Generation of specific antibodies against the rap1A, rap1B and rap2 small GTP-binding proteins. Analysis of rap and ras proteins in membranes from mammalian cells

Specific antibodies against rap1A and rap1B small GTP-binding proteins were generated by immunization of rabbits with peptides derived from the C-terminus of the processed proteins. Immunoblot analysis of membranes from several mammalian cell lines and human thrombocytes with affinity-purified antibodies against rap1A or rap1B demonstrated the presence of multiple immunoreactive proteins in the 22-23 kDa range, although at strongly varying levels. Whereas both proteins were present in substantial amounts in membranes from myelocytic HL-60, K-562 and HEL cells, they were hardly detectable in membranes from lymphoma U-937 and S49.1 cyc- cells. Membranes from human thrombocytes and 3T3-Swiss Albino fibroblasts showed strong rap1B immunoreactivity, whereas rap1A protein was present in much lower amounts. In the cytosol of HL-60 cells, only small amounts of rap1A and rap1B proteins were detected, unless the cells were treated with lovastatin, an inhibitor of hydroxymethylglutaryl-coenzyme A reductase, suggesting that both proteins are isoprenylated. By comparison with recombinant proteins, the ratio of rap1A/ras proteins in membranes from HL-60 cells was estimated to be about 4:1. An antiserum directed against the C-terminus of rap2 reacted strongly with recombinant rap2, but not with membranes from tested mammalian cells. In conclusion, rap1A and rap1B proteins are distributed differentially among membranes from various mammalian cell types and are isoprenylated in HL-60 cells

The multi-stripe travelling salesman problem

In the classical Travelling Salesman Problem (TSP), the objective function sums the costs for travelling from one city to the next city along the tour. In the q-stripe TSP with q ≥ 1, the objective function sums the costs for travelling from one city to each of the next q cities along the tour. The resulting q-stripe TSP generalizes the TSP and forms a special case of the quadratic assignment problem. We analyze the computational complexity of the q-stripe TSP for various classes of specially structured distance matrices. We derive NP-hardness results as well as polyomially solvable cases. One of our main results generalizes a well-known theorem of Kalmanson from the classical TSP to the q-stripe TSP

Size and position of intervening sequences are critical for the splicing efficiency of pre-mRNA in the yeast Saccharomyces cerevisiae.

The size of the 309 bp actin gene intron of the yeast Saccharomyces cerevisiae was enlarged by inserting DNA fragments of different lengths and sequence. Enlarging the intron above 551 bp, the largest known yeast intron, led to a decrease in splicing efficiency. The effect on transcript splicing was dependent on the length of the inserted fragments rather than their sequence. It was also observed that insertion of the actin gene intron into different regions of the normally unsplit yeast YP2 gene, significantly influenced the efficiency of splicing of the resulting transcripts. The splicing efficiency of splicing of with the increase of the distance between the mRNA cap site and the intervening sequence