understanding of the mechanisms implicated in the transport of inner nuclear membrane and nuclear lamina proteins from the endoplasmic reticulum to the nucleus in interphase cells and mitosis, with special attention to A-type lamins.     

H-thymidine incorporation into nuclear DNA of leaf cells

The incorporation of ³H-thymidine into nuclear DNA of leaf cells of Nanthium pennsylvanicum was studied as a function of concentration and specific activity of the radioisotope. From the assessment of the average number of grains per nucleus and the percent of labeled nuclei, it was concluded that the incorporation was a linear function of concentration of the exogenous radioisotopic solution and a logarithmic function of the incubation time. Ten microcuries per milliliter on the average yielded 20% of labeled nuclei with 18 grains per nucleus. Seven-fold increase in concentration only doubled the amount of ³H-thymidine incorporated. The lamina regions near the vein incorporated a significantly greater amount of the radioisotope than the lamina region at some distance from the vein. The specific activities of 2, 3.35, 6.7 and 15.3 c/mmole had no effect upon the amount of ³H-thymidine incorporated, if the amount of microcuries of the incubation solution was the same in each activity. Considering the total number of molecules, the estimated rates of incorporation indicated that at the activity of 2 c/mmole, the system operated with about 7 times higher rates as compared with the activity of 15.3 c/mmole.     

Towards understanding selenocysteine incorporation into bacterial proteins

In bacteria, UGA stop codons can be recoded to direct the incorporation of selenocysteine into proteins on the ribosome. Recoding requires a selenocysteine incorporation sequence (SECIS) downstream of the UGA codon, a specialized translation factor SelB, and the non-canonical Sec-tRNASec, which is formed from Ser-tRNASec by selenocysteine synthase, SelA, using selenophosphate as selenium donor. Here we describe a rapid-kinetics approach to study the mechanism of selenocysteine insertion into proteins on the ribosome. Labeling of SelB, Sec-tRNASec and other components of the translational machinery allows direct observation of the formation or dissociation of complexes by monitoring changes in the fluorescence of single dyes or fluorescence resonance energy transfer between two fluorophores. Furthermore, the structure of SelA was studied by electron cryomicroscopy (cryo-EM). We report that intact SelA from the thermophilic bacterium Moorella thermoacetica (mthSelA) can be vitrified for cryo-EM using a controlled-environment vitrification system. Two-dimensional image analysis of vitrified mthSelA images shows that SelA can adopt the wide range of orientations required for high-resolution structure determination by cryo-EM. The results indicate that mthSelA forms a homodecamer that has a ring-like structure with five bilobed wings, similar to the structure of the E. coli complex determined previously.     

Kinetics of amino acid incorporation into serum proteins

1. The effect of varying body temperature on the rate of amino acid incorporation into serum protein does not give support to the idea that the rate of this process is adjusted in vivo to restore those protein molecules destroyed by thermal denaturation. The experimentally observed Q₁₀ was about 3.9. 2. When amino acids are injected into the blood of animals in a steady state of serum protein turnover, a period of time elapses before these amino acids can be found in the serum proteins. This has been called transit time. At a given temperature (31°) it is the same in rabbits, turtles, and Limulus (1 hour). In rabbits and turtles it has a Q₁₀ of 3.2. It appears to be specifically related to the process of synthesis (or release) of serum proteins. 3. It was not possible to affect the transit time or the incorporation rate by the administration of amino acid analogues.     

The incorporation of Na2 75SeO3 into sheep erythrocytes

Injection of Na₂ ⁷⁵SeO₃ into untreated, phlebotomized, and phenylhydrazine treated sheet has shown that the rate of⁷⁵SeO₃-incorporation into erythrocytes is dependent on the degree of stimulation. Analysis of labeled erythrocytes by gel filtration and twodimensional electrophoresis has indicated that the transient labeling of a hemoglobin-like peptide is the only protein labeled in addition to glutathione peroxidase.     

Phosphate incorporation into secretory protein of Chironomus salivary glands occurs during translation

The Balbiani rings (BR) in Chironomus salivary gland cells code for giant secretory proteins, the sp-I family. During normal growth conditions the phosphorylated proteins sp-Ia and sp-Ib are formed with most phosphate present as phosphoserine. We can show that most if not all incorporation of ³²P into sp-I occurs in parallel with the incorporation of [³⁵S]-methionine in the giant polysomes that form sp-I and contain BR-derived mRNA. We suggest that the main function of phosphorylation of sp-Ia and sp-Ib is to provide charge neutralization of an excess of lysine and arginine residues and is therefore required during early stages of protein folding. This view is supported by the previous observation that glutamic (and aspartic) acid largely substitute for phosphoserine in a non-phosphorylated member of the sp-I family, sp-Ic, which is produced during phosphate starvation.     

Selenocysteine biosynthesis and mechanism of incorporation into growing proteins

The universal genetic code codes for the 20 canonical amino acids, while selenocysteine (Sec) is encoded by UGA, one of the three well-known stop codons. Selenocysteine is of particular interest of molecular biology, principally differing in the mechanism of incorporation into growing polypeptide chains from the other 20 amino acids. The process involves certain cis- and trans-active factors, such as the Sec insertion sequence (SECIS). The SECIS is in the 3′-untranslated mRNA region in eukaryotes and within the open reading frame located immediately downstream of the Sec UGA codon in bacteria, the difference leading to differences in the mechanism of Sec incorporation between the two domains of life. The trans-active factors include Sec-tRNA^[Ser]Sec, which is synthesized by a unique system; the Sec-specific elongation factor EFsec; and a SECIS-binding protein (SBP2). Thus, many additional molecules are to be synthesized in the cell to allow Sec incorporation during translation. The fact makes Sec-containing proteins rather “expensive” and emphasizes their crucial role in metabolism.