Conflicting genomes, the demic theory & biodiversity

Organismic-centered Darwinism, in order to use direct phenotypes to measure natural selection's effect, necessitates genome's harmony and uniform coherence plus large population sizes. However, modern gene-centered Darwinism has found new interpretations to data that speak of genomic incoherence and disharmony. As a result of these two conflicting positions a conceptual crisis in Biology has arisen. My position is that the presence of small, even pocket-size, demes is instrumental in generating divergence and phenotypic crisis. Moreover, the presence of parasitic genomes as in acanthocephalan worms, which even manipulate suicidal behavior in their hosts; segregation distorters that change meiosis and Mendelian ratios; selfish genes and selfish whole chromosomes, such as the case of B-chromosomes in grasshoppers; P-elements in Drosophila; driving Y-chromosomes that manipulate sex ratios making males more frequent, as in Hamilton's X-linked drive; male strategists and outlaw genes, are eloquent examples of the presence of real conflicting genomes and of a non-uniform phenotypic coherence and genome harmony. Thus, we are proposing that overall incoherence and disharmony generate disorder but also more biodiversity and creativeness. Finally, if genes can manipulate natural selection, they can multiply mutations or undesirable characteristics and even lethal or detrimental ones, hence the accumulation of genetic loads. Outlaw genes can change what is adaptively convenient even in the direction of the trait that is away from the optimum. The optimum can be "negotiated" among the variants, not only because pleiotropic effects demand it, but also, in some cases, because selfish, outlaw, P-elements or extended phenotypic manipulation require it. With organismic Darwinism the genome in the population and in the individual was thought to act harmoniously without conflicts, and genotypes were thought to march towards greater adaptability. Modern Darwinism has a gene-centered vision in which genes, as natural selection's objects can move in dissonance in the direction which benefits their multiplication. Thus, we have greater opportunities for genomes in permanent conflict.

Genetic interactions involving major genes at the dwarf locus in egg-type chickens

Three egg-type stocks segregating dwarf (dw) and bantam (dwB) genes in female progeny were produced from the same 18 heterozygous (dwB/dw) sires used to inseminate dams of three different genotypes: normal (dw+), dwarf (dw) and bantam (dwB) dams. The heritability of 8-week body weight estimated from full-sibs of the same phenotype of progeny was 0.40, and that estimated from paternal half-sibs of the same phenotype (dwarf or bantam), and from the same genotype of dam was 0.38. Therefore, maternal and non-additive effects within genotypic classes of dam made little contribution to the genetic variance for 8-week body weight among their progeny. The interaction of sires (S) with genotypes (dw+, dw and dwB) of dam (G) was significant at the 5% level. This indicates that the rankings of the sires within each one of the three genotypes of dam were not the same, probably due to non-additive genetic variation among genotypes of dams. The evidence indicated that in general the genes from individual sires combined differently with each type of dam (G). Those genes which combined well with the genes from normal (dw+) dams combined poorly with both the genes from the dwarf (dw) and the genes from the bantam (dwB) dams. The interaction of sires (S) with phenotypes (dwarf and bantam) of progeny (P) was significant at the 10% level. The results indicated a probable gene x genotype interaction for 8-week weight between genes at the dwarf locus (dw and dwB) and the background genotype (single and/or polygenes). The correlation among paternal half-sibs was influenced more by the S x G than by the S x P interaction, but the effects tended to be cumulative

Mini-Mu insertions in the tetracycline resistance determinant from Proteus mirabilis

The inducible tetracycline resistance determinant isolated from Proteus mirabilis cloned into the plasmid pACYC177 was mutagenized by insertion of a mini-Mu-lac phage in order to define the regions in the cloned sequences encoding the structural and regulatory proteins. Three different types of mutants were obtained: one lost the resistance phenotype and became Lac+; another expressed the resistance at lower levels and constitutively; the third was still dependent on induction but showed a lower minimal inhibitory concentration. The mutant phenotypes and the locations of the insertions indicate that the determinant is composed of a repressor gene and a structural gene which are not transcribed divergently as are other known tetracycline determinants isolated from Gram-negative bacteria

Isoenzyme analysis of Arthrobotrys, a nematode-trapping fungus

Extraction and isoenzyme analysis of four isolates of Arthrobotrys including A. musiformis, A. robusta and A. conoides were conducted. Among the 14 enzymes studied by starch gel electrophoresis, using morpholine-citrate as gel/electrode buffer, the following nine enzymes showed interpretable banding patterns: a-esterase, fumarase, hexokinase, isocitrate dehydrogenase, leucine aminopeptidase, malate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucomutase and phosphoglucoisomerase. All isolates studied displayed typical isoenzyme phenotypes for each species. Two isolates of A. conoides differed in their a-isoesterase banding patterns, but no differences were observed for the other enzymes. The assay was satisfactory for enzyme extraction and resolution of Arthrobotrys and could be used in future taxonomic and genetic studies of this organism

Rabies infection and specific effect of vaccination in mice selected for high and low immunobiological parameters

Innate and acquired resistance to rabies infection was investigated in mice genetically selected for high (H) or low (L) antibody responsiveness from selections I, III and IV and in mice selected for maximal (AIRmax) or minimal (AIRmin) acute inflammatory reaction. These mouse lines were infected intramuscularly with different virus dilutions and the LD50 was determined. The HIII and HIV mouse lines were more susceptible than the LIII and LIV lines and the HI line showed a discrete but higher resistance than the LI line. Analysis of the interline (H x L) F1 hybrids from selections III and IV indicated different dominance effects on the "resistant" and" susceptible" phenotypes when the route of vaccination was changed. No differences were observed between the AIRmax and AIRmin mice, suggesting that inflammation plays a minor role in the resistance to rabies virus. The comparison of LD50 in mice vaccinated by distinct routes showed that the highest interline difference occurred after intramuscular vaccination (250-fold between H and L and 800-fold between F1 and L). These results indicate that different mechanisms may participate in acquired antirabies resistance

Gender-related pathways for behavior problems in the offspring of alcoholic fathers

The objective of the present study was to examine gender differences in the influence of paternal alcoholism on children's social-emotional development and to determine whether paternal alcoholism is associated with a greater number of externalizing symptoms in the male offspring. From the Mannheim Study of Risk Children, an ongoing longitudinal study of a high-risk population, the developmental data of 219 children [193 (95 boys and 98 girls) of non-alcoholic fathers, non-COAs, and 26 (14 boys, 12 girls) of alcoholic fathers, COAs] were analyzed from birth to the age of 11 years. Paternal alcoholism was defined according to the ICD-10 categories of alcohol dependence and harmful use. Socio-demographic data, cognitive development, number and severity of behavior problems, and gender-related differences in the rates of externalizing and internalizing symptoms were assessed using standardized instruments (IQ tests, Child Behavior Checklist questionnaire and diagnostic interviews). The general linear model analysis revealed a significant overall effect of paternal alcoholism on the number of child psychiatric problems (F = 21.872, d.f. = 1.217, P < 0.001). Beginning at age 2, significantly higher numbers of externalizing symptoms were observed among COAs. In female COAs, a pattern similar to that of the male COAs emerged, with the predominance of delinquent and aggressive behavior. Unlike male COAs, females showed an increase of internalizing symptoms up to age 11 years. Of these, somatic complaints revealed the strongest discriminating effect in 11-year-old females. Children of alcoholic fathers are at high risk for psychopathology. Gender-related differences seem to exist and may contribute to different phenotypes during development from early childhood to adolescence.

The anti-IRBP IgG1 and IgG2a response does not correlate with susceptibility to experimental autoimmune uveitis

Susceptibility to experimental autoimmune uveitis (EAU) in inbred mice has been associated with a dominant Th1 response. Elevated anti-inter-photoreceptor retinoid-binding protein (anti-IRBP) IgG2a/IgG1 antibody ratios have been implicated as candidate markers to predict disease severity. In the present study, both the anti-IRBP antibody isotype and severity of EAU phenotypes were examined in 4 non-isogenic genetically selected mouse lines to determine if they can be used as general markers of disease. Mice between 8 and 12 weeks old selected for high (H III) or low (L III) antibody response and for maximum (AIR MAX) or minimum (AIR MIN) acute inflammatory reaction (AIR) were immunized with IRBP. Each experiment was performed with at least 5 mice per group. EAU was evaluated by histopathology 21 days after immunization and the minimal criterion was inflammatory cell infiltration of the ciliary body, choroid and retina. Serum IgG1- and IgG2a-specific antibodies were determined by ELISA. EAU was graded by histological examination of the enucleated eyes. The incidence of EAU was lower in AIR MIN mice whereas in the other strains approximately 40% of the animals developed the disease. Low responder animals did not produce anti-IRBP IgG2a antibodies or interferon-gamma. No correlation was observed between susceptibility to EAU and anti-IRBP isotype profiles. Susceptibility to EAU is related to the intrinsic capacity to mount higher inflammatory reactions and increased production of anti-IRBP IgG2a isotype is not necessarily a marker of this immunologic profile.

High frequency of mutation G377S in Brazilian type 3 Gaucher disease patients

Gaucher disease (GD), the most prevalent lysosome storage disorder, presents an autosomal recessive mode of inheritance. It is a paradigm for therapeutic intervention in medical genetics due to the existence of effective enzyme replacement therapy. We report here the analysis of GD in 262 unrelated Brazilian patients, carried out in order to establish the frequency of the most common mutations and to provide prognostic information based on genotype-phenotype correlations. Among 247 type 1 GD patients, mutation N370S was detected in 47% of all the alleles, but N370S/N370S homozygosity was found in only 10% of the patients, a much lower frequency than expected, suggesting that most individuals presenting this genotype may not receive medical attention. Recombinant alleles were detected at a high frequency: 44% of the chromosomes bearing mutation L444P had other mutations derived from the pseudogene sequence, present in 25% of patients. Three neuronopathic type 2 patients were homozygous for L444P, all presenting additional mutations (E326K or recombinant alleles) that probably lead to the more severe phenotypes. Six children, classified as type 1 GD patients, had a L444P/L444P genotype, showing that neuronopathic symptoms may only manifest later in life. This would indicate the need for a higher treatment dose during enzyme replacement therapy. Finally, mutation G377S was present in 4 homozygous type 1 patients and also in compound heterozygosity in 5 (42%) type 3 patients. These findings indicate that G377S cannot be unambiguously classified as mild and suggest an allele-dose effect for this mutation.

Inducing mutations in the mouse genome with the chemical mutagen ethylnitrosourea

When compared to other model organisms whose genome is sequenced, the number of mutations identified in the mouse appears extremely reduced and this situation seriously hampers our understanding of mammalian gene function(s). Another important consequence of this shortage is that a majority of human genetic diseases still await an animal model. To improve the situation, two strategies are currently used: the first makes use of embryonic stem cells, in which one can induce knockout mutations almost at will; the second consists of a genome-wide random chemical mutagenesis, followed by screening for mutant phenotypes and subsequent identification of the genetic alteration(s). Several projects are now in progress making use of one or the other of these strategies. Here, we report an original effort where we mutagenized BALB/c males, with the mutagen ethylnitrosourea. Offspring of these males were screened for dominant mutations and a three-generation breeding protocol was set to recover recessive mutations. Eleven mutations were identified (one dominant and ten recessives). Three of these mutations are new alleles (Otop1mlh, Foxn1sepe and probably rodador) at loci where mutations have already been reported, while 4 are new and original alleles (carc, eqlb, frqz, and Sacc). This result indicates that the mouse genome, as expected, is far from being saturated with mutations. More mutations would certainly be discovered using more sophisticated phenotyping protocols. Seven of the 11 new mutant alleles induced in our experiment have been localized on the genetic map as a first step towards positional cloning.

Epidemiological characterization of resistance and PCR typing of Shigella flexneri and Shigella sonnei strains isolated from bacillary dysentery cases in Southeast Brazil

Shigella spp are Gram-negative, anaerobic facultative, non-motile, and non-sporulated bacilli of the Enterobacteriaceae family responsible for "Shigellosis" or bacillary dysentery, an important cause of worldwide morbidity and mortality. However, despite this, there are very few epidemiological studies about this bacterium in Brazil. We studied the antibiotic resistance profiles and the clonal structure of 60 Shigella strains (30 S. flexneri and 30 S. sonnei) isolated from shigellosis cases in different cities within the metropolitan area of Campinas, State of São Paulo, Brazil. We used the following well-characterized molecular techniques: enterobacterial repetitive intergenic consensus, repetitive extragenic palindromic, and double-repetitive element-polymerase chain reaction to characterize the bacteria. Also, the antibiotic resistance of the strains was determined by the diffusion disk method. Many strains of S. flexneri and S. sonnei were found to be multi-resistant. S. flexneri strains were resistant to ampicillin in 83.3% of cases, chloramphenicol in 70.0%, streptomycin in 86.7%, sulfamethoxazole in 80.0%, and tetracycline in 80.0%, while a smaller number of strains were resistant to cephalothin (3.3%) and sulfazotrim (10.0%). S. sonnei strains were mainly resistant to sulfamethoxazole (100.0%) and tetracycline (96.7%) and, to a lesser extent, to ampicillin (6.7%) and streptomycin (26.7%). Polymerase chain reaction-based typing supported the existence of specific clones responsible for the shigellosis cases in the different cities and there was evidence of transmission between cities. This clonal structure would probably be the result of selection for virulence and resistance phenotypes. These data indicate that the human sanitary conditions of the cities investigated should be improved.

Effects of a PPARG gene variant on obesity characteristics in Brazil

The contribution of genetic factors to the development of obesity has been widely recognized, but the identity of the genes involved has not yet been fully clarified. Variation in genes involved in adipocyte differentiation and energy metabolism is expected to have a role in the etiology of obesity. We assessed the potential association of a polymorphism in one candidate gene, peroxisome proliferator-activated receptor-gamma (PPARGg), involved in these pathways and obesity-related phenotypes in 335 Brazilians of European descent. All individuals included in the sample were adults. Pregnant women, as well as those individuals with secondary hyperlipidemia due to renal, liver or thyroid disease, and diabetes, were not invited to participate in the study; all other individuals were included. The gene variant PPARG Pro12Ala was studied by a PCR-based method and the association between this genetic polymorphism and obesity-related phenotypes was evaluated by analysis of covariance. Variant allele frequency was PPARG Ala12 = 0.09 which is in the same range as described for European and European-derived populations. No statistically significant differences were observed for mean total cholesterol, LDL cholesterol, HDL cholesterol, or triglyceride levels among PPARG genotypes in either gender. In the male sample, an association between the PPARG Pro12Ala variant and body mass index was detected, with male carriers of the Ala variant presenting a higher mean body mass index than wild-type homozygotes (28.3 vs 26.2 kg/m², P = 0.037). No effect of this polymorphism was detected in women. This finding suggests that the PPARG gene has a gender-specific effect and contributes to the susceptibility to obesity in this population.

Lack of evidence for monosomy 1p36 in patients with Prader-Willi-like phenotype

Monosomy 1p36 is the most common subtelomeric microdeletion syndrome with an incidence rate estimated to be 1 in 5000 births. A hypothesis of a similarity between patients with 1p36 deletion and those with Prader-Willi syndrome and the existence of two different phenotypes for 1p36 microdeletion has been suggested. The main objective of the present study was to determine the existence of 1p36 microdeletion in a sample of patients with mental retardation, obesity and hyperphagia who tested negative by the methylation test for Prader-Willi syndrome. Sixteen patients (7 females, 9 males), 16-26 years old, were evaluated with high-resolution cytogenetic analysis at 550-850 band levels and with 11 polymorphic microsatellite markers located in the 1p36 region. All patients had normal cytogenetic and molecular results. The results obtained by high-resolution cytogenetic methodology were confirmed by the molecular analyses. We did not detect a 1p36 microdeletion in 16 subjects with the Prader-Willi-like phenotype, which reinforces that no correlation seems to exist between Prader-Willi-like phenotype and the 1p36 microdeletion syndrome.

Reduced folate carrier 1 (RFC1) is associated with cleft of the lip only

In this report, we have reanalyzed genotyping data in a collection of families from South America based on maternal origin. Genotyping analysis was performed at the Craniofacial Anomalies Research Center at the University of Iowa. These genotypes were derived from genomic DNA samples obtained from blood spots from children born with isolated orofacial clefts in 45 hospitals located in eight countries (Argentina, Bolivia, Brazil, Chile, Ecuador, Paraguay, Uruguay, and Venezuela) collaborating with ECLAMC (Latin American Collaborative Studies of Congenital Malformations) between January 1998 and December 1999. Dried blood samples were sent by regular mail to the Laboratory of Congenital Malformations, Federal University of Rio de Janeiro. Previous findings suggested that mitochondrial haplotype D is more commonly found among cleft cases born in South America. We hypothesized that association of certain genes may depend upon the ethnic origin, as defined by population-specific markers. Therefore, we tested if markers in MTHFR (5,10-methylenetetrahydrofolate reductase) and RFC1 (reduced folate carrier 1) were associated with oral clefts, depending on the maternal origin defined by the mitochondrial haplotype. Transmission distortion of alleles in MTHFR C677T and RFC1 G80A polymorphic variants was tested in 200 mother/affected child pairs taking into consideration maternal origin. RFC1 variation was over-transmitted to children born with cleft lip only (P = 0.017) carrying mitochondrial DNA haplotypes other than haplotype D. Our results provide a new indication that variation in RFC1 may contribute to cleft lip only. Future studies should investigate the association between oral clefts and RFC1 based on more discrete phenotypes.

Asthma: where is it going?

Asthma is characterized by reversible airway obstruction, airway hyperresponsiveness, and airway inflammation. Although our understanding of its pathophysiological mechanisms continues to evolve, the relative contributions of airway hyperresponsiveness and inflammation are still debated. The first mechanism identified as important for asthma was bronchial hyperresponsiveness. In a second step, asthma was recognized also as an inflammatory disease, with chronic inflammation inducing structural changes or remodeling. However, persistence of airway dysfunction despite inflammatory control is observed in chronic severe asthma of both adults and children. More recently, a potential role for epithelial-mesenchymal communication or transition is emerging, with epithelial injury often resulting in a self-sustaining phenotype of wound repair modulation by activation/reactivation of the epithelial-mesenchymal trophic unit, suggesting that chronic asthma can be more than an inflammatory disease. It is noteworthy that the gene-environmental interactions critical for the development of a full asthma phenotype involve processes similar to those occurring in branching morphogenesis. In addition, a central role for airway smooth muscle in the pathogenesis of the disease has been explored, highlighting its secretory function as well as different intrinsic properties compared to normal subjects. These new concepts can potentially shed light on the mechanisms underlying some asthma phenotypes and improve our understanding of the disease in terms of the therapeutic strategies to be applied. How we understand asthma and its mechanisms along time will be the focus of this overview.

Do Caucasian and Asian clocks tick differently?

The Period 3 and Clock genes are important components of the mammalian molecular circadian system. Studies have shown association between polymorphisms in these clock genes and circadian phenotypes in different populations. Nevertheless, differences in the pattern of allele frequency and genotyping distribution are systematically observed in studies with different ethnic groups. To investigate and compare the pattern of distribution in a sample of Asian and Caucasian populations living in Brazil, we evaluated two well-studied polymorphisms in the clock genes: a variable number of tandem repeats (VNTR) in PER3 and a single nucleotide polymorphism (SNP) in CLOCK. The aim of this investigation was to search for clues about human evolutionary processes related to circadian rhythms. We selected 109 Asian and 135 Caucasian descendants. The frequencies of the shorter allele (4 repeats) in the PER3 gene and the T allele in the CLOCK gene among Asians (0.86 and 0.84, respectively) were significantly higher than among Caucasians (0.69 and 0.71, respectively). Our results directly confirmed the different distribution of these polymorphisms between the Asian and Caucasian ethnic groups. Given the genetic differences found between groups, two points became evident: first, ethnic variations may have implications for the interpretation of results in circadian rhythm association studies, and second, the question may be raised about which evolutionary conditions shaped these genetic clock variations.