Common chelatase design in the branched tetrapyrrole pathways of heme and anaerobic cobalamin synthesis.

Prosthetic groups such as heme, chlorophyll, and cobalamin (vitamin B(12)) are characterized by their branched biosynthetic pathway and unique metal insertion steps. The metal ion chelatases can be broadly classed either as single-subunit ATP-independent enzymes, such as the anaerobic cobalt chelatase and the protoporphyrin IX (PPIX) ferrochelatase, or as heterotrimeric, ATP-dependent enzymes, such as the Mg chelatase involved in chlorophyll biosynthesis. The X-ray structure of the anaerobic cobalt chelatase from Salmonella typhimurium, CbiK, has been solved to 2.4 A resolution. Despite a lack of significant amino acid sequence similarity, the protein structure is homologous to that of Bacillus subtilis PPIX ferrochelatase. Both enzymes contain a histidine residue previously identified as the metal ion ligand, but CbiK contains a second histidine in place of the glutamic acid residue identified as a general base in PPIX ferrochelatase. Site-directed mutagenesis has confirmed a role for this histidine and a nearby glutamic acid in cobalt binding, modulating metal ion specificity as well as catalytic efficiency. Contrary to the predicted protoporphyrin binding site in PPIX ferrochelatase, the precorrin-2 binding site in CbiK is clearly defined within a large horizontal cleft between the N- and C-terminal domains. The structural similarity has implications for the understanding of the evolution of this branched biosynthetic pathway.     

Perceptual and neural olfactory similarity in honeybees

The question of whether or not neural activity patterns recorded in the olfactory centres of the brain correspond to olfactory perceptual measures remains unanswered. To address this question, we studied olfaction in honeybees Apis mellifera using the olfactory conditioning of the proboscis extension response. We conditioned bees to odours and tested generalisation responses to different odours. Sixteen odours were used, which varied both in their functional group (primary and secondary alcohols, aldehydes and ketones) and in their carbon-chain length (from six to nine carbons).The results obtained by presentation of a total of 16 × 16 odour pairs show that (i) all odorants presented could be learned, although acquisition was lower for short-chain ketones; (ii) generalisation varied depending both on the functional group and the carbon-chain length of odours trained; higher generalisation was found between long-chain than between short-chain molecules and between groups such as primary and secondary alcohols; (iii) for some odour pairs, cross-generalisation between odorants was asymmetric; (iv) a putative olfactory space could be defined for the honeybee with functional group and carbon-chain length as inner dimensions; (v) perceptual distances in such a space correlate well with physiological distances determined from optophysiological recordings of antennal lobe activity. We conclude that functional group and carbon-chain length are inner dimensions of the honeybee olfactory space and that neural activity in the antennal lobe reflects the perceptual quality of odours.     

Expression of polypeptide GalNAc-transferases in stratified epithelia and squamous cell carcinomas: immunohistological evaluation using monoclonal antibodies to three members of the GalNAc-transferase family

Mucin-type O-glycosylation is initiated by a large family of UDP- GalNAc: polypeptide N -acetyl-galactosaminyltransferases (GalNAc- transferases). Individual GalNAc-transferases appear to have different functions and Northern analysis indicates that they are differently expressed in different organs. This suggests that O-glycosylation may vary with the repertoire of GalNAc-transferases expressed in a given cell. In order to study the repertoire of GalNAc-transferases in situ in tissues and changes in tumors, we have generated a panel of monoclonal antibodies (MAbs) with well defined specificity for human GalNAc-T1, -T2, and -T3. Application of this panel of novel antibodies revealed that GalNAc- transferases are differentially expressed in different cell lines, in spermatozoa, and in oral mucosa and carcinomas. For example, GalNAc-T1 and -T2 but not -T3 were highly expressed in WI38 cells, and GalNAc-T3 but not GalNAc-T1 or -T2 was expressed in spermatozoa. The expression patterns in normal oral mucosa were found to vary with cell differentiation, and for GalNAc-T2 and -T3 this was reflected in oral squamous cell carcinomas. The expression pattern of GalNAc-T1 was on the other hand changed in tumors to either total loss or expression in cytological poorly differentiated tumor cells, where the normal undifferentiated cells lacked expression. These results demonstrate that the repertoire of GalNAc-transferases is different in different cell types and vary with cellular differentiation, and malignant transformation. The implication of this is not yet fully understood, but it suggests that specific changes in sites of O-glycosylation of proteins may occur as a result of changes in the repertoire of GalNAc-transferases.      

Knockdown of CENH3 in Arabidopsis reduces mitotic divisions and causes sterility by disturbed meiotic chromosome segregation

The histone H3 variant (CENH3) of centromeric nucleosomes is essential for kinetochore assembly and thus for chromosome segregation in eukaryotes. The mechanism(s) that determine centromere identity, assembly and maintenance of kinetochores are still poorly understood. Although the role of CENH3 during mitosis has been studied in several organisms, little is known about its meiotic function. We show that RNAi-mediated CENH3 knockdown in Arabidopsis thaliana caused dwarfism as the result of a reduced number of mitotic divisions. The remaining mitotic divisions appeared to be error-free. CENH3 RNAi transformants had reduced fertility because of frequently disturbed meiotic chromosome segregation. N-terminally truncated EYFP-CENH3(C) is deposited to and functional within Arabidopsis centromeres of mitotic chromosomes, but cannot be loaded onto centromeres of meiotic nuclei. Thus the N-terminal part is apparently required for CENH3 loading during meiosis. EYFP-CENH3(C) expression reduces the amount of endogenous CENH3, thus mimicking the effect of RNAi. The consequences of reduced endogenous CENH3 and lack of meiotic incorporation of EYFP-CENH3(C) are reduced fertility caused by insufficient CENH3 loading to the centromeres of meiotic chromosomes, subsequent lagging of chromosomes and formation of micronuclei.     

Carbon Dioxide Fixation in Roots and Nodules of Alnus glutinosa: I. Role of Phosphoenolpyruvate Carboxylase and Carbamyl Phosphate Synthetase in Dark CO(2) Fixation, Citrulline Synthesis, and N(2) Fixation

Detached roots and nodules of the N₂-fixing species, Albus glutinosa (European black alder), actively assimilate CO₂. The maximum rates of dark CO₂ fixation observed for detached nodules and roots were 15 and 3 micromoles CO₂ fixed per gram dry weight per hour, respectively. The net incorporation of CO₂ in these tissues was catalyzed by phosphoenolpyruvate carboxylase which produces organic acids, some of which are used in the synthesis of the amino acids, aspartate, glutamate, and citrulline and by carbamyl phosphate synthetase. The latter accounts for approximately 30 to 40% of the CO₂ fixed and provides carbamyl phosphate for the synthesis of citrulline. Results of labeling studies suggest that there are multiple pools of malate present in nodules. The major pool is apparently metabolically inactive and of unknown function while the smaller pool is rapidly utilized in the synthesis of amino acids. Dark CO₂ fixation and N₂ fixation in nodules decreased after treatment of nodulated plants with nitrate while the percentage of the total ¹⁴C incorporated into organic acids increased. Phosphoenolpyruvate carboxylase and carbamyl phosphate synthetase play key roles in the synthesis of amino acids including citrulline and in the metabolism of N₂-fixing nodules and roots of alder.     

C-banding pattern and polymorphism of Aegilops caudata and chromosomal constitutions of the amphiploid T. aestivum — Ae. caudata and six derived chromosome addition lines

Summary C-banding patterns were analysed in 19 different accessions of Aegilops caudata (= Ae. markgrafii, = Triticum dichasians) (2n = 14, genomically CC) from Turkey, Greece and the USSR, and a generalized C-banded karyotype was established. Chromosome specific C-bands are present in all C-genome chromosomes, allowing the identification of each of the seven chromosome pairs. While only minor variations in the C-banding pattern was observed within the accessions, a large amount of polymorphic variation was found between different accessions. C-banding analysis was carried out to identify Ae. caudata chromosomes in the amphiploid Triticum aestivum cv Alcedo — Ae. caudata and in six derived chromosome addition lines. The results show that the amphiploid carries the complete Ae. Caudate chromosome complement and that the addition lines I, II, III, IV, V and VIII carry the Ae. caudata chromosome pairs B, C, D, F, E and G, respectively. One of the two SAT chromosome pairs (A) is missing from the set. C-banding patterns of the added Ae. caudata chromosomes are identical to those present in the ancestor species, indicating that these chromosomes are not structurally rearranged. The results are discussed with respect to the homoeologous relationships of the Ae. caudata chromosomes.     

Double-strand break toxicity is chromatin context independent

Cells respond to double-strand breaks (DSBs) by activating DNA damage response pathways, including cell cycle arrest. We have previously shown that a single double-strand break generated via CRISPR/Cas9 is sufficient to delay cell cycle progression and compromise cell viability. However, we also found that the cellular response to DSBs can vary, independent of the number of lesions. This implies that not all DSBs are equally toxic, and raises the question if the location of a single double-strand break could influence its toxicity. To systematically investigate if DSB-location is a determinant of toxicity we performed a CRISPR/Cas9 screen targeting 6237 single sites in the human genome. Next, we developed a data-driven framework to design CRISPR/Cas9 sgRNA (crRNA) pools targeting specific chromatin features. The chromatin context was defined using ChromHMM states, Lamin-B1 DAM-iD, DNAseI hypersensitivity, and RNA-sequencing data. We computationally designed 6 distinct crRNA pools, each containing 10 crRNAs targeting the same chromatin state. We show that the toxicity of a DSB is highly similar across the different ChromHMM states. Rather, we find that the major determinants of toxicity of a sgRNA are cutting efficiency and off-target effects. Thus, chromatin features have little to no effect on the toxicity of a single CRISPR/Cas9-induced DSB.