Search for unstable DNA in schizophrenia families with evidence for genetic anticipation.

Evidence for genetic anticipation has recently become an important subject of research in clinical psychiatric genetics. Renewed interest in anticipation was evoked by molecular genetic findings of a novel type of mutation termed "unstable DNA." The unstable DNA model can be construed as the "best fit" for schizophrenia twin and family epidemiological data. We have performed a large-scale Southern blot hybridization, asymmetrical PCR-based, and repeat expansion-detection screening for (CAG)n/(CTG)n and (CCG)n/(CGG)n expansions in eastern Canadian schizophrenia multiplex families demonstrating genetic anticipation. There were no differences in (CAG)n/(CTG)n and (CCG)n/(CGG)n pattern distribution either between affected and unaffected individuals or across generations. Our findings do not support the hypothesis that large (CAG)n/(CTG)n or (CCG)n/(CGG)n expansions are the major etiologic factor in schizophrenia. A separate set of experiments directed to the analysis of small (30-130 trinucleotides), Huntington disease-type expansions in individual genes is required in order to fully exclude the presence of (CAG)n/(CTG)n- or (CCG)n/(CGG)n-type unstable mutation.     

Interspecific dominance does not exclude sub-dominant blennies from offshore petroleum platforms

Interspecific interactions can determine the abundance and distributions of animals. Seaweed blennies, Parablennius marmoreus, and tesselated blennies, Hypsoblennius invemar, are found in barnacle cavities on offshore petroleum platforms in the northern Gulf of Mexico. I measured the interspecific resource defense interactions between these fishes in aquaria. Seaweed blennies were dominant over tesselated blennies when equal-sized fishes were tested. No difference in dominance was found when tesselated blennies had a 10% advantage in size. However, tesselated blennies were able to successfully defend cavities against equal-sized competitors when given the advantage of prior residence. This prior residence advantage persisted despite seaweed blennies having the advantage of past experience. Seaweed blennies attain a larger size on petroleum platforms, but empty barnacle cavities are common in this environment. Tesselated blennies are able to colonize and successfully spawn because they can enter an empty barnacle cavity, gain the advantage of prior residence, and successfully defend this cavity.     

When no pollen does not mean no trees

The question of what signal, if any, appears in the pollen record when trees are present in the vegetation without producing pollen or with no pollen being recorded, is addressed. Four scenarios are envisaged: (i) the number of trees in the landscape are very few and scattered, (ii) the trees are too young to produce pollen, (iii) climate conditions are unfavourable for the trees to produce pollen and (iv) the trees are cut or damaged so that they do not flower. Each of these is considered in terms of pollen accumulation rates (PARs) and present theories and models of pollen dispersal. Examples are provided for the forest limit areas of the northern boreal trees in Finnish Lapland using data of pollen deposition monitored by pollen traps and results from the high temporal resolution (near annual) analyses of peat profiles. The relevance of the results to questions such as finds of spruce macrofossils in the Swedish mountains, the 8200 cal b.p. cold event, the migration of species/vegetation succession, and widespread damage to trees are all considered. It is concluded that although these situations are sometimes ‘invisible’ or misrepresented when pollen assemblages are expressed in the traditional percentage manner, they are often revealed by PARs. The fact that the pollen assemblage reflects a much wider regional area than is often understood can strengthen signals which have a regional impact, such as those which are climate induced, but may obscure events which affect only a limited spatial area or occur as small patches in the landscape.Communicated by Pim van der Knaap     

Habituation of plant cells does not mean insensitivity to plant growth regulators

Summary Fully habituated organogenic and nonorganogenic sugarbeet calluses reacted to application of the synthetic auxin [3-benzo(b) selenienyl] acetic acid by changes in growth and ethylene production. Treatment of fully habituated cells of periwinkle with 2,4-dichlorophenoxyacetic acid led to the decrease of free cytokinin contents (isopentenyl adenine, zeatin riboside, and zeatin) during the late exponential phase of growth. The polyamine contents were also modified and the capacity to biotransform secologanin into ajmalicine was decreased. Treatment of the habituated periwinkle cells with zeatin greatly increased the amount of a polypeptide of 16 kDa; this response was more marked than that displayed by the auxin-dependent line. These data show that hormone-independent calluses and cell suspensions can retain some sensitivity to growth hormones. However, differences of responses were observed between the auxin-dependent lines and the habituated lines.     

With selection for fecundity the mean fitness does not necessarily increase

A population with two alleles at one locus is considered. It is assumed that there is random mating of adults and that matings in which a particular pair of genotypes is involved may have a different mean number of offspring, or fecundity, than other types of matings. There is assumed to be no other selection. It is shown that the genotypic frequencies that maximize the mean fecundity of the population are not necessarily the same as the stable equilibrium frequencies. Thus, examples can be found for which the mean fecundity decreases from one generation to the next, and one such example is presented. An example in which there is no stable equilibrium, and the mean fecundity oscillates, is also given.     

Low abundance does not mean less importance in cysteine metabolism

The cysteine molecule plays an essential role in cells because it is part of proteins and because it functions as a reduced sulfur donor molecule. In addition, the cysteine molecule may also play a role in the redox signaling of different stress processes. Even though the synthesis of cysteine by the most abundant of the isoforms of O-acetylserine(thiol) lyase in the chloroplast, the mitochondria and the cytosol is relatively well-understood, the role of the other less common isoforms homologous to O-acetylserine(thiol)lyase is unknown. Several studies on two of these isoforms, one located in the cytosol and the other one in the chloroplast, have shown that while one isoform operates with a desulfhydrase activity and is essential to regulate the homeostasis of cysteine in the cytosol, the other, located in the chloroplast, synthesizes S-sulfocysteine. This metabolite appears to be essential for the redox regulation of the chloroplast under certain lighting conditions.Key words: cysteine, S-sulfocysteine, desulfhydrase, sulfur metabolism, redox regulation, ArabidopsisCysteine occupies a central position in the plant primary and secondary metabolism due to its biochemical functions. Cysteine is the first organic compound with reduced sulfur synthesized by the plant in the photosynthetic primary sulfate assimilation. The importance of cysteine for plants derives from its role as an amino acid in proteins but also because of its functions as a precursor for a huge number of essential bio-molecules, such as many plant defense compounds formed in response to different environmental adverse conditions.^1,2 All of these bio-molecules contain sulfur moieties that act as functional groups and are derived from cysteine, and therefore, are intimately linked via their biosynthetic pathways.In addition to the final destination of the reduced sulfur atom in the primary and secondary metabolism of cells, the thiol residue of the cysteine molecule is a functional group that translates the physico-chemical signal (redox) of ROS and RNS into a functional signal, altering the properties of small molecules such as GSH or proteins whose enzymatic or functional properties depend on the redox state of its cysteine residues.³Sulfate is the major sulfur form available to plants. Sulfate is taken up to plant cells through specific sulfate transporters and is activated to adenosine 5′-phosphosulfate (APS). The reduction of the activated sulfate form, APS, is linked to plastids and the photosynthetic activity; therefore, APS is reduced to sulfite by the APS reductase using two GSH molecules as donors of the two electrons required in this step. Sulfite is further reduced to sulfide by the sulfite reductase that uses photosynthetically reduced ferredoxine (Fd) as an electron donor of the six required electrons. The biosynthesis of cysteine is further accomplished by the sequential reaction of two enzymes: First, the serine acetyltransferase (SAT) synthesizes the intermediary product, O-acetylserine (OAS), from acetyl-CoA and serine; and second, the O-acetylserine(thiol)lyase (OASTL) incorporates the sulfide to OAS producing the cysteine. Recently, much progress has been made toward understanding the action of the O-acetylserine(thiol)lyase (OASTL) enzyme, one of the enzymes responsible for the biosynthesis of cysteine, using as a model system the plant Arabidopsis thaliana. The focus of the research has been mainly placed on the most abundant enzymes based on their involvement in the primary sulfate assimilation pathway. Biochemical and molecular analysis of the major OASTL knockout mutants in Arabidopsis thaliana revealed that part of the produced sulfide is incorporated to O-acetylserine to form cysteine in the chloroplast with the assistance of the OAS-B isoform. However, most of the chloroplastic sulfide overflows and escapes into the cytosol and the mitochondria, where it is also assimilated into cysteine by the OAS-A1 and OAS-C isoforms, respectively.^4–6The three major OASTL isoforms seem to be redundant under normal growth conditions. However, our investigations on the major cytosolic isoform, the OAS-A1, revealed new insights on the function of this enzyme as a determinant of the antioxidative capacity of the cytosol.⁷ The OASTL homolog, CYS-C1, exhibits OASTL activity, but in fact, it is a β-cyanoalanine synthase enzyme that uses cysteine to detoxify cyanide within the mitochondria.⁸ Furthermore, Arabidopsis cells contain four additional O-acetylserine(thiol)lyase isoforms encoded by the CYS-D1 (At3g04940), CYS-D2 (At5g28020), CS26 (At3g03630) and CS-LIKE (At5g28030) genes with unknown function. Are these four isoforms authentic OASTL and are, therefore, redundant enzymes or do they have different activities and, therefore, different functions?Our recent research on the less-common isoforms, CS-like and CS26, shed light on this issue, and we are decoding two important aspects of the sulfur metabolism in plants.^9,10 The CS-LIKE protein was identified by sequence homology upon the completion of the sequencing of the Arabidopsis genome. Because of its cytosolic localization, it is thought to have an auxiliary function with respect to the major cytosolic isoform, the OAS-A1. The characterization of the purified recombinant protein has shown that the CS-LIKE isoform catalyzes the desulfuration of L-cysteine to sulfide plus ammonia and pyruvate; thus, CS-LIKE is a novel L-cysteine desulfhydrase (EC 4.4.1.1), and it is designated as DES1 (Fig. 1). This enzyme is important for maintaining the homeostasis of cysteine in the cell, and the loss of function of this protein in knockout mutant plants results in higher levels of cysteine and glutathione. This increased level of soluble thiols results also in a higher antioxidant capacity of the plant, which, in turn, becomes more resistant to abiotic stress phenomena such as the presence of heavy metals or hydrogen peroxide. This observation may indicate that the regulation of this enzyme may be a key component of the plant physiological processes that involve redox reactions. Cytosolic cysteine degrading enzymes with desulfhydrase activity has been found in plants, but the protein responsible for this activity remained unisolated until now that it is revealed with our investigation on DES1.¹¹ From the standpoint of biotechnology, plants with this modified enzyme may result in abiotic stress-resistant lines that deserve to be studied.Open in a separate windowFigure 1Biosynthesis of cysteine and S-sulfocysteine in the chloroplast and cytosol of Arabidopsis and subcellular localization of the responsible enzymes. The cytosolic and plastidial O-acetylserine(thiol)lyase, L-cysteine desulfhydrase and S-sulfocysteine synthase are shown in red. A single representative of a grana thylakoid is shown as a grey oval compartment.The other less common enzyme studied, called CS26 and localized in the chloroplast, has proved to be an enzyme with S-sulfocysteine synthase activity.¹⁰ This enzyme synthesizes the incorporation of thiosulfate to O-acetylserine to form S-sulfocysteine (RSSO₃⁻). This activity, discovered for the first time in plants, was previously reported in bacteria where the biosynthesis of cysteine can be accomplished by two enzymes encoded by the cysK and cysM genes.^12,13 This enzyme activity is essential for the chloroplast function under long-day growing conditions but seems to be superfluous under short-day conditions. Morphologic and biochemical phenotype comparisons of the knockout oas-b and cs26 highlight the importance of the metabolite S-sulfocysteine and not the cysteine in the redox control of the chloroplast. Under long-day growth conditions, the cs26 mutants exhibit a reduction in size and show leaf paleness, have reductions in the chlorophyll content and photosynthetic activity, and are not able to properly detoxify reactive oxygen species, which are accumulated to high levels. None of these changes are observed in the oas-b mutant.Although we do not know the function of the S-sulfocysteine molecule in the chloroplast, two aspects are important to note. On the one hand, the enzyme CS26 can be located in the chloroplast''s lumen in opposition to the enzyme OAS-B, which is located in the stroma. The second aspect is the difference in chemical reactivity of S-sulfocysteine and cysteine. The S-sulfocysteine has two sulfur atoms with different degrees of oxidation, −1 and +5; therefore, it may act as an oxidant molecule by reacting with reduced thiols forming a disulfide bridge and releasing sulfite.¹⁴ We have suggested that a putative target of S-sulfocysteine can be the STN7 kinase, which contains a transmembrane region that separates its catalytic kinase domain on the stromal side from its N-terminal end in the thylakoid lumen with two conserved cysteines that are critical for its activity. A disulfide bridge between these two cysteines is required for the kinase activity, but how the redox states of these two cysteines are regulated in the lumen remains an open question.¹⁵ In general, how the thiol oxidation of proteins located in the thylakoid lumen takes place is still unclear because no sulfhydryl oxidases have been identified in this compartment. In fact, this process is highly important because the chaperones and peptidyl-prolyl cis-trans isomerases, such as the AtFKBP13, need to be oxidized in order to be functional in the lumen and to regulate the folding of the Rieske protein.^16–18     

Tetramethylammonium does not universally neutralize sequence dependent DNA stability.

Effects of different concentrations of Tetramethylammonium on the thermostability of six DNA dumbbells with similar well defined sequences have been investigated. Each molecule has a 16 base pair duplex stem linked on both ends by T4 single strand loops. Only the sequence of the four central base pairs distinguishes one molecule from the next. The distinguishing central sequences are, [A-T-A-T], [T-A-T-A], [A-A-A-A], [C-G-C-G], [G-C-G-C] and [G-G-G-G] situated between the sequences: 5'-G-T-A-T-C-C-[]-G-G-A-T-A-C-3' which are the same in all molecules. Optical melting curves collected on these molecules as a function of TMA concentration over the range from 0.09 M to 4.5 M revealed there is no single concentration of TMA where all these molecules exhibit the same melting temperature.     

Eukaryotic DNA does not form nucleosomes as readily as some prokaryotic DNA.

Nucleosomal-length DNA was prepared from the genomic DNA of various prokaryotic and eukaryotic organisms by limited nuclease digestion after reconstitution with core histones. The DNAs ranged in base composition from 26.5% to 72% guanosine-plus-cytosine (%GC). The nucleosomal-length DNAs were then used in a competitive reconstitution assay in order to quantitatively determine their relative abilities to form nucleosomes. The results of the assay indicate a linear dependence of the free energy of nucleosome formation on base composition and, surprisingly, show that several prokaryotic DNAs form nucleosomes as well as or better than eukaryotic DNAs.     

Reproductive CRISPR does not cure disease

Given recent advancements in CRISPR‐Cas9 powered genetic modification of gametes and embryos, both popular media and scientific articles are hailing CRISPR’s life‐saving, curative potential for people with serious monogenic diseases. But claims that CRISPR modification of gametes or embryos, a form of germline engineering, has therapeutic value are deeply mistaken. This article explains why reproductive uses of CRISPR, and germline engineering more generally, do not treat or save lives that would otherwise have a genetic disease. Reproductive uses of CRISPR create healthy people whose existence is not inevitable in the first place. Creating healthy lives has distinct and lesser moral value from saving or curing lives that would otherwise have genetic disease. The real value in reproductive uses of CRISPR is in helping a very limited population of people have healthy, genetically related children. This diminished value cannot compete with the concerns in opposition to germline engineering, nor is it worth the investment of research money.     

Caged DNA does not aggregate in high ionic strength solutions.

The assembly of DNA into compact particles that do not aggregate in physiologic salt solution occurs naturally in chromatin and viral particles but has been challenging to duplicate using artificial constructs. Cross-linking amino-containing polycations in the presence of DNA with bisimidoester cross-linker leads to the formation of caged DNA particles that are stable in salt solutions. This first demonstration of caged DNA provides insight into how natural condensation processes avoid aggregation and a promising avenue for developing nonviral gene therapy vectors.