Differential enhancement of spontaneous transition mutations in the lacI gene of an Ung- strain of Escherichia coli

In this communication, the contribution of cytosine deamination to spontaneous mutagenesis in the lacI gene of E. coli was examined. In a wild-type strain, 75% of the amber mutations recovered were G:C----A:T transitions and 60% of these were at the 5-methylcytosine spontaneous hotspots Am6, Am15 and Am34. In a strain deficient for uracil-DNA glycosylase (Ung-), 96% of the amber mutations were G:C----A:T transitions while only 15% of these occurred at the hotspot sites. This shift in the mutational distribution demonstrates that cytosine deamination is a potent mutagenic process, which is enhanced in the absence of glycosylase. Moreover, some amber sites were greatly enhanced in the Ung- strain while others were only slightly enhanced. This result suggests that the rate of cytosine deamination at individual sites may be influenced by surrounding base composition. Therefore, we examined the neighboring sequences and found a strong correlation between the fold-increase in mutation and the A/T richness of the surrounding sequence. It is suggested that A/T-rich regions denature more often, forming transient single strands in which cytosine residues would be expected to deaminate more readily.     

Missense mutation in the lacI gene of Escherichia coli. Inferences on the structure of the repressor protein

The lac repressor has been studied extensively but a precise three-dimensional structure remains unknown. Studies using mutational data can complement other information and provide insight into protein structure. We have been using the lacI gene-repressor protein system to study the mutational specificity of spontaneous and induced mutation. The sequencing of over 6000 lacI- mutations has revealed 193 missense mutations generating 189 amino acid replacements at 102 different sites within the lac repressor. Replacement sites are not distributed evenly throughout the protein, but are clustered in defined regions. Almost 40% of all sites and over one-half of all substitutions found occur within the amino-terminal 59 amino acid residues, which constitute the DNA-binding domain. The core domain (residues 60 to 360) is less sensitive to amino acid replacement. Here, substitution is found in regions involved in subunit aggregation and at sites surrounding residues that are implicated in sugar-binding. The distribution and nature of missense mutational sites directs attention to particular amino acid residues and residue stretches.     

Kin-structured migration and the rate of advance of an advantageous gene

Hemoglobin E, an allele generally considered to confer malarial resistance in heterozygotes, is found in high frequencies across a wide area of Southeast Asia. Apparently it originated as a single-point mutation which was spread by gene flow. The rate of diffusion of this adaptive allele is studied using four computer simulation models. It is shown that in small populations deterministic equations for gene flow may overestimate rates of diffusion. Other aspects of population structure, however, such as kin-structuring of migrant groups, increase the rate of advance. Finally, population growth coupled with the spread of the allele leads to much more rapid diffusion. These results suggest that population structure can be an important factor affecting the diffusion of advantageous genes.     

Effects of the isothiocyanate sulforaphane on TGF-β1-induced rat cardiac fibroblast activation and extracellular matrix interactions

An important step in many pathological conditions, particularly tissue and organ fibrosis, is the conversion of relatively quiescent cells into active myofibroblasts. These are highly specialized cells that participate in normal wound healing but also contribute to pathogenesis. These cells possess characteristics of smooth muscle cells and fibroblasts, have enhanced synthetic activity secreting abundant extracellular matrix components, cytokines, and growth factors, and are capable of generating contractile force. As such, these cells have become potential therapeutic targets in a number of disease settings. Transforming growth factor β (TGF-β) is a potent stimulus of fibrosis and myofibroblast formation and likewise is an important therapeutic target in several disease conditions. The plant-derived isothiocyanate sulforaphane has been shown to have protective effects in several pathological models including diabetic cardiomyopathy, carcinogenesis, and fibrosis. These studies suggest that sulforaphane may be an attractive preventive agent against disease progression, particularly in conditions involving alterations of the extracellular matrix and activation of myofibroblasts. However, few studies have evaluated the effects of sulforaphane on cardiac fibroblast activation and their interactions with the extracellular matrix. The present studies were carried out to determine the potential effects of sulforaphane on the conversion of quiescent cardiac fibroblasts to an activated myofibroblast phenotype and associated alterations in signaling, expression of extracellular matrix receptors, and cellular physiology following stimulation with TGF-β1. These studies demonstrate that sulforaphane attenuates TGF-β1-induced myofibroblast formation and contractile activity. Sulforaphane also reduces expression of collagen-binding integrins and inhibits canonical and noncanonical TGF-β signaling pathways.     

Using the Hard, Randy, and Rittler Test to Evaluate Color Vision in Capuchins (Cebus libidinosus)

The identification of color vision types in primates is fundamental to understanding the evolution and biological function of color perception. The Hard, Randy, and Rittler (HRR) pseudoisochromatic test categorizes human color vision types successfully. Here we provide an experimental setup to employ HRR in a nonhuman primate, the capuchin (Cebus libidinosus), a platyrrhine with polymorphic color vision. The HRR test consists of plates with a matrix composed of gray circles that vary in size and brightness. Differently colored circles form a geometric shape (X, O, or Δ) that is discriminated visually from the gray background pattern. The ability to identify these shapes determines the type of dyschromatopsy (deficiency in color vision). We tested six capuchins in their own cages under natural sunlight. The subjects chose between two HRR plates in each trial: one with the gray pattern only and the other with a colored shape, presented on the left or right side at random. We presented the test 40 times and calculated the 95?% confidence limits for chance performance based on the binomial test. We also genotyped all subjects for exons 3 and 5 of the X-linked opsin genes. The HRR test diagnosed two subjects as protan dichromats (missing or defective L-cone), three as deutan dichromats (missing or defective M-cone), and one female as trichromat. Genetic analysis supported the behavioral data for all subjects. These findings show that the HRR test can be applied to diagnose color vision in nonhuman primates.     

Mechanisms of Action of Rabbit CAP18 on Monolayers and Liposomes Made from Endotoxins or Phospholipids

We have investigated the mechanism of action of the cationic antimicrobial protein (18 kDa) CAP18 on liposomes and monolayers made from phospholipids and enterobacterial lipopolysaccharides (LPS). CAP18 intercalates into lipid matrices composed of LPS from sensitive strains, weaker into those made of LPS from a resistant strain (Proteus mirabilis strain R45) or negatively charged phospholipids, but not into those composed of neutral phosphatidylcholine. From the combination of data obtained with fluorescence resonance energy transfer and Fourier-transform infrared spectroscopy and film balance measurements, it can be concluded that structural differences in the LPS determine the depth of intercalation of CAP18 into the respective lipid matrices. Thus, we identified the L-Arap4N linked to the first Kdo of the LPS of P. mirabilis strain R45 to be responsible for the CAP18 resistance of this strain. These data provide insight into CAP18-mediated effects on the integrity of the outer membrane of Gram-negative bacteria and led to an improved model for rabbit CAP18 membrane interaction. Received: 14 January 2000/Revised: 20 April 2000     

Red-green color vision impairment in Duchenne muscular dystrophy

The present study evaluated the color vision of 44 patients with Duchenne muscular dystrophy (DMD) (mean age 14.8 years; SD 4.9) who were submitted to a battery of four different color tests: Cambridge Colour Test (CCT), Neitz Anomaloscope, Ishihara, and American Optical Hardy-Rand-Rittler (AO H-R-R). Patients were divided into two groups according to the region of deletion in the dystrophin gene: upstream of exon 30 (n=12) and downstream of exon 30 (n=32). The control group was composed of 70 age-matched healthy male subjects with no ophthalmological complaints. Of the patients with DMD, 47% (21/44) had a red-green color vision defect in the CCT, confirmed by the Neitz Anomaloscope with statistical agreement (P<.001). The Ishihara and the AO H-R-R had a lower capacity to detect color defects--5% and 7%, respectively, with no statistical similarity between the results of these two tests nor between CCT and Anomaloscope results (P>.05). Of the patients with deletion downstream of exon 30, 66% had a red-green color defect. No color defect was found in the patients with deletion upstream of exon 30. A negative correlation between the color thresholds and age was found for the controls and patients with DMD, suggesting a nonprogressive color defect. The percentage (66%) of patients with a red-green defect was significantly higher than the expected <10% for the normal male population (P<.001). In contrast, patients with DMD with deletion upstream of exon 30 had normal color vision. This color defect might be partially explained by a retina impairment related to dystrophin isoform Dp260.     

Neighboring base identity affects N-ethyl-N-nitrosourea-induced mutagenesis in Escherichia coli

This study investigated the influence of different neighboring base contexts on the production of base substitutions generated by N-ethyl-N-nitrosourea (ENU). A set of bacterial strains having all possible bases neighboring an ochre (TAA) nonsense mutation in the tyrA gene of Escherichia coli were employed and true reversions of the nonsense mutation were induced by two separate doses of ENU. Base substitution mutations were investigated by direct sequencing methods. These studies revealed that 1) mutations occurring at 5'-purine-T sites were produced better, on average, than mutations involving 5'-pyrimidine-T sites, and 5'-TT sites contributed the least to the formation of mutations, 2) the order of preference for A:T to G:C transitions was 5'-GT>5'-AT, 5'-CT>5'-TT, and 3) A:T to C:G transversions at the first position of the codon (GAA mutations) were produced best at 5'-AT sites, while A:T to T:A transversions at the third position (TAT mutations) occurred more often at 5'-GT sites. These findings suggest that the occurrence of a specific mutation may reflect the sequence-dependent probability of DNA damage at a particular site as well as factors involving preferential DNA repair or differential base selection by DNA polymerase.     

Colonization models and initial genetic diversity in the Americas

The mode and tempo of colonization of the Americas established the initial pattern of continental genetic diversity. Despite a long history of study, the process of settlement remains controversial in terms of date, rate, and pattern. While there is agreement that Asia was the source population, several different models have been proposed for the colonization process. A classic model postulates a rapid spread of population ("blitzkrieg") from a small band of hunters entering through the corridor between the continental ice sheets circa 11,000 years B.P. Colonization occurred as a wave of expansion across the land masses of North and South America. An alternative model envisions the original colonists initially limiting settlement to the coastline, using boats, and entering the Americas at an earlier date, circa 13,500 B.P. Range expansion along this linear habitat from North to South America could be rapid without requiring population saturation of entire continental regions. These models have markedly different implications for genetic variation among Native Americans. The blitzkrieg colonization process would have generated multiple founder effects leading to extreme loss of genetic variation. Computer simulation of this model shows nearly complete fixation in 30 generations. Simulation of the coastal model, on the other hand, requires less extreme demographic assumptions and maintains substantial genetic variability after 100 generations. Although with the coastal model continental interiors are occupied less rapidly than with the blitzkrieg model, the coastal model allows earlier entry and rapid expansion to the southern limits of the hemisphere.