A role for U2/U6 helix Ib in 5' splice site selection

Selection of pre-mRNA splice sites is a highly accurate process involving many trans-acting factors. Recently, we described a role for U6 snRNA position G52 in selection of the first intron nucleotide (+1G). Because some U2 alleles suppress U6-G52 mutations, we investigated whether the corresponding U2 snRNA region also influenced 5' splice site selection. Our results demonstrate that U2 snRNAs mutated at position U23, but not adjacent nucleotides, specifically affect 5' splice site cleavage. Furthermore, all U2 position U23 mutations are synthetic lethal with the thermosensitive U6-G52U allele. Interestingly, the U2-U23C substitution has an unprecedented hyperaccurate splicing phenotype in which cleavage of introns with a +1G substitution is reduced, whereas the strain grows with wild-type kinetics. U2 position U23 forms the first base pair with U6 position A59 in U2/U6 helix Ib. Restoration of the helical structure suppresses 5' splice site cleavage defects, showing an important role for the helix Ib structure in 5' splice site selection. U2/U6 helix Ib and helix II have recently been described as being functionally redundant. This report demonstrates a unique role for helix Ib in 5' splice site selection that is not shared with helix II.     

Dermatofibroma. A clinico-pathologic classification scheme

Yeast strains lacking functional copies of the two genes SSA1 and SSA2, which encode cytosolic molecular chaperones, are temperature-sensitive. In this report, we describe the isolation of a high-copy suppressor of this temperature sensitivity, UBP3, which encodes a de-ubiquitinating enzyme. We show that ubp3 mutant yeast strains have a mild slow-growth phenotype and accumulate ubiquitin-protein conjugates. We propose a model in which Ubp3p acts in vivo to reverse the ubiquitination of substrate proteins, allowing temporarily misfolded proteins an opportunity to fold correctly.     

High-dose-rate brachytherapy as the primary treatment of medically inoperable stage I-II endometrial carcinoma

A protein fold recognition method was tested by the blind prediction of the structures of a set of proteins. The method evaluates the compatibility of an amino acid sequence with a three-dimensional structure using the four evaluation functions: side-chain packing, solvation, hydrogen-bonding, and local conformation functions. The structures of 14 proteins containing 19 sequences were predicted. The predictions were compared with the experimental structures. The experimental results showed that 9 of the 19 target sequences have known folds or portions of known folds. Among them, the folds of Klebsiella aerogenes urease beta subunit (KAUB) and pyruvate phosphate dikinase domain 4 (PPDK4) were successfully recognized; our method predicted that KAUB and PPDK4 would adopt the folds of macromomycin (Ig-fold) and phosphoribosylanthranilate isomerase:indoleglycerol-phosphate synthase (TIM barrel), respectively, and the experimental structure revealed that they actually adopt the predicted folds. The predictions for the other targets were not successful, but they often gave secondary structural patterns similar to those of the experimental structures.     

The phenotypes En(a-), Wr(a-b-), and En(a+), Wr(a+b-), and further studies on the Wright and En blood group systems

In 1975, we showed 18, 19 an En(a-) blood sample to be phenotypically Wr(a-b-). In the current report, we describe tests that show that three En(a-) members of a single family, not believed to be related to the family of the previously tested En(a-) person, are also Wr(a-b-). They have red blood cells that neither react with nor adsorb anti-Wra or anti-Wrb. In addition, we have shown that the red blood cells of six EnaEn heterozygotes, in the family tested, are Wr(a-b+) but carry only a single dose of Wrb antigen. Tests on anti-Ena have shown conclusively that one example is a mixture of separable anti-Ena and anti-Wrb and that a second example may well contain the same two antibodies. By various methods, we have demonstrated that the red blood cells of the only known Wr(a+b-) individual are En(a+) and do not display any of the physicochemical abberations of the En(a-) phenotype. It is further shown that neuraminidase and trypsin do not denature the Wra or Wrb antigens in vitro, but that the protease ficin does have a limited ability to denature Wrb. Additional observations on the first reported example of anti-Wrb are included. These various findings have been considered in the light of gene linkage of, or gene interaction between, the En and Wright system genes. It is concluded that the evidence does not exclude the possibility that En is a silent allele at the WraWrb locus so that the genotype EnEn (or WrWr) might result in the phenotype En(a-), Wr(a-b-). However, it is also pointed out that the evidence equally well supports the postulation that the Wra and Wrb genes are unable to function in the absence of an Ena gene. If this latter theory is proved correct, the interaction between Ena and the Wright genes can be thought of as similar to that between the H and ABO or X1r and CDE genes. It is pointed out that if En is a silent allele at the MN locus (current evidence on this point is not conclusive,) En and Wr cannot be synonymous for it is known that the Wra and M and N genes segregate independently. Location of En at the MN locus would not, however, refute the theory that Wra and Wrb cannot function in the absence of En. Finally, it is pointed out that the supposed anti-Wrb is probably just what its name implies but that even if this assumption is later disproved, the high incidence antigen defined by the antibody presently called anti-Wrb is unequivocally associated with Ena.