Persistence of neutral polymorphisms in Lake Victoria cichlid fish

Phylogenetic trees for groups of closely related species often have different topologies, depending on the genes used. One explanation for the discordant topologies is the persistence of polymorphisms through the speciation phase, followed by differential fixation of alleles in the resulting species. The existence of transspecies polymorphisms has been documented for alleles maintained by balancing selection but not for neutral alleles. In the present study, transspecific persistence of neutral polymorphisms was tested in the endemic haplochromine species flock of Lake Victoria cichlid fish. Putative noncoding region polymorphisms were identified at four randomly selected nuclear loci and tested on a collection of 12 Lake Victoria species and their putative riverine ancestors. At all loci, the same polymorphism was found to be present in nearly all the tested species, both lacustrine and riverine. Different polymorphisms at these loci were found in cichlids of other East African lakes (Malawi and Tanganyika). The Lake Victoria polymorphisms must have therefore arisen after the flocks now inhabiting the three great lakes diverged from one another, but before the riverine ancestors of the Lake Victoria flock colonized the Lake. Calculations based on the mtDNA clock suggest that the polymorphisms have persisted for about 1.4 million years. To maintain neutral polymorphisms for such a long time, the population size must have remained large throughout the entire period.

Semirational design of active tumor suppressor p53 DNA binding domain with enhanced stability

We have designed a p53 DNA binding domain that has virtually the same binding affinity for the gadd45 promoter as does wild-type protein but is considerably more stable. The design strategy was based on molecular evolution of the protein domain. Naturally occurring amino acid substitutions were identified by comparing the sequences of p53 homologues from 23 species, introducing them into wild-type human p53, and measuring the changes in stability. The most stable substitutions were combined in a multiple mutant. The advantage of this strategy is that, by substituting with naturally occurring residues, the function is likely to be unimpaired. All point mutants bind the consensus DNA sequence. The changes in stability ranged from +1.27 (less stable Q165K) to −1.49 (more stable N239Y) kcal mol−1, respectively. The changes in free energy of unfolding on mutation are additive. Of interest, the two most stable mutants (N239Y and N268D) have been known to act as suppressors and restored the activity of two of the most common tumorigenic mutants. Of the 20 single mutants, 10 are cancer-associated, though their frequency of occurrence is extremely low: A129D, Q165K, Q167E, and D148E are less stable and M133L, V203A and N239Y are more stable whereas the rest are neutral. The quadruple mutant (M133LV203AN239YN268D), which is stabilized by 2.65 kcal mol−1 and Tm raised by 5.6°C is of potential interest for trials in vivo.

Structure and function in rhodopsin: Rhodopsin mutants with a neutral amino acid at E134 have a partially activated conformation in the dark state*

The Glu-134–Arg-135 residues in rhodopsin, located near the cytoplasmic end of the C helix, are involved in G protein binding, or activation, or both. Furthermore, the charge-neutralizing mutation Glu-134 to Gln-134 produces hyperactivity in the activated state and produces constitutive activity in opsin. The Glu/Asp-Arg charge pair is highly conserved in equivalent positions in other G protein-coupled receptors. To investigate the structural consequences of charge-neutralizing mutations at Glu-134 and Arg-135 in rhodopsin, single spin-labeled side chains were introduced at sites in the cytoplasmic domains of helices C (140), E (227), F (250), or G (316) to serve as “molecular sensors” of the local helix bundle conformation. In each of the spin-labeled rhodopsins, a Gln substitution was introduced at either Glu-134 or Arg-135, and the electron paramagnetic resonance spectrum of the spin label was used to monitor the structural response of the helix bundle. The results indicate that a Gln substitution at Glu-134 induces a photoactivated conformation around helices C and G even in the dark state, an observation of potential relevance to the hyperactivity and constitutive activity of the mutant. In contrast, little change is induced in helix F, which has been shown to undergo a dominant motion upon photoactivation. This result implies that the multiple helix motions accompanying photoactivation are not strongly coupled and can be induced to take place independently. Gln substitution at Arg-135 produces only minor structural changes in the dark- or light-activated conformation, suggesting that this residue is not a determinant of structure in the regions investigated, although it may be functionally important.

A point mutation in the MET oncogene abrogates metastasis without affecting transformation

The MET oncogene encodes the tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF), known to stimulate invasive growth of epithelial cells. MET is overexpressed in a significant percentage of human cancers and is amplified during the transition between primary tumors and metastasis. To investigate whether this oncogene is directly responsible for the acquisition of the metastatic phenotype, we exploited a single-hit oncogenic version of MET, able to transform and to confer invasive and metastatic properties to nontumorigenic cells, both in vitro and in nude mice. We mutagenized the signal transducer docking site of Met (Y1349VHVX3Y1356VNV), which has the uncommon property of binding and activating multiple src homology region 2 (SH2)-containing intracellular effectors. Notably, a point mutation (H1351 → N) increased the transforming ability of the oncogene but abolished its metastatic potential. This mutation duplicates the Grb2 binding site, super-activating the Ras pathway and preventing the binding of the other intracellular transducers. Complementation in trans with another nonmetastatic mutant (N1358 → H), recruiting all the transducers downstream to Met except Grb2, rescued the invasive–metastatic phenotype. It is concluded that the metastatic potential of the MET oncogene relies on the properties of its multifunctional docking site, and that a single point mutation affecting signal transduction can dissociate neoplastic transformation from metastasis.

Disruption of RB/E2F-1 interaction by single point mutations in E2F-1 enhances S-phase entry and apoptosis.

The retinoblastoma protein (RB) has been proposed to function as a negative regulator of cell proliferation by complexing with cellular proteins such as the transcription factor E2F. To study the biological consequences of the RB/E2F-1 interaction, point mutants of E2F-1 which fail to bind to RB were isolated by using the yeast two-hybrid system. Sequence analysis revealed that within the minimal 18-amino acid peptide of E2F-1 required for RB binding, five residues, Tyr (position 411), Glu (419), and Asp-Leu-Phe (423-425), are critical. These amino acids are conserved among the known E2F family members. While mutation of any of these five amino acids abolished binding to RB, all mutants retained their full transactivation potential. Expression of mutated E2F-1, when compared with that of wild-type, significantly accelerated entry into S phase and subsequent apoptosis. These results provide direct genetic evidence for the biological significance of the RB/E2F interaction and strongly suggest that the interplay between RB and E2F is critical for proper cell cycle progression.