The role of the head and tail domain in lamin structure and assembly: analysis of bacterially expressed chicken lamin A and truncated B2 lamins.

Nuclear lamins like cytoplasmic intermediate filament proteins exhibit a characteristic tripartite domain structure with a segmented alpha-helical rod domain flanked by an N-terminal head and a C-terminal tail domain. To examine the influence of the head and tail domains on the structure and assembly properties of nuclear lamins, we have engineered "headless," "tailless," and "rod" chicken lamin B2 cDNAs and expressed them in Escherichia coli. A full-length chicken lamin A cDNA was also expressed in E. coli, and the recombinant protein compared with the structure and assembly properties of full-length chicken lamin B2 (E. Heitlinger et al. (1991) J. Cell Biol. 113, 485-495). As with lamin B2, at their first level of structural organization, lamin A and the headless lamin B2 formed myosin-like dimers consisting of a 51- to 52-nm-long tail flanked by two globular heads at one end. Similarly, the tailless and rod lamin B2 fragments formed tropomyosin-like dimers consisting of a 51 to 52-nm-long rod. In contrast to the lateral mode of association of cytoplasmic IF dimers into four-chain tetramers, at their second level of structural organization, lamin A dimers, just as lamin B2 dimers (E. Heitlinger et al. (1991) J. Cell Biol. 113, 485-495), associated longitudinally to form polar head-to-tail polymers. Whereas dimers made of the truncated B2 headless and rod lamins had lost their propensity to associate head-to-tail, tailless lamin B2 dimers revealed an enhanced head-to-tail association. Finally, at their third level of structural organization, rather than assembling into stable 10-nm filaments, both lamin A and the three truncated B2 lamins formed paracrystalline arrays exhibiting distinct transverse banding patterns with axial repeats of either 24 or 48-49 nm depending on the species.     

Genetic Analysis of Rap1p/Sir3p Interactions in Telomeric and Hml Silencing in Saccharomyces Cerevisiae

We have identified three SIR3 suppressors of the telomeric silencing defects conferred by missense mutations within the Rap1p C-terminal tail domain (aa 800-827). Each SIR3 suppressor was also capable of suppressing a rap1 allele (rap1-21), which deletes the 28 aa C-terminal tail domain, but none of the suppressors restored telomeric silencing to a 165 amino acid truncation allele. These data suggest a Rap1p site for Sir3p association between the two truncation points (aa 664-799). In SIR3 suppressor strains lacking the Rap1p C-terminal tail domain, the presence of a second intragenic mutation within the rap1s domain (aa 727-747), enhanced silencing 30-300-fold. These data suggest a competition between Sir3p and factors that interfere with silencing for association in the rap1(s) domain. rap1-21 strains containing both wild-type Sir3p and either of the Sir3 suppressor proteins displayed a 400-4000-fold increase in telomeric silencing over rap1-21 strains carrying either Sir3p suppressor in the absence of wild-type Sir3p. We propose that this telomere-specific synergism is mediated in part through stabilization of Rap1p/Sir3p telomeric complexes by Sir3p-Sir3p interactions.     

Consistencies of individual DNA base-amino acid interactions in structures and sequences.

Amino acid-amino acid interaction energies have been derived from crystal structure data for a number of years. Here is reported the first derivation of normalized relative interaction from binding data for each of the four bases interacting with a specific amino acid, utilizing data from combinatorial multiplex DNA binding of zinc finger domains [Desjarlais, J. R. and Berg, J. M. (1994) Proc. Natl. Acad. Sci. USA, 91, 11099-11103]. The five strongest interactions are observed for lysine-guanine, lysine-thymine, arginine-guanine, aspartic acid-cytosine and asparagine-adenine. These rankings for interactions with the four bases appear to be related to base-amino acid partial charges. Also, similar normalized relative interaction energies are derived by using DNA binding data for Cro and lambda repressors and the R2R3 c-Myb protein domain [Takeda, Y., Sarai, A. and Rivera, V. M. (1989) Proc. Natl. Acad. Sci. USA, 86, 439-443; Sarai, A. and Takeda, Y. (1989) Proc. Natl. Acad. Sci. USA, 86, 6513-6517; Ogata, K. et al. (1995) submitted]. These energies correlate well with the combinatorial multiplex energies, and the strongest cases are similar between the two sets. They also correlate well with similar relative interaction energies derived directly from frequencies of bases in the bacteriophage lambda operator sequences. These results suggest that such potentials are general and that extensive combinatorial binding studies can be used to derive potential energies for DNA-protein interactions.     

Changes in blood lymphocyte numbers with age in vivo and their association with the levels of cytokines/cytokine receptors

Background

Alterations in the number and composition of lymphocytes and their subsets in blood are considered a hallmark of immune system aging. However, it is unknown whether the rates of change of lymphocytes are stable or change with age, or whether the inter-individual variations of lymphocyte composition are stable over time or undergo different rates of change at different ages. Here, we report a longitudinal analysis of T- and B-cells and their subsets, and NK cells in the blood of 165 subjects aged from 24 to 90 years, with each subject assessed at baseline and an average of 5.6 years follow-up.

Results

The rates of change of T-(CD4⁺ and CD8⁺) and B-cells, and NK cells were relative stable throughout the adult life. A great degree of individual variations in numbers of lymphocytes and their subsets and in the rates of their changes with age was observed. Among them, CD4⁺ T cells exhibited the highest degree of individual variation followed by NK cells, CD8⁺ T cells, and B cells. Different types of lymphocytes had distinct trends in their rates of change which did not appear to be influenced by CMV infection. Finally, the rates of CD4⁺, CD8⁺ T cells, naive CD4⁺ and naïve CD8⁺ T cells were closely positively correlated.

Conclusion

Our findings provide evidence that the age-associated changes in circulating lymphocytes were at relative stable rates in vivo in a highly individualized manner and the levels of selected cytokines/cytokine receptors in serum might influence these age-associated changes of lymphocytes in circulation.

     

Rearrangement of Con A and WGA receptors on murine mastocytoma cell membranes: relationship to cell cycle

Incubation of interphase murine mastocytoma cells with fluorescein-labeled wheat germ agglutinin (FITC-WGA) or with rhodamine-labeled concanavalin A (Rh-Con A) shows binding of the lectins to all the cells in a ring form distribution. Treatment of the cells with trypsin before addition of FITC-WGA, or with colchicine before addition of Rh-Con A, promoted rearrangement of the lectin binding sites into cap formation in 27-29% of the cells. Trypsin had no effect on cap formation by Rh-Con A, and colchicine had no effect on cap formation by FITC-WGA. When the same cells were pretreated consecutively with trypsin and colchicine followed by incubation with FITC-WGA and Rh-Con A, no increase in the percentage of cells bearing caps was observed. Moreover, the same cells showed co-capping with both lectins. Experiments with synchronous cultures of mastocytoma cells have indicated that capping induced by either lectin is maximal in the G2 phase and minimal in the G1 and S phases of the cell cycle. At the G2 phase 60% of the cells show cap formation either after trypsin-WGA treatment or after colchicine-Con A treatment. These data indicate that rearrangement of membrane receptors for Con-A and WGA in murine mastocytoma cells can occur only at certain phases of the cell cycle.     

Membrane mobility agent alters the consequences of lectin-cell interaction in a malignant cell membrane

The membrane mobility agent 2-(2-methoxyethoxy)-ethyl 8-(cis-2-n-octylcyclopropyl)-octanoate promotes cap formation from wheat germ agglutinin-receptor combinations at the expense of agglutination in membranes of malignant mastocytoma cells.     

Codon reading and translational error. Reading of the glutamine and lysine codons during protein synthesis in vitro

The reading of glutamine and lysine codons during protein synthesis in vitro has been investigated using an MS2-RNA-programed system derived from Escherichia coli. Under conditions when either glutaminyl-tRNA1Gln (s2UUG) or glutaminyl-tRNA2Gln (CUG) was the only source of glutamine for protein synthesis both tRNAs were able to read the glutamine codons CAA and CAG as indicated by the incorporation of labeled glutamine into the pertinent coat protein tryptic peptides. On the other hand, when the two glutamine tRNAs competed for the codon CAA the reading efficiency of the anticodon s2UUG, which reads the codon according to the wobble rules, was almost 40 times higher than that of the competing anticodon CUG, which reads the codon by "two out of three," i.e. it cannot form a regular base pair with the third codon position. In reading the codon CAG the anticodon CUG was approximately eight times more efficient than the anticodon s2UUG. The lysyl-tRNA1Lys (CUU) could not alone sustain any detectable coat protein synthesis in the MS2 system indicating that there was no significant reading of the lysine codon AAA. This conclusion is supported by the outcome of experiments where lysyl-tRNA1Lys (CUU) and lysyl-tRNA2Lys (s2UUU) competed for the codon AAA. The reading efficiency of the anticodon CUU was less than 1% of that of the competing s2UUU which represents the limit of resolution of our experimental system. When the two lysine tRNAs competed for the codon AAG the anticodon CUU was about four times more efficient than s2UUU. These results are discussed in the context of the two out of three hypothesis, which attempts to relate the frequency of such reading to the hydrogen bonding properties of the codon nucleotides.     

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