The ATP-dependent synthesis of factor 390 by cell-free extracts of Methanobacterium thermoautotrophicum (strain ΔH)

Abstract Cell-free extracts of Methanobacterium thermoautotrophicum (strain ΔH) converted the 8-OH-5-deazaflavin coenzyme F₄₂₀ to factor 390, a 8-adenylyl derivative (F₄₂₀-AMP). Activity was only observed upon exposure of the crude cell-free extract to oxygen. The ability to synthesize F₃₉₀ was lost when crude cell-free extract was subsequently brought to an anaerobic reducing environment. The enzymatic reaction used ATP and oxidized coenzyme F₄₂₀ as substrates and inorganic pyrophosphate was formed next to F₃₉₀. GTP could be used instead of ATP resulting in a guanylylated derivative. The crude cell-free extract showed K _m values of 154 μM for coenzyme F₄₂₀ and 2.4 mM for ATP. A partially purified enzyme preparation exhibited a K _eq of 0.32. In accordance, coenzyme F₄₂₀ and ATP could be synthesized from F₃₉₀ and PPi by the reverse reaction.     

INHERITANCE OF RESISTANCE TO CYHALOTHRIN IN THE HOUSEFLY (DIPTERA: MUSCIDAE)

Abstract The genetic inheritance of resistance to cyhalothrin in housefly, Musca domstica (L) was investigated.
Reciprocal crosses between susceptible (S) and resistant (R) strains were used to determine the characteristics of resistance. Analysis of probit line from the F₁ generation and F₂ generation obtained by inbreeding the F₁ hybrids indicated that cyhalothrin resistance was controlled by more than one factors and degree of resistance dominance to cyhalothrin was -0.10, indicating cyhalothrin resistance is conferred by incompletely recessive gene(s). The realized heritability of resistance to cyhalothrin cyhalothrin calculated from data collected routinely from laboratory selection was 0.12.     

IN VIVO METHYLATION AND TURNOVER OF RAT BRAIN HISTONES

Abstract— The turnover of the different histone components from brain nuclei was studied after the administration of l -[³H]lysine and l -[¹⁴C-methyl]methionine to newborn rats. The radioactivities of the different histone subfractions as well as other proteins were determined over a 280-day period. Biphasic type decay curves (³H and ¹⁴C) were obtained for total brain histones and all the subfractions. From 6 to 40 days of age the half life of total brain histones was 19 days. After reaching brain maturity the half life was 132 days. The lysine rich histone (F₁) was found to turnover the fastest of all the histones, having half lives of 13 and 112 days, respectively. The decay curve for the slightly lysine rich histones (F_2a2, F_2b) gave half lives of 25 days up to 40 days of age and 189 days after reaching brain maturity. The arginine rich histones (F_2a1, F₃) gave a half life of 32 days up to 40 days of age, while no turnover was observed after maturity. The turnover rates of the methyl groups and/or methionyl residues did not vary significantly from the turnover rates of the lysyl residues in the F₂ and F₃ histones. The lysine-rich histones did not contain significant amounts of methionyl residues or methyl groups.
Amino acid analysis of the brain histones revealed that about 3·6 per cent of the lysyl residues in the slightly lysine rich histones were methylated, mainly as ε-N-dimethyllysine. About 13 per cent of the lysyl residues in the arginine rich histones were methylated, mainly as ε-N-monomethyllysine and ε-N-dimethyllysine.     

Fertility of roach × bream hybrids, Rutilus rutilus (L.) ×Abramis brama (L.), and their identification

Adult roach, bream and their presumed F₁ hybrid from an Anglian Water reservoir were identified on the basis of morphological and meristic characteristics. The hybrid was clearly intermediate. Four hybrid breeding crosses were induced to spawn by hypophysis. A bream × roach cross (female named first) failed to produce fertile eggs, whereas F₁ hybrid × roach, roach × F₁ hybrid and F₁ hybrid × F₁ hybrid all produced fry. Fertility (defined as survival of eggs to hatching) was high for the F₁ hybrid × roach back-cross (56%) but low for the others (<2%), in comparison to the pure species controls (roach 69%, bream 76%). Progeny from these crosses were reared until anal fin rays could be counted. These counts indicated intermediacy between the parents and back-crossed individuals, and similarity between F₁ hybrids and their F₂ progeny.     

Artificial hybridization within Saxifraga pentadactylis (Saxifragaceae)

Saxifraga pentadactylis subsp. almanzorii , an endemic to the subalpine nucleus of Sierra de Gredos (central Spain), differs from its closest relative, subsp. willkommiana , by its less showy petals. An artificial crossing program was carried out in order to assess the degree of reproductive isolation between the subspecies. To facilitate interpretation of the results, the program was extended to 10 other interspecific hybrid combinations within sect. Saxifraga . All the data gathered are congruent with the occurrence of two evolutionary scenarios. Interspecific crossings, rendering moderate to high seed-set (in obtaining the F₁), and vigorous but relatively sterile F₁ offspring, reveal reproductive barriers at the level of the F₁ fertility, probably originated as a byproduct of divergent evolution. In contrast, intraspecific crossings within S. pentadactylis resulted in seed-set values lower than expected (in obtaining the F₁), in a majority of weak non-viable F₁ offspring but also in a few fertile F₁ hybrid specimens which were able to originate F₂ offspring. This second pattern reveals reproductive barriers at the level of the F₁ vitality, probably arisen in a quite abrupt fashion. The lower P/O for subsp. almanzorii as compared to subsp. willkommiana , together with the rest of the evidence suggest that the reproductive barriers between them might be the product of active selection against hybridization achieved by incrementing the levels of autogamy in the former.     

Inbreeding depression in the convict cichlid, Cichlasoma nigrofasciatum (Baird and Girard)

Convict cichlids, Cichlasoma nigrofasciatum , inbred for four and five generations exhibited moderate and severe morphological deformities. Deformed fishes were characterized by a more vertically sloping forehead, irregular jaw, hyoid, opercula and fin structure, reduced growth and irregular swimming behaviour. Inbred fish (F₄, F₅) did not differ from F₁s in their age of maturation or the average size of their first broods. Brood survival was 18.7% in F₅ inbred fish, compared to 75.5% in F₁s.     

Calcium taste preferences: genetic analysis and genome screen of C57BL/6J x PWK/PhJ hybrid mice

To characterize the genetic basis of voluntary calcium consumption, we tested C57BL/6J mice (B6; with low avidity for calcium), PWK/PhJ mice (PWK; with high avidity for calcium) and their F₁ and F₂ hybrids. All mice received a series of 96-h two-bottle preference tests with a choice between water and the following: 50 m m CaCl₂, 50 m m calcium lactate, 50 m m MgCl₂, 100 m m KCl, 100 m m NH₄Cl, 100 m m NaCl, 5 m m citric acid, 30 μ m quinine hydrochloride and 2 m m saccharin. Most frequency distributions of the parental and F₁ but not F₂ groups were normally distributed, and there were few sex differences. Reciprocal cross analysis showed that B6 × PWK F₁ mice had a non-specific elevation of fluid intake relative to PWK × B6 F₁ mice. In the F₂ mice, trait correlations were clustered among the divalent salts and the monovalent chlorides. A genome screen involving 116 markers showed 30 quantitative trait loci (QTLs), of which six involved consumption of calcium chloride or lactate. The results show pleiotropic controls of calcium and magnesium consumption that are distinct from those controlling consumption of monovalent chlorides or exemplars of the primary taste qualities.     

Population differentiation in an annual legume: genetic architecture

Abstract. The presence or absence of epistasis, or gene interaction, is explicitly assumed in many evolutionary models. Although many empirical studies have documented a role of epistasis in population divergence under laboratory conditions, there have been very few attempts at quantifying epistasis in the native environment where natural selection is expected to act. In addition, we have little understanding of the frequency with which epistasis contributes to the evolution of natural populations. In this study we used a quantitative genetic design to quantify the contribution of epistasis to population divergence for fitness components of a native annual legume, Chamaecrista fasciculata . The design incorporated the contrast of performance of F₂ and F₃ segregating progeny of 18 interpopulation crosses with the F₁ and their parents. Crosses were conducted between populations from 100 m to 2000 km apart. All generations were grown for two seasons in the natural environment of one of the parents. The F₁ often outperformed the parents. This F₁ heterosis reveals population structure and suggests that drift is a major contributor to population differentiation. The F₂ generation demonstrated that combining genes from different populations can sometimes have unexpected positive effects. However, the F₃ performance indicated that combining genes from different populations decreased vigor beyond that due to the expected loss of heterozygosity. Combined with previous data, our results suggest that both selection and drift contribute to population differentiation that is based on epistatic genetic divergence. Because only the F₃ consistently expressed hybrid breakdown, we conclude that the epistasis documented in our study reflects interactions among linked loci.     

Electron transfer reactions for the reduction of NADP+ in Methanosphaera stadtmanae

Abstract Methanosphaera stadtmanae , a member of the Methanobacteriales reduces methanol, but not CO₂ with H₂ or 2-propanol to produce methane. In cell-free extracts of M. stadtmanae the activities of several enzymes involved in electron transfer were measured. The activities of an F₄₂₀-nonreactive hydrogenase, NADP⁺: F₄₂₀ oxidoreductase, NADP⁺-dependent 2-propanol dehydrogenase, and a methyl viologen dependent F₄₂₀ dehydrogenase were observed. Based on the presence of these particular enzyme activities, their cofactor requirements and the absence of F₄₂₀-dependent hydrogenase activity, a model of the electron transport pathway through the coenzyme F₄₂₀ to provide electrons for biosynthesis, was formulated.     

Can Extensive Reticulation and Concerted Evolution Result in a Cladistically Structured Molecular Data Set?

Hierarchy is the main criterion for informativeness in a data set, even if no explicit reference to evolution as a causal process is provided. Sequence data (nuclear ribosomal DNA ITS) from Armeria (Plumbaginaceae) contains a certain amount of hierarchical structure as suggested by data decisiveness and distribution of tree lengths. However, ancillary evidence suggests that extensive gene flow and biased concerted evolution in these multicopy regions have significantly shaped the ITS data set. This argument is discussed using parsimony analysis of four data sets, constructed by combining wild sequences with those from different generations of artificial hybrids (wild + F₁, F₂, and backcrosses; wild + backcrosses; wild + F₁; wild + F₂). Compared to the F₁ hybrids, F₂ show a certain degree of homogenization in polymorphic sites. This effect reduces topological disruption caused by F₁ and is considered to be illustrative of how extensive gene flow and biased concerted evolution may have modeled the wild ITS data. The possibility that hierarchy has arisen as a result of—or despite a significant contribution from—those two such potentially perturbing forces raises the question of what kind of signal are we recovering from this molecular data set.     

Biosynthesis of Prostacyclin in Rat Cerebral Microvessels and the Choroid Plexus

Abstract: Microvessels, predominantly capillaries, were isolated from rat cerebrum by a modification of published procedures. The morphology and purity of the preparations were monitored by light and electron microscopy and by enrichment in alkaline phosphatase, γ-glutamyl transpeptidase, and prostacyclin synthetase. A reversed-phase high-pressure liquid chromatographic method was used in the purification of prostaglandins after extraction from aqueous incubation solutions. Prostacyclin synthesis in brain is localized in cerebral blood vessels and capillaries. The endogenous biosynthetic capacity of the isolated cerebral capillary fractions for prostacyclin, measured as its chemically stable breakdown product, 6-keto-prostaglandin F_1α, was 11 ng/mg protein/10 min. Choroid plexus and intact surface vessels synthesized 6-keto-prostaglandin F_1α at 37 and 35 ng/mg protein/10 min, respectively. The prostacyclin-synthesizing enzyme of the cerebral capillaries also converted the exogenously added prostaglandin endoperoxides to 6-keto-prostaglandin F_1α. Comparison of the synthesis of prostaglandins 6-keto-F_1α, E₂, and F_2α showed that 6-keto-prostaglandin F_1α was the major prostaglandin formed in the microvessels, in the larger surface vessels, and in the choroid plexus. Prostaglandin D₂ was not detected. Prostacyclin synthesis by the cerebral vasculature is similar to that in other blood vessels and cultured human endothelial cells. Possible physiological roles of prostacyclin in the cerebral microvasculature are discussed with special regard to the autoregulation of cerebral blood flow.     

The F1FO ATP synthase genes in Methanosarcina acetivorans are dispensable for growth and ATP synthesis

There is a long-standing discussion in the literature, based on biochemical and genomic data, whether some archaeal species may have two structurally and functionally distinct ATP synthases in one cell: the archaeal A₁A_O together with the bacterial F₁F_O ATP synthase. To address a potential role of the bacterial F₁F_O ATP synthase, we have exchanged the F₁F_O ATPase gene cluster in Methanosarcina acetivorans against a puromycin resistance cassette. Interestingly, the mutant was able to grow with no difference in growth kinetics to the wild type, and cellular ATP contents were identical in the wild type and the mutant. These data demonstrate that the F₁F_O ATP synthase is dispensable for the growth of M. acetivorans .     

Genetic and in vitro molecular hybridization of malate dehydrogenase isozymes in interspecific bass (Micropterus) hybrids

Supernatant malate dehydrogenase (MDH) isozymes (as visualized by starch gel electrophoresis) are encoded by two distinct gene loci in both the largemouth and smallmouth bass. When an interspecific F₁ hybrid is formed between these two fish, a unique MDH isozyme is generated. The results of freeze-thaw molecular hybridization (which is the first application of this technique to MDH) indicate that this unique isozyme in the F₁ hybrid is a heterodimer composed of one subunit of each parental type. The F₁ hybrids produced F₂ hybrids which in turn formed the F₃ hybrid population. The inheritance of alleles at the MDH-B locus is consistent with a single Mendelian autosomal locus. Furthermore, there is no evidence of linkage between the lactate dehydrogenase-E locus and the MDH-B locus.     

A comprehensive analysis of QTL for abdominal fat and breast muscle weights on chicken chromosome 5 using a multivariate approach

Quantitative trait loci (QTL) influencing the weight of abdominal fat (AF) and of breast muscle (BM) were detected on chicken chromosome 5 (GGA5) using two successive F₂ crosses between two divergently selected 'Fat' and 'Lean' INRA broiler lines. Based on these results, the aim of the present study was to identify the number, location and effects of these putative QTL by performing multitrait and multi-QTL analyses of the whole available data set. Data concerned 1186 F₂ offspring produced by 10 F₁ sires and 85 F₁ dams. AF and BM traits were measured on F₂ animals at slaughter, at 8 (first cross) or 9 (second cross) weeks of age. The F₀, F₁ and F₂ birds were genotyped for 11 microsatellite markers evenly spaced along GGA5. Before QTL detection, phenotypes were adjusted for the fixed effects of sex, F₂ design, hatching group within the design, and for body weight as a covariable. Univariate analyses confirmed the QTL segregation for AF and BM on GGA5 in male offspring, but not in female offspring. Analyses of male offspring data using multitrait and linked-QTL models led us to conclude the presence of two QTL on the distal part of GGA5, each controlling one trait. Linked QTL models were applied after correction of phenotypic values for the effects of these distal QTL. Several QTL for AF and BM were then discovered in the central region of GGA5, splitting one large QTL region for AF into several distinct QTL. Neither the 'Fat' nor the 'Lean' line appeared to be fixed for any QTL genotype. These results have important implications for prospective fine mapping studies and for the identification of underlying genes and causal mutations.     

Peroxidase and polyphenol oxidase activities in Cyperus esculentus leaves following glyphosate applications

Following a 2-week treatment with glyphosate [N-(phosphonomethyl)glycine] changes in peroxidase (EC 1.11.1.7) and polyphenol oxidase (PPO; EC 1.14.18.1) activities of yellow nutsedge ( Cyperus esculentus L.) plants, were determined. Glyphosate caused significant increases of both activities. Isoelectric focusing gave 3 species (F₁, F₂ and F₃) of peroxidase activity, at pl 3.8, 4.4 and 4.8, and 4 species (F_a, F_b, F_c and F_d) of PPO activity at pl 7.0, 7.5, 7.8 and 9.5. The activity of the 4 active forms of PPO increased with increasing glyphosate dose up to 10⁻² M . The effect of the herbicide on the 3 fractions with peroxidase activity was to change their relative activities. Highest F₁ activity was found in control plants whereas the F₂ fraction was the predominant form in the plants treated with glyphosate at 10⁻² M and the highest F₃ activity occurred in plants treated with 5 × 10⁻³ M glyphosate. The increased PPO activity could produce phytotoxic o -quinones, and variations in peroxidase isoenzymes activity could enhance isoperoxidases with lignin biosynthetic activity.     

ASYMMETRIC VIABILITY OF RECIPROCAL-CROSS HYBRIDS BETWEEN CRESTED AND MARBLED NEWTS (TRITURUS CRISTATUS AND T. MARMORATUS)

Hybridization between divergent lineages often results in reduced hybrid viability. Here we report findings from a series of independent molecular analyses over several seasons on four life stages of F₁ hybrids between the newts Triturus cristatus and T. marmoratus . These two species form a bimodal hybrid zone of broad overlap in France, with F₁ hybrids making up about 4% of the adult population. We demonstrate strong asymmetry in the direction of the cross, with one class ( cristatus -mothered) making up about 90% of F₁ hybrids. By analyzing embryos and hatchlings, we show that this asymmetry is not due to prezygotic effects, as both classes of hybrid embryos are present at similar frequencies, implicating differential selection on the two hybrid classes after hatching. Adult F₁ hybrids show a weak Haldane effect overall, with a 72% excess of females. The rarer marmoratus -mothered class, however, consists entirely of males. The absence of females from this class of adult F₁ hybrids is best explained by an incompatibility between the cristatus X chromosome and marmoratus cytoplasm. It is thus important to distinguish the two classes of reciprocal-cross hybrids before making general statements about whether Haldane's rule is observed.     

Possible Involvement of Interleukin-1 in Cyclooxygenase-2Induction After Spinal Cord Injury in Rats

Abstract : A standardized compression injury of rat spinal cord brought about a time-dependent biphasic production of thromboxane A₂ (detected as thromboxane B₂) and prostaglandin I₂ (detected as 6-ketoprostaglandin F_1α. Thromboxane B₂ was predominant during the first 1 h, whereas the 6-ketoprostaglandin F_1α level exceeded that of thromboxane B₂ at 8 h postinjury. As examined by inhibitor experiments and northern blotting, cyclooxygenase-1 was responsible for the first phase, and cyclooxygenase-2 was involved in the second phase. On compression injury the levels of interleukin-1α and -1β detected as mRNA and protein increased and peaked at 2-4 h. Injection of exogenous interleukin-1 α into the spinal cord resulted in an increase of cyclooxygenase-2 mRNA content and a predominant production of 6-ketoprostaglandin F_1α resembling the second phase of eicosanoid production. Concomitantly, extravascular migration of polymorphonuclear leukocytes was enhanced after the interleukin-1α injection. These cells together with vascular endothelial cells and glial cells were stained positively with an anti-cyclooxygenase-2 antibody. The results suggest that the immediate eicosanoid synthesis after spinal cord injury was due to the constitutive cyclooxygenase-1 and the delayed synthesis of eicosanoids was attributable to the induction of cyclooxygenase-2 mediated by interleukin-1 α.     

Time-resolved microfluorescence for measuring coenzyme F420 in Methanobacterium thermoautotrophicum

Abstract The time course of photobleaching and the nanosecond fluorescence decay have been measured from microscopic samples of methanogenic bacteria, to our knowledge the first application of these methods in this field. Decay times of about 1 ns and 3 ns were obtained for the specific coenzymes F₄₂₀ and 7-methylpterin, respectively. In contrast to methylpterin and other fluorescent compounds the intensity of F₄₂₀ fluorescence was reduced selectively due to photobleaching. This effect, as well as the different decay time constants could be used to discriminate F₄₂₀ from other fluorescent components. In addition, active and inactive bacterial cells could be differentiated following the course of photobleaching.     

Chloroplast ultrastructure in two Crassulacean species and an F1 hybrid with differing biomass δ13C values

Abstract. The chloroplasts of two species of the Crassulaceae and their F₁ hybrid were compared by electron microscopy. The two species had contrasting leaf tissue δ¹³C values of −25°/_∞ ( Sedum greggii ) and −13°/_∞ ( Cremnophila linguifolia ), and the F₁ hybrid had a value of − 18°/_∞. S. greggii had a mean of 8.9 thylakoids per granum in contrast to C. linguifolia which had a mean of only 3.8 thylakoids per granum. The F₁ hybrid had a mean of 6.4 thylakoids per granum. Crystaloids were observed in S. greggii and the hybrid but not in C. linguifolia     

Non-association between Rfp-Y major histocompatibility complex-like genes and susceptibility to Marek's disease virus-induced tumours in 63× 72 F2 intercross chickens

Marek's disease (MD) is a lymphoproliferative disease caused by a member of the herpesvirus family, and the best understood genetic resistance to MD involves the chicken major histocompatibility complex (MHC) B -complex. Preliminary observations have suggested that MHC-like Rfp-Y genes might also influence the incidence of MD. This study describes the differentiation and definition of unique Rfp-Y genes in inbred lines 6₃ and 7₂, lines that possess identical B -complex genes, but that are resistant or susceptible to MD, respectively. To assess if Rfp-Y genes affect susceptibility to MD, 265 6₃× 7₂ F₂ chickens were challenged with the JM strain of MD virus at 1 week of age and were evaluated for MD lesions at up to 10 weeks of age. Genotyping of the F₂ chickens for Rfp-Y haplotypes was performed by restriction fragment length polymorphism analysis of genomic DNA using Taq I and a B-FIV probe. Analysis of variance and interval mapping procedures were used to determine association between the Rfp-Y haplotypes and the phenotypic MD values of the F₂ chickens. The cosegregation analysis of 265 F₂ chickens indicated that there was no association between Rfp-Y haplotypes and MD susceptibility. Furthermore, the fact that the Rfp-Y haplotypes fit the 1:2:1 segregation ratio and the Rfp-Y allele frequencies did not differ significantly from 0·5 in the full population or in selected subpopulations (of either 40 MD-resistant or 39 MD-susceptible chickens) also indicated that Rfp-Y haplotypes do not significantly influence MD susceptibility. We conclude that Rfp-Y haplotypes do not play a major role in determining the genetic susceptibility to MD in 6₃× 7₂ F₂ White Leghorn chickens.