The Bag320 satellite DNA family in Bacillus stick insects (Phasmatodea): different rates of molecular evolution of highly repetitive DNA in bisexual and parthenogenic taxa

The Bag320 satellite DNA (satDNA) family was studied in seven populations of the stick insects Bacillus atticus (parthenogenetic, unisexual) and Bacillus grandii (bisexual). It was characterized as widespread in all zymoraces of B. atticus and in all subspecies of B. grandii. The copy number of this satellite is higher in the bisexual B. grandii (15%-20% of the genome) than in the parthenogenetic B. atticus (2%-5% of the genome). The nucleotide sequences of 12 Bag320 clones from B. atticus and 17 from B. grandii differed at 13 characteristic positions by fixed nucleotide substitutions. Thus, nucleotide sequences from both species cluster conspecifically in phylogenetic dendrograms. The nucleotide sequences derived from B. grandii grandii could be clearly discriminated from those of B. grandii benazzii and B. grandii maretimi on the basis of 25 variable sites, although all taxa come from Sicily. In contrast, the Bag320 sequences from B. atticus could not be discriminated accordingly, although they derive from geographically quite distant populations of its three zymoraces (the Italian and Greek B. atticus atticus, the Greek and Turkish B. atticus carius, and the Cyprian B. atticus cyprius). The different rate of evolutionary turnover of the Bag320 satDNA in both species can be related to their different modes of reproduction. This indicates that meiosis and chromosome segregation affect processes in satDNA diversification.      

Satellite sequence turnover in parthenogenetic systems: the apomictic triploid hybrid Bacillus lynceorum (Insecta, Phasmatodea)

In the genus Bacillus (Insecta, Phasmatodea) the Bag320 satellite DNA family is present in the bisexual B. grandii and in the related automictic nonhybrid B. atticus; it is lacking in the other bisexual taxon of the genus, B. rossius. This family of highly repeated sequences was analyzed for 11 populations of the apomictic triploid hybrid B. lynceorum. In the neighbor-joining dendrogram, B. lynceorum nucleotide sequences distribute, regardless of geographical origin, among two clusters, one also including all clones of the three B. atticus races, and the other including sequences of the B. grandii grandii subspecies. Thus, B. lynceorum is a trihybrid taxon: as the molecular approach definitively demonstrates, it embodies one haploid complement each of both B. grandii grandii and B. atticus, which must be added to that of B. rossius. The contribution of the latter species has already been assessed on karyological and allozymic grounds. A statistical analysis performed on p-distances shows that for the parental taxa, nucleotide substitution values are of comparable magnitudes at the population level but differ at the subspecific level, being higher for the bisexual taxon. In the apomictic hybrid, atticus- and grandii grandii- like sequences coexist with significantly different p-distance values. For three clones, the nucleotide compositions at the diagnostic loci suggest that gene conversion can occur between atticus- and grandii grandii-like monomers. On the whole, this supports bisexuality as a driving force in variant fixation and suggests that in Bacillus, different gametogenetic processes and different origins of the unisexuals are mirrored in genomic turnover rates of satellite DNA.      

The mitochondrial cytochrome oxidase II gene in Bacillus stick insects: ancestry of hybrids, androgenesis, and phylogenetic relationships

Sequencing of a cytochrome oxidase II (COII) gene fragment in Bacillus taxa provided evidence that the bisexual B. rossius is the maternal ancestor of the hybridogenetic B. rossius-grandii strains and revealed the same ancestry for both parthenogenetic hybrids: the diploid B. whitei (B. rossius/grandii grandii) and the triploid B. lynceorum (B. rossius/grandii grandii/atticus). Present data clearly demonstrate that all Bacillus unisexuals arose through asymmetrical hybridization events and realized a paraphyletic derivation from the B. rossius redtenbacheri subspecies. The invention of B. rossius mitochondrial DNA haplotypes in specimens with B. grandii grandii nuclear genomes revealed the occurrence of androgenesis in nature. Natural androgens represent a peculiar escape from hybridity and can help maintain the hybridogenetic system through the production of the fathering taxon via hybrid females. Results from the COII gene support the phyletic relationships among taxa suggested by previous taxonomical approaches, but also indicate a departure of B. grandii subspecies from the established taxonomy. Assuming the existence of a molecular clock, the evaluated substitution rate brings the splitting between B. rossius and B. grandii/B. atticus back to 22.79 +/- 2.65 myr before present, while the origin of hybrids appears to be much more recent (1.06 +/- 0.53 myr).     

New DAPI and FISH findings on egg maturation processes in related hybridogenetic and parthenogenetic Bacillus hybrids (Insecta, Phasmatodea)

Bacillus stick insects have proved adequate for studying a wide array of reproductive modes: sexual, parthenogenetic, hybridogenetic, androgenetic. Hybridogenetic strains (B. rossius-grandii) were thought to discard the paternal "grandii" haploset during first meiotic division and keep the "rossius" hemiclone, whereas the clonal B. whitei (=rossius/grandii) would maintain its hybrid structure by fusing back two nonsister nuclei-each derived from previously segregated heterospecific complements-by the end of the 2(nd) meiotic division. New investigations on laid eggs and ovariole squashes, either DAPI stained or FISH labeled, revealed that in hybridogens the "grandii" set is excluded from the germ line prior to meiosis and that a DNA extra-synthesis should occur to produce hemiclonal eggs after two cytologically normal meiotic divisions. On the other hand, in B. whitei eggs no genome segregation appears to occur and an intrameiotic DNA extra-synthesis must take place to produce 2n tetrachromatidic oocytes I; these divide twice and give unreduced clonal eggs. The new findings bring hybridogenetic oogenesis of Bacillus to be coincident with that of the known hemiclonal organisms and point to an independent onset of B. whitei from hemiclonal strains. In addition, B. whitei gains a closer resemblance to B. lynceorum owing to the sharing of a cytologically identical egg maturation mechanism, of the same maternal ancestor and of peculiar chromosomal features. It is here suggested that B. lynceorum originated from the incorporation of an "atticus" genome into a B. whitei egg, according to a pathway of repeated hybridization often occurred with other polyploid hybrids.     

Unisexuality and Molecular Drive: Bag320 Sequence Diversity in <Emphasis Type="Italic">Bacillus</Emphasis> Taxa (Insecta Phasmatodea)

Satellite DNA variability follows a pattern of concerted evolution through homogenization of new variants by genomic turnover mechanisms and variant fixation by chromosome redistribution into new combinations with the sexual process. Bacillus taxa share the same Bag320 satellite family and their reproduction ranges from strict bisexuality (B. grandii) to automictic (B. atticus) and apomictic (B. whitei = rossius/ grandii; B. lynceorum = rossius/grandii/atticus) unisexuality. Thelytokous reproduction clearly allows uncoupling of homogenization from fixation. Both trends and absolute values of satellite variability were analyzed in all Bacillus taxa but B. rossius, on 906 sequenced monomers at all level of comparisons: intraspecimen, intrapopulation, interpopulation, intersubspecies, and interspecies. For unisexuals, allozymic and mitochondrial clones were also taken into account. Different reproductive modes (sexual/parthenogenetic) appear to explain observed variability trends, supporting Dover's hypothesis of sexuality acting as a driving force in the fixation of sequence variants, but the present analyses also highlight current spreading of new variants in B. grandii maretimi specimens and point to a biased sequence inheritance at the time of hybrid onset in the apomictic hybrids B. whitei and B. lynceorum. Evidence of biased gene conversion events suggests that, given enough time, sequence homogenization can take place in a unisexual such as B. lynceorum. On the contrary, the absolute values of sequence diversity in each taxon are linked to the species' range, time of divergence, and repeat copy number and, possibly, to transposon features. Satellite dynamics appears therefore to be the outcome of both general molecular processes and specific organismal traits.     

Amplification of the nucleolus organizer region during the sexual phase of Neurospora crassa

Previously we have shown that the nucleolus organizer region (NOR) of Neurospora crassa displays frequent size changes during crosses. In these initial studies, we observed that decreases in NOR size are far more common than increases. Here, we have investigated the inheritance of NOR size in a strain with an unusually small NOR. We call this strain SNO for small nuclelous organizer. We found that progeny that inherit their rDNA from SNO receive either an NOR that is larger than that of SNO or, rarely, the same size, but never an NOR that is smaller than that of SNO. The number of progeny that inherit their NOR from SNO is not significantly different from the number that inherit their NOR from the other parent in the cross. This argues against the idea that the failure to find progeny with NORs smaller than that of SNO is due to inviability of spores carrying such an NOR, or that it is due to unconscious bias by the experimenter against isolating such spores. These results can most easily be explained by a combination of unequal sister chromatid exchanges in the rDNA, or sister chromatid conversion, coupled with selection againts nuclei harboring small NORs during the premeiotic phase of the Neurospora life cycle. Other, less conventional, explanations are also possible, such as directed increase in the target NOR without corresponding loss at some other NOR.     

Genetic structure and phyletic relationships of eastern mediterraneanBacillus atticus brunner (insecta phasmatodea): A biochemical study

The allozymic characterization of several new Croatian, Greek, and Turkish samples thought to belong to different subspecies ofBacillus atticus or toatticus-like taxa is given. Several allelic combinations (zymotypes) were observed among both diploid and triploid samples; the occurrence of highly different levels of heterozygosity for the same locus among populations is also common. The biochemical-genetic features of the numerous zymotypes are interpreted on the basis of the recently assessed cytology of their parthenogenetic reproduction. Biochemical and meiotic features also allow one to suggest that both diploid and triploid cytotypes ofB. atticus are more likely interracial hybrids in origin. The new triploid Greek samples show only small genetic distances from the Turkish triploid and diploid ones; also, they do not show clear-cut morphological differences, so that all triploids and Turkish diploid samples are together referred to asB. a. carius. On the other hand, all Croatian, Greek, and Italian diploids appear to belong to the same electrophoretic cluster, biochemically differentiated at a subspecific level fromB. a. carius. This newly defined comprehensive group of diploid samples, which also morphologically show gradual patterns of variation, is referred to asB. a. atticus.     

Replication banding patterns of the diploid-tetraploid treefrogs Hyla chrysoscelis and H. versicolor

Populations of the diploid-tetraploid treefrogs Hyla chrysoscelis and H. versicolor can be defined by the polymorphic positions of the nucleolar organizing regions (NORs) on their chromosomes. Evidence from NOR positions and interstitial telomere sequence data shows that gene flow between H. chrysoscelis populations appears to be restricted, with contact occurring only in narrow "hybrid" zones. Hyla versicolor appears to have had multiple origins from H. chrysoscelis populations, and this, too, is reflected in the NOR positions. We used replication banding to determine if genetic isolation of H. chrysoscelis populations was accompanied by karyotype evolution in the populations or in contact zones. We also sought to detect karyotype alteration or replication differences associated with polyploidy in H. versicolor. Homologous chromosome pairs of all H. chrysoscelis studied displayed no differences in replication banding patterns, nor did they differ from those of H. versicolor. Although NOR positions differed between the populations studied, no disturbance of the replication banding patterns was found, indicating that structural rearrangements were not involved in creating the multiple NOR positions seen in populations of H. versicolor and H. chrysoscelis.     

Mutations within the P2 domain of norovirus capsid affect binding to human histo-blood group antigens: evidence for a binding pocket

Noroviruses (NORs) are an important cause of acute gastroenteritis. Recent studies of NOR receptors showed that different NORs bind to different histo-blood group antigens (HBGAs), and at least four distinct binding patterns were observed. To determine the structure-function relationship for NORs and their receptors, two strains representing two of the four binding patterns were studied. Strain VA387 binds to HBGAs of A, B, and O secretors, whereas strain MOH binds to HBGAs of A and B secretors only. Using multiple sequence alignments, homology modeling, and structural analysis of NOR capsids, we identified a plausible "pocket" in the P2 domain that may be responsible for binding to HBGA receptors. This pocket consists of a conserved RGD/K motif surrounded by three strain-specific hot spots (N(302), T(337), and Q(375) for VA387 and N(302), N(338), and E(378) for MOH). Subsequent mutagenesis experiments demonstrated that all four sites played important roles in binding. A single amino acid mutation at T(337) (to A) in VA387 or a double amino acid mutation at RN(338) (to TT) in MOH abolished binding completely. Change of the entire RGD motif to SAS abolished binding in case of VA387, whereas single amino acid mutations in that motif did not have an apparent effect on binding to A and B antigens but decreased binding to H antigen. Multiple mutations at the RGK motif of MOH (SIRGK to TFRGD) completely knocked out the binding. Mutation of N(302) or Q(375) in VA387 affected binding to type O HBGA only, while switch mutants with three amino acid changes at either site from MOH to VA387 resulted in a weak binding to type O HBGAs. A further switch mutant with three amino acid changes at E(378) from MOH to VA387 diminished the binding to type A HBGA only. Taken together, our data indicate that the binding pocket likely exists on NOR capsids. Direct evidence of this hypothesis requires crystallography studies.     

C-banding patterns in the AustralianBulbine (Liliaceae): The annual group,B. semibarbata s. lato

Chromosome C-band patterns have been studied in 34 populations of the Australian annualBulbine group, which comprises 4x (2n = 26, 28), 8x (2n = 52, 54) and 12x (2n = 78) populations. The 2n = 26B. semibarbata populations have a simple, low heterochromatin pattern with very minor polytypic variation. The 2n = 28 populations, corresponding morphologically to a group given separate status asB. alata, are similar in pattern but exhibit pronounced enhancement of telomeric and, more particularly, centromeric dot bands. NOR heterochromatin and satellites are difficult to identify inB. alata but appear to occur in different positions from the 26-chromosome karyotype. Eastern Australian 8 x patterns are consistent with a proposed hybrid ancestry,B. semibarbata ×B. alata. Annual and perennial C-band profiles in the AustralianBulbine are discussed briefly in relation to the additive and transformation models of heterochromatin evolution and to the possible adaptive significance of variation in heterochromatin content.Cytoevolution in the AustralianBulbine 2; for part 1 see Pl. Syst. Evol.157, 201–217.     

Characterization of Bacillus strains of marine origin.

A total of twenty aerobic endospore-forming bacilli, isolated from marine invertebrates and sea water of different areas of the Pacific Ocean, were taxonomically characterized. Most of the bacilli (11 strains) of marine origin belonged to the species Bacillus subtilis, according to their phenotypic characteristics, antibiotic susceptibility profiles, and fatty acids patterns. A group of four alkaliphilic strains formed a separate cluster that was tentatively classified as B. horti. One isolate, KMM 1717, associated with a sponge from the Coral Sea was identified as B. pumilus. Two strains, Bacillus KMM 1916 and KMM 1918, showed antibiotic sensitivity profiles similar to B. licheniformis, but they had a distinct fatty acid composition and peculiar phenotypic traits. The taxonomic affiliation of KMM 1810 and KMM 1763 remained unclear since their fatty acid composition and antibiotic sensitivity patterns were not resembled with none of these obtained for Bacillus strains.     

Two-dimensional RFLP analyses reveal megabase-sized clusters of rRNA gene variants in Arabidopsis thaliana, suggesting local spreading of variants as the mode for gene homogenization during concerted evolution

Eukaryotic genes encoding the precursor of 18S, 5.8S and 25S ribosomal RNA (rRNA genes or rDNA) are virtually identical within a species, yet they evolve rapidly between species, a phenomenon known as concerted evolution. The mechanisms by which sequence homogenization and fixation of new rRNA gene variants occurs within a genome are not clear. In diploid Arabidopsis thaliana , approximately 1500 rRNA genes are tandemly arrayed at two nucleolus organizer regions, one on chromosome 2 ( NOR2 ), the other on chromosome 4 ( NOR4 ). This paper shows that NOR2 and NOR4 are similar in size, each spanning approximately 3.5–4.0 Mbp. Using two-dimensional mapping techniques involving a combination of pulsed-field and conventional gel electrophoresis, the distributions of four distinct rRNA gene variants at NOR2 and NOR4 have been determined. rRNA genes at NOR4 are homogeneous with respect to a Hin dIII site occurring once per gene. In contrast, fewer than 10% of the rRNA genes at NOR2 are Hin dIII-bearing variants. A single intergenic spacer length is found among rRNA genes at NOR2 but three classes of spacer length variants are present at NOR4 . The NOR4 variants are not intermingled with one another; instead, they are highly clustered over distances as large as 1.5 Mbp. These data suggest that in the concerted evolution of rRNA genes, homogenization is a consequence of local spreading of new rRNA gene variants.     

Genes within the Idd5 and Idd9/11 diabetes susceptibility loci affect the pathogenic activity of B cells in nonobese diabetic mice

Autoreactive T cells clearly mediate the pancreatic beta cell destruction causing type 1 diabetes (T1D). However, studies in NOD mice indicate that B cells also contribute to pathogenesis because their ablation by introduction of an Igmunull mutation elicits T1D resistance. T1D susceptibility is restored in NOD.Igmunull mice that are irradiated and reconstituted with syngeneic bone marrow plus NOD B cells, but not syngeneic bone marrow alone. Thus, we hypothesized some non-MHC T1D susceptibility (Idd) genes contribute to disease by allowing development of pathogenic B cells. Supporting this hypothesis was the finding that unlike those from NOD donors, engraftment with B cells from H2g7 MHC-matched, but T1D-resistant, nonobese-resistant (NOR) mice failed to restore full disease susceptibility in NOD.Igmunull recipients. T1D resistance in NOR mice is mainly encoded within the Idd13, Idd5.2, and Idd9/11 loci. B cells from NOD congenic stocks containing Idd9/11 or Idd5.1/5.2-resistance loci, respectively, derived from the NOR or C57BL/10 strains were characterized by suppressed diabetogenic activity. Immature autoreactive B cells in NOD mice have an impaired ability to be rendered anergic upon Ag engagement. Interestingly, both Idd5.1/5.2 and Idd9/11-resistance loci were found to normalize this B cell tolerogenic process, which may represent a mechanism contributing to the inhibition of T1D.     

Obligatory parthenogenesis and TE load: Bacillus stick insects and the R2 non‐LTR retrotransposon

Transposable elements (TEs) are selfish genetic elements whose self‐replication is contrasted by the host genome. In this context, host reproductive strategies are predicted to impact on both TEs load and activity. The presence and insertion distribution of the non‐LTR retrotransposon R2 was here studied in populations of the strictly bisexual Bacillus grandii maretimi and of the obligatory parthenogenetic Bacillus atticus atticus. Furthermore, data were also obtained from the offspring of selected B. a. atticus females. At the population level, the gonochoric B. g. maretimi showed a significantly higher R2 load than the obligatory parthenogenetic B. a. atticus. The comparison with bisexual and unisexual Bacillus rossius populations showed that their values were higher than those recorded for B. a. atticus and similar, or even higher, than those of B. g. maretimi. Consistently, an R2 load reduction is scored in B. a. atticus offspring even if with a great variance. On the whole, data here produced indicate that in the obligatory unisexual B. a. atticus R2 is active and that mechanisms of molecular turnover are effective. Furthermore, progeny analyses show that, at variance of the facultative parthenogenetic B. rossius, the R2 activity is held at a lower rate. Modeling parental‐offspring inheritance, suggests that in B. a. atticus recombination plays a major role in eliminating insertions rather than selection, as previously suggested for unisexual B. rossius progeny, even if in both cases a high variance is observed. In addition to this, mechanisms of R2 silencing or chances of clonal selection cannot be ruled out.     

Sister chromatid differential staining and NOR activity of BrdU-resistant sublines

Summary Two 30 g/ml BrdU-resistant sublines and two 60 g/ml BrdU-resistant sublines are induced from a Chinese hamster cell line Wg3h (HGPRT^–) by one-step and two-step selections, respectively. By inoculating the cells into BrdU-free medium or by adding more BrdU into the culture medium for 26–27 h, it was found that the two BrdU-resistant sublines analysed have very clear sister chromatid differential (SCD) staining patterns. This indicates that some of the nuclear DNA of the BrdU-resistant cells incorporate with BrdU to reach a kinetic balance. Frequencies of sister chromatid exchange (SCE) of the resistant cells are twice to four times as high as those of the Wg3h cells, depending on which BrdU-resistant subline is analysed. The SCE frequencies of the resistant cells also increase with the BrdU concentration in the medium. Analysis of silver-stained nucleolar organizer regions (NORs) indicates that the NOR activity of three out of the four BrdU-resistant sublines is significantly suppressed, i.e., averages of the Ag-NOR number and number of the chromosomes bearing Ag-NORs per cell decrease significantly. The degree of suppression for different BrdU-resistant sublines may be quite different. The suppressed NOR activity of the resistant cells can gradually be restored when the cells are inoculated into BrdU-free medium, but the recovery speed is far lower than that of the Wg3h cells. The suppression of the NOR activity of the BrdU-resistant sublines should be due to BrdU toxicity.     

Structure and variation of the Nucleolus Organizer Region in turtles

Differential staining techniques were used to study the structure and variation of the NORs of 27 species of cryptodiran turtles. No species or individuals had more than a single pair of NORs. Extensive variation in NOR structure and chromosomal location was found among higher taxa and individual variation in NOR size was common. Thirty eight percent of all individuals studied were heterozygous for the size of the NOR. However, interspecific variation in chromosomal location and structure of the NOR within major taxa was relatively rare. It is concluded that ( I ) the pattern of variation of NORs is consistent with patterns of chromosomal evolution in turtles; (2) Turtles have only a single pair of NORs whereas other animals, such as some mammals, possess numerous NORs; (3) The heterochromatin associated with the NOR is involved in the structure of the nucleolus.