Distribution, expression, and motif variability of ankyrin domain genes in Wolbachia pipientis

The endosymbiotic bacterium Wolbachia pipientis infects a wide range of arthropods, in which it induces a variety of reproductive phenotypes, including cytoplasmic incompatibility (CI), parthenogenesis, male killing, and reversal of genetic sex determination. The recent sequencing and annotation of the first Wolbachia genome revealed an unusually high number of genes encoding ankyrin domain (ANK) repeats. These ANK genes are likely to be important in mediating the Wolbachia-host interaction. In this work we determined the distribution and expression of the different ANK genes found in the sequenced Wolbachia wMel genome in nine Wolbachia strains that induce different phenotypic effects in their hosts. A comparison of the ANK genes of wMel and the non-CI-inducing wAu Wolbachia strain revealed significant differences between the strains. This was reflected in sequence variability in shared genes that could result in alterations in the encoded proteins, such as motif deletions, amino acid insertions, and in some cases disruptions due to insertion of transposable elements and premature stops. In addition, one wMel ANK gene, which is part of an operon, was absent in the wAu genome. These variations are likely to affect the affinity, function, and cellular location of the predicted proteins encoded by these genes.

Wolbachia pipientis: intracellular infection and pathogenesis in Drosophila

Wolbachia pipientis is a vertically transmitted, obligate intracellular symbiont of arthropods. The bacterium is best known for its ability to manipulate host reproductive biology where it can induce cytoplasmic incompatibility, parthenogenesis, feminization and male-killing. In addition to the various reproductive phenotypes it generates through interaction with host reproductive tissue it is also known to infect somatic tissues. However, relatively little is known about the consequences of infection of these tissues with the exception that in some hosts Wolbachia acts as a classical mutualist and in others a pathogen, dramatically shortening adult insect lifespan. Manipulation experiments have demonstrated that the severity of Wolbachia-induced effects on the host is determined by a combination of host genotype, Wolbachia strain, host tissue localization, and interaction with the environment. The recent completion of the whole genome sequence of Wolbachia pipientis wMel strain indicates that it is likely to use a type IV secretion system to establish and maintain infection in its host. Moreover, an unusual abundance of genes encoding proteins with eukaryotic-like ankyrin repeat domains suggest a function in the various described phenotypic effects in hosts.

Wolbachia: why these bacteria are important to genome research

Intracellular bacteria of the genus Wolbachia were first discovered in mosquitoes in the 1920s. Their superficial similarity to pathogenic rickettsia initially raised interest in them as potential human pathogens. However, injection experiments with mice showed that they were non-pathogenic, and they were subsequently classified as symbionts of insects. Until the 1970s, Wolbachia was considered to infect a limited number of species of mosquitoes. It is now clear that Wolbachia is an extremely common intracellular agent of invertebrates, infecting nearly all the major groups of arthropods and other terrestrial invertebrates. Its wide host range and abundance can be attributed partly to the unusual phenotypes it exerts on the host it infects. These include the induction of parthenogenesis (the production of female offspring from unmated mothers) in certain insects, the feminization of genetic male crustaceans to functional phenotypic females, and the failure of fertilization in hosts when males and females have a different infection status (cytoplasmic incompatibility). All of these phenotypes favor maternal transmission of the intracellular Wolbachia. In the last year, Wolbachia has also been shown to be a widespread symbiont of filarial nematodes. It appears that Wolbachia is needed by the adult worm for normal fertility, indicating that Wolbachia is behaving like a classic mutualist in this case. This discovery exemplifies that the extent of the host range of Wolbachia and its associated phenotypes is still far from fully understood.

Evolution of Wolbachia pipientis transmission dynamics in insects

Wolbachia pipientis is an intracellular bacterial parasite of arthropods that enhances its transmission by manipulating host reproduction, most commonly by inducing cytoplasmic incompatibility. The discovery of isolates with modified cytoplasmic incompatibility phenotypes and others with novel virulence properties is an indication of the potential breadth of evolutionary strategies employed by Wolbachia.

In vitro cultivation of Wolbachia pipientis in an Aedes albopictus cell line

A continuous cell line, Aa23, was established from eggs of a strain of the Asian tiger mosquito, Aedes albopictus, naturally infected with the intracellular symbiont Wolbachia pipientis. The resulting cell line was shown to be persistently infected with the bacterial endosymbiont. Treatment with antibiotics cured the cells of the infection. In the course of establishing this cell line it was noticed that RFLPs in the PCR products of two Wolbachia genes from the parental mosquitoes were fixed in the infected cell line. This indicates that the mosquito host was naturally superinfected with different Wolbachia strains, whereas the infected cell line derived from these mosquitoes only contained one of the original Wolbachia strains. The development of anin vitroculture system for this fastidious microorganism should facilitate molecular analysis of the reproduction distorting phenotypes it induces in natural arthropod hosts.

Modification of arthropod vector competence via symbiotic bacteria

Some of the world's most devastating diseases are transmitted by arthropod vectors. Attempts to control these arthropods are currently being challenged by the widespread appearance of insecticide resistance. It is therefore desirable to develop alternative strategies to complement existing methods of vector control. In this review, Charles Beard, Scott O'Neill, Robert Tesh, Frank Richards and Serap Aksoy present an approach for introducing foreign genes into insects in order to confer refractoriness to vector populations, ie. the inability to transmit disease-causing agents. This approach aims to express foreign anti-parasitic or anti-viral gene products in symbiotic bacteria harbored by insects. The potential use of naturally occurring symbiont-based mechanisms in the spread of such refractory phenotypes is also discussed.

Molecular development of the olfactory nerve pathway

There are, at least, two major questions concerning the molecular development of the olfactory nerve pathway. First, what are the molecular cues responsible for guiding axons from the nasal cavity to the olfactory bulb? Second, what is the molecular basis of axon targeting to specific glomeruli once axons reach the olfactory bulb? Studies in the primary olfactory pathway have focused on the role of the extracellular matrix and ensheathing cells in establishing an initial substrate for growth of pioneer axons between the periphery and brain. The primary axons also express a multitude of cell adhesion molecules that regulate fasciculation of axons and hence may play a role in fascicle formation in the olfactory nerve. Although the olfactory neuroepithelium principally consists of a morphologically homogeneous class of primary olfactory neurons, there are numerous subpopulations of olfactory neurons expressing chemically distinct phenotypes. In particular, numerous subpopulations have been characterized by expression of unique carbohydrate residues and olfactory receptor proteins. Some of these molecules have recently been implicated in axon guidance and targeting to specific glomeruli.

Structural interpretation of mutations in phenylalanine hydroxylase protein aids in identifying genotype-phenotype correlations in phenylketonuria

Phenylalanine hydroxylase (PAH) is the enzyme that converts phenylalanine to tyrosine as a rate-limiting step in phenylalanine catabolism and protein and neurotransmitter biosynthesis. Over 300 mutations have been identified in the gene encoding PAH that result in a deficient enzyme activity and lead to the disorders hyperphenylalaninaemia and phenylketonuria. The determination of the crystal structure of PAH now allows the determination of the structural basis of mutations resulting in PAH deficiency. We present an analysis of the structural basis of 120 mutations with a 'classified' biochemical phenotype and/or available in vitro expression data. We find that the mutations can be grouped into five structural categories, based on the distinct expected structural and functional effects of the mutations in each category. Missense mutations and small amino acid deletions are found in three categories:'active site mutations', 'dimer interface mutations', and 'domain structure mutations'. Nonsense mutations and splicing mutations form the category of 'proteins with truncations and large deletions'. The final category, 'fusion proteins', is caused by frameshift mutations. We show that the structural information helps formulate some rules that will help predict the likely effects of unclassified and newly discovered mutations: proteins with truncations and large deletions, fusion proteins and active site mutations generally cause severe phenotypes; domain structure mutations and dimer interface mutations spread over a range of phenotypes, but domain structure mutations in the catalytic domain are more likely to be severe than domain structure mutations in the regulatory domain or dimer interface mutations.

Shared and unique environmental factors determine the ecology of methanogens in humans and rats

OBJECTIVE: This study ascertains the relative contributions of genetics and environment in determining methane emission in humans and rats. There is considerable interest in the factors determining the microbial species that inhabit the colon. Methanogens, which are archaebacteria, are an easily detected colonic luminal bacteria because they respire methane. They are present in some but not all human colons and lower animal hindguts. Opinion varies on the nature of the factors influencing this ecology with some studies proposing the existence of host genetic influences. METHODS: Methane emission was measured in human twin pairs by gas chromatography, and structural equation modeling was used to determine the proportion of genetic and environmental determinants. The importance of the timing of environmental effects and rat strain on the trait of methane emission were ascertained by experiments with cohabiting methanogenic and nonmethanogenic rats. RESULTS: Analysis of breath samples from 274 adolescent twin pairs and their families indicated that the major influences on the trait of methane emission are the result of shared (53%, 95% confidence interval 39-61) and unique environmental (47%, 95% confidence interval 38-56) effects. No significant autosomal genetic effects were detected, but as observed in other studies, men (37%) were less likely to excrete methane in their breath than women (63%). Investigation of methane emission in rats indicated that environmental effects in this animal are most potent during the weaning period, with stable gut microbial ecology thereafter for some but not all rat strains. CONCLUSIONS: These results are consistent with shared and unique environmental factors being the main determinants of the ecology of this colonic microbe. (Am J Gastroenterol 2000;95:2872-2879. (C) 2000 by Am. Coll. of Gastroenterology).

Allele and genotype frequencies of polymorphic cytochromes P4502D6, 2C19 and 2E1 in Aborigines from Western Australia

The polymorphisms of the important xenobiotic metabolizing enzymes CYP2D6, CYP2C19 and CYP2E1 have been studied extensively in a large number of populations and show significant heterogeneity in the frequency of different alleles/genotypes and in the prevalence of the extensive and poor metabolizer phenotypes, Understanding of inter-ethnic differences in genotypes is important in prediction of either beneficial or adverse effects from therapeutic agents and other xenobiotics. Since no data were available for Australian Aborigines, we investigated the frequencies of alleles and genotypes for CYP2D6, CYP2C19 and CYP2E1 in a population living in the far north of Western Australia. Because of its geographical isolation, this population can serve as a model to study the impact of evolutionary forces on the distribution of different alleles for xenobiotic metabolizing enzymes. Twelve CYP2D6 alleles were analysed, The wild-type allele *1 was the most frequent (85.8%) and the non-functional alleles (*4, *5, *16) had an overall frequency of less than 10%. Only one subject (0.4%) was a poor metabolizer for CYP2D6 because of the genotype *5/*5, For CYP2C19, the frequencies of the *1 (wild-type) and the non-functional (*2 and *3) alleles were 50.2%, 35.5% and 14.3%, respectively. The combined CYP2C19 genotypes (*2/*2, *2/*3 or *3/*3) correspond to a predicted frequency of 25.6% for the CYP2C19 poor metabolizer phenotype, For CYP2E1, only one subject had the rare c2 allele giving an overall allele frequency of 0.2%. For CYP2D6 and CYP2C19, allele frequencies and predicted phenotypes differed significantly from those for Caucasians but were similar to those for Orientals indicating a close relationship to East Asian populations. Differences between Aborigines and Orientals in allele frequencies for CYP2D6*10 and CYP2E1 c2 may have arisen through natural selection, or genetic drift, respectively, Pharmacogenetics 11:69-76 (C) 2001 Lippincott Williams & Wilkins.

Mutant GABA(A) receptor gamma 2-subunit in childhood absence epilepsy and febrile seizures

Epilepsies affect at least 2% of the population at some time in life, and many forms have genetic determinants(1,2). We have found a mutation in a gene encoding a GABA, receptor subunit in a large family with epilepsy. The two main phenotypes were childhood absence epilepsy (CAE) and febrile seizures (FS), There is a recognized genetic: relationship between FS and CAE, yet the two syndromes have different ages of onset, and the physiology of absences and convulsions is distinct. This suggests the mutation has age-dependent effects on different neuronal networks that influence the expression of these clinically distinct, but genetically related, epilepsy phenotypes. We found that the mutation in GABRG2 (encoding the gamma2-subunit) abolished in vitro sensitivity to diazepam, raising the possibility that endozepines do in fact exist and have a physiological role in preventing seizures.

Generalized epilepsy with febrile seizures plus: Mutation of the sodium channel subunit SCN1B

Generalized epilepsy with febrile seizures plus (GEFS(+)) is an important childhood genetic epilepsy syndrome with heterogeneous phenotypes, including febrile seizures (FS) and generalized epilepsies of variable severity. Forty unrelated GEFS(+) and FS patients were screened for mutations in the sodium channel beta-subunits SCN1B and SCN2B, and the second GEFS(+) family with an SCN1B mutation is described here. The family had 19 affected individuals: 16 with typical GEFS(+) phenotypes and three with other epilepsy phenotypes. Site-specific mutation within SCN1B remains a rare cause of GEFS(+), and the authors found no evidence to implicate SCN2B in this syndrome.

X-linked myoclonic epilepsy with spasticity and intellectual disability - Mutation in the homeobox gene ARX

Objective: To describe a new syndrome of X-linked myoclonic epilepsy with generalized spasticity and intellectual disability (XMESID) and identify the gene defect underlying this disorder. Methods: The authors studied a family in which six boys over two generations had intractable seizures using a validated seizure questionnaire, clinical examination, and EEG studies. Previous records and investigations were obtained. Information on seizure disorders was obtained on 271 members of the extended family. Molecular genetic analysis included linkage studies and mutational analysis using a positional candidate gene approach. Results: All six affected boys had myoclonic seizures and TCS; two had infantile spasms, but only one had hypsarrhythmia. EEG studies show diffuse background slowing with slow generalized spike wave activity. All affected boys had moderate to profound intellectual disability. Hyperreflexia was observed in obligate carrier women. A late-onset progressive spastic ataxia in the matriarch raises the possibility of late clinical manifestations in obligate carriers. The disorder was mapped to Xp11.2-22.2 with a maximum lod score of 1.8. As recently reported, a missense mutation (1058C>T/P353L) was identified within the homeodomain of the novel human Aristaless related homeobox gene (ARX). Conclusions: XMESID is a rare X-linked recessive myoclonic epilepsy with spasticity and intellectual disability in boys. Hyperreflexia is found in carrier women. XMESID is associated with a missense mutation in ARX. This disorder is allelic with X-linked infantile spasms (ISSX; MIM 308350) where polyalanine tract expansions are the commonly observed molecular defect. Mutations of ARX are associated with a wide range of phenotypes; functional studies in the future may lend insights to the neurobiology of myoclonic seizures and infantile spasms.

Childhood absence epilepsy and febrile seizures: A family with a GABAA receptor mutation

Although several genes for idiopathic epilepsies from families with simple Mendelian inheritance have been found, genes for the common idiopathic generalized epilepsies, where inheritance is complex, presently are elusive. We studied a large family with epilepsy where the two main phenotypes were childhood absence epilepsy (CAE) and febrile seizures (FS), which offered a special opportunity to identify epilepsy genes. A total of 35 family members had seizures over four generations. The phenotypes comprised typical CAE (eight individuals); FS alone (15), febrile seizures plus (FS+) (three); myoclonic astatic epilepsy (two); generalized epilepsy with tonic-clonic seizures alone (one); partial epilepsy (one); and unclassified epilepsy despite evaluation (two). In three remaining individuals, no information was available. FS were inherited in an autosomal dominant fashion with 75% penetrance. The inheritance of CAE in this family was not simple Mendelian, but suggestive of complex inheritance with the involvement of at least two genes. A GABA(A) receptor gamma2 subunit gene mutation on chromosome 5 segregated with FS, FS+ and CAE, and also occurred in individuals with the other phenotypes. The clinical and molecular data suggest that the GABA(A) receptor subunit mutation alone can account for the FS phenotype. An interaction of this gene with another gene or genes is required for the CAE phenotype in this family. Linkage analysis for a putative second gene contributing to the CAE phenotype suggested possible loci on chromosomes 10, 13, 14 and 15. Examination of these loci in other absence pedigrees is warranted.

Sodium channel alpha 1-subunit mutations in severe myoclonic epilepsy of infancy and infantile spasms

Background: Mutations in SCN1A, the gene encoding the alpha1 subunit of the sodium channel, have been found in severe myoclonic epilepsy of infancy (SMEI) and generalized epilepsy with febrile seizures plus (GEFS(+)). Mutations in SMEI include missense, nonsense, and frameshift mutations more commonly arising de novo in affected patients. This finding is difficult to reconcile with the family history of GEFS(+) in a significant proportion of patients with SMEI Infantile spasms (IS), or West syndrome, is a severe epileptic encephalopathy that is usually symptomatic. In some cases, no etiology is found and there is a family history of epilepsy. Method: The authors screened SCN1A in 24 patients with SMEI and 23 with IS. Results: Mutations were found in 8 of 24 (33%) SMEI patients, a frequency much lower than initial reports from Europe and Japan. One mutation near the carboxy terminus was identified in an IS patient. A family history of seizures was found in 17 of 24 patients with SMEI. Conclusions: The rate of SCN1A mutations in this cohort of SMEI patients suggests that other factors may be important in SMEI. Less severe mutations associated with GEFS(+) could interact with other loci to cause SMEI in cases with a family history of GEFS(+). This study extends the phenotypic heterogeneity of mutations in SCN1A to include IS.