Constraints on amino acid substitutions in the N-terminal helix of cytochrome c explored by random mutagenesis.

The interaction of the N- and C-terminal helices is a hallmark of the cytochrome c family. Oligodeoxyribonucleotide-directed random mutagenesis within the gene encoding the C102T protein variant of Saccharomyces cerevisiae iso-1-cytochrome c was used to generate a library of mutations at the evolutionary invariant residues Gly-6 and Phe-10 in the N-terminal helix. Transformation of this library (contained on a low-copy-number yeast shuttle phagemid) into a yeast strain lacking a functional cytochrome c, followed by selection for cytochrome c function, reveals that 4-10% of the 400 possible amino acid substitutions are compatible with function. DNA sequence analysis of phagemids isolated from transformants exhibiting the functional phenotype elucidates the requirements for a stable helical interface. Basic residues are not tolerated at position 6 or 10. There is a broad volume constraint for amino acids at position 6. The amino acid substitutions observed to be compatible with function at Phe-10 show that the hydrophobic effect alone is sufficient to promote helical association. There are severe constraints that limit the combinations consistent with function, but the number of functionally consistent combinations observed exemplifies the plasticity of proteins.     

Population genetics and phylogenetics of DNA sequence variation at multiple loci within the Drosophila melanogaster species complex

Two regions of the genome, a 1-kbp portion of the zeste locus and a 1.1- kbp portion of the yolk protein 2 locus, were sequenced in six individuals from each of four species: Drosophila melanogaster, D. simulans, D. mauritiana, and D. sechellia. The species and strains were the same as those of a previous study of a 1.9-kbp region of the period locus. No evidence was found for recent balancing or directional selection or for the accumulation of selected differences between species. Yolk protein 2 has a high level of amino acid replacement variation and a low level of synonymous variation, while zeste has the opposite pattern. This contrast is consistent with information on gene function and patterns of codon bias. Polymorphism levels are consistent with a ranking of effective population sizes, from low to high, in the following order: D. sechellia, D. melanogaster, D.mauritiana, and D. simulans. The apparent species relationships are very similar to those suggested by the period locus study. In particular, D. simulans appears to be a large population that is still segregating variation that arose before the separation of D. mauritiana and D. sechellia. It is estimated that the separation of ancestral D. melanogaster from the other species occurred 2.5-3.4 Mya. The separations of D. sechellia and D. mauritiana from ancestral D. simulans appear to have occurred 0.58- 0.86 Mya, with D. mauritiana having diverged from ancestral D. simulans 0.1 Myr more recently than D. sechellia.      

Design, synthesis, expression, and characterization of the genes for mouse Fc gamma RIIb1 and Fc gamma RIIb2 cytoplasmic regions.

The cytoplasmic regions of the mouse low-affinity Fc gamma RII isoforms, mFc gamma RIIb1, and mFc gamma RIIb2, play a key role in signal transduction by mediating different cellular functions. mFc gamma RIIb1 has a 94-residue cytoplasmic region, whereas mFc gamma RIIb2 has a 47-residue cytoplasmic region. Genes encoding the cytoplasmic regions of mFc gamma RIIb1 (b1-94) and mFc gamma RIIb2 (b2-47) were designed, synthesized, and expressed as fusion proteins in Escherichia coli. A sequence-specific protease, thrombin, was used to release the b1-94 peptide, which was purified by using HPLC. The b2-47 peptide was synthesized chemically. CD spectropolarimetry was employed to examine the secondary structures of b1-94 and b2-47. These studies were conducted in aqueous solution, in mixtures of water and trifluoroethanol or methanol, and as a function of temperature. The results indicate that the b1-94 and b2-47 structures are sensitive functions of the solvent environment, and that nonaqueous solvents induce significant alpha-helical structure.     

Digital image analysis of AgNORs in cervical smears of women with premalignant and malignant lesions of the uterine cervix

We performed a hospital-based, unmatched case-control study to investigate the association between progressive stages of cervical neoplasia and digital analysis of cell proliferation by silver stained nucleolus organizer region associated proteins (AgNORs). We measured cell proliferation levels in the cervical epithelial cells of 10 women with low grade squamous intraepithelial lesions (LG-SIL), eight with high grade squamous intraepithelial lesions (HG-SIL), 11 with cervical cancer (CC) and eight with no cervical lesions (controls) using the AgNORs technique. Cell proliferation was measured by digital image analysis (DIA). DIA revealed increased total areas of AgNORs in HG-SIL and CC compared to LG-SIL and control patients. AgNORs with a kidney or cluster shape exhibited greater areas than those with a spherical or long shape. We propose a cut-off of 118 pixels to differentiate benign (control and LG-SIL) from malignant (HG-SIL and CC) lesions. DIA of AgNORs is a simple and inexpensive method for studying proliferation. The increased total area of AgNORs in malignant lesions provides information regarding cell behavior and may be related to cervical carcinogenesis; however, further validation studies are required to establish its usefulness in cytological analysis.     

The cytoplasmic region of mouse Fc gamma RIIb1, but not Fc gamma RIIb2, binds phospholipid membranes

The cytoplasmic regions of the mouse low-affinity Fc gamma RII isoforms, Fc gamma RIIb1 and Fc gamma RIIb2, play key roles in signal transduction by mediating different cellular functions. The Fc gamma RIIb1 (94 residues) and Fc gamma RIIb2 (47 residues) cytoplasmic regions are generated by differential mRNA splicing in which a single aspartic acid residue in Fc gamma RIIb2 is replaced by a 48-residue insert in Fc gamma RIIb1. In previous work, quantities of the mFc gamma RIIb1 and mFc gamma RIIb2 cytoplasmic regions were generated, and their secondary structures were examined in different solutions with circular dichroism [Chen, L., Thompson, N. L., and Pielak, G. J. (1997) Protein Sci. 6, 1038-1046]. In the work described here, steady-state light scattering was used to investigate possible interactions of the two isolated cytoplasmic regions with phospholipid vesicles. Three phospholipid compositions were examined: phosphatidylserine/phosphatidylcholine (PS/PC) (25/75, mol/mol); phosphatidylinositol bisphosphate/phosphatidylcholine (PIP2/PC) (25/75, mol/mol); and pure phosphatidylcholine (PC). Binding was examined in the presence and absence of Ca2+. The mFc gamma RIIb1 cytoplasmic peptide binds PS/PC vesicles weakly in the absence of Ca2+ and more strongly in the presence of Ca2+. For PIP2/PC vesicles, the behavior is reversed; binding is weak in the presence of Ca2+ and stronger in its absence. The mFc gamma RIIb1 peptide also weakly binds pure PC vesicles, in a Ca2+-independent manner. The mFc gamma RIIb2 cytoplasmic peptide does not bind, in the presence or absence of Ca2+, to PS/PC, PIP2/PC, or PC vesicles. The implications of these results for the mechanisms of signal transduction mediated by the two mFc gamma RII cytoplasmic regions are discussed.     

Peroxidative aggregation of alpha-synuclein requires tyrosines

Alpha-synuclein is the main component of the intracellular protein aggregates in neurons of patients with Parkinson's disease. The occurrence of the disease is associated with oxidative damage. Although it is known that peroxidative chemistry leads to the aggregation of alpha-synuclein in vitro, the specific amino acid types of alpha-synuclein involved in this type of aggregation have not been identified. We show, using human cytochrome c plus H(2)O(2) as the source oxidative stress, that the tyrosines of alpha-synuclein are required for aggregation. The studies reveal the chemical basis for a crucial step in the aggregation process.