A group of α-1,4-glucan lyase genes from the fungi Morchella costata,M. vulgaris and Peziza ostracoderma. Cloning,complete sequencing and heterologous expression

We here report genes encoding a newly discovered class of starch- and glycogen-degrading enzyme, -1,4-glucan lyase (EC 4.2.2.13), which degrades starch and glycogen to 1,5-anhydro-D-fructose. Two lyases were purified and partially sequenced from the macrofungi Morchella costata and M. vulgaris. The obtained lyase amino acid sequences were used to generate PCR primers, which were further used to probe the fungal genomic libraries. Two lyase genes (Agll1;Mo.cos and Agll1;Mo.vul) from the two fungi were fully sequenced and found to contain a coding region of 3201 bp and 3213 bp, respectively. A total of 13 small introns were found in each of the two genes with identical positions. The two lyase genes share 86% identity at the amino acid level. They encode mature lyases with 1066 and 1070 amino acids, respectively. The deduced molecular masses of 121530 and 121971 Da agree with the values found for the two purified lyases. A structure analysis of the promoter regions of the lyase genes revealed a number of putative regulatory DNA elements, such as the AREA and CREA sites, which are related to nitrogen and carbon metabolism, respectively, and the CCAAT/CAAT boxes, which are related to basal expression of genes. A third lyase gene (Agll1;Pe.ost) from the fungus Peziza ostracoderma was partially sequenced to 557 bp. The amino acid sequence deduced from this nucleotide fragment shares 76% identity with the M. costata lyase. Heterologous expression of the M. costata lyase gene was achieved intracellularly in Pichia pastoris and Aspergillus niger.     

Positive supercoiling is generated in the presence of Escherichia coli SeqA protein

In Escherichia coli, the SeqA protein is known as a negative regulator of chromosome replication. This protein is also suggested to have a role in chromosome organization. SeqA preferentially binds to hemi-methylated DNA and is by immunofluorescence microscopy seen as foci situated at the replication factories. Loss of SeqA leads to increased negative supercoiling of the DNA. We show that purified SeqA protein bound to fully methylated, covalently closed or nicked circular DNA generates positive supercoils in vitro in the presence of topoisomerase I or ligase respectively. This means that binding of SeqA changes either the twist or the writhe of the DNA. The ability to affect the topology of DNA suggests that SeqA may take part in the organization of the chromosome in vivo. The topology change performed by SeqA occurred also on unmethylated plasmids. It is, however, reasonable to suppose that in vivo the major part of such activity is performed on hemi-methylated DNA at the replication factories and presumably forms the basis for the characteristic SeqA foci observed by fluorescence microscopy.     

Active MHC class Ib genes in rat are pseudogenes in the mouse

The most telomeric class I region of the MHC in rat and mouse is the M region, which contains about 20 class I genes or gene fragments. The central part carries three class I genes—M4, M5, and M6—which are orthologous between the two species. M4 and M6 are pseudogenes in the mouse but transcribed, intact genes in the rat. To analyze the pseudogene status for the mouse genes in more detail, we have sequenced the respective exons in multiple representative haplotypes. The stop codons are conserved in all mouse strains analyzed, and, consistent with the pseudogene status, all strains show additional insertions and deletions, taking the genes further away from functionality. Thus, M4 and M6 indeed have a split status. They are silent in the mouse but intact in the closely related rodent, the rat.GenBank accession numbers: AF057065 to AF057072 (exon 3 of H2-M4 of reported mouse strains), AF057976 to AF057985 (exon 3 of RT1.M4 of reported rat strains), AF058923 and AF058924 (exon 2 of RT1.M4 of strains PVG and BN), AY286080 to AY286092 (exon 4 of H2-M6 of reported mouse stains), and AY303772 (full-length genomic sequence of RT1.M6-1^l)     

Cyanobacterial protease inhibitor microviridin J causes a lethal molting disruption in Daphnia pulicaria

Laboratory experiments identified microviridin J as the source of a fatal molting disruption in Daphnia species organisms feeding on Microcystis cells. The molting disruption was presumably linked to the inhibitory effect of microviridin J on daphnid proteases, suggesting that hundreds of further cyanobacterial protease inhibitors must be considered potentially toxic to zooplankton.     

Structural features and mechanisms for sensing high osmolarity in microorganisms

During their lifetime, most organisms experience osmotic stress, mostly due to fluctuating external osmolarities, but also as a result of desiccation or freezing. Under these conditions, the ratio of osmolytes to water and macromolecules in the cells is significantly altered. To survive, cells must continuously sense these alterations and adapt accordingly. Osmolarity is a physico-chemical parameter that causes pleiotropic alterations in cell physiology. Recent research has revealed various mechanisms to sense high external osmolarity, based on monitoring cellular changes that are associated with the altered environment.     

Optimized expression of Plasmodium falciparum erythrocyte membrane protein 1 domains in Escherichia coli

BACKGROUND: Removal of exhaled air from total body emanations or artificially standardising carbon dioxide (CO2) outputs has previously been shown to eliminate differential attractiveness of humans to certain blackfly (Simuliidae) and mosquito (Culicidae) species. Whether or not breath contributes to between-person differences in relative attractiveness to the highly anthropophilic malaria vector Anopheles gambiae sensu stricto remains unknown and was the focus of the present study. METHODS: The contribution to and possible interaction of breath (BR) and body odours (BO) in the attraction of An. gambiae s.s. to humans was investigated by conducting dual choice tests using a recently developed olfactometer. Either one or two human subjects were used as bait. The single person experiments compared the attractiveness of a person's BR versus that person's BO or a control (empty tent with no odour). His BO and total emanations (TE = BR+BO) were also compared with a control. The two-person experiments compared the relative attractiveness of their TE, BO or BR, and the TE of each person against the BO of the other. RESULTS: Experiments with one human subject (P1) as bait found that his BO and TE collected more mosquitoes than the control (P = 0.005 and P < 0.001, respectively), as did his BO and the control versus his BR (P < 0.001 and P = 0.034, respectively). The TE of P1 attracted more mosquitoes than that of another person designated P8 (P < 0.021), whereas the BR of P8 attracted more mosquitoes than the BR of P1 (P = 0.001). The attractiveness of the BO of P1 versus the BO of P8 did not differ (P = 0.346). The BO from either individual was consistently more attractive than the TE from the other (P < 0.001). CONCLUSIONS: We demonstrated for the first time that human breath, although known to contain semiochemicals that elicit behavioural and/or electrophysiological responses (CO2, ammonia, fatty acids) in An. gambiae also contains one or more constituents with allomonal (~repellent) properties, which inhibit attraction and may serve as an important contributor to between-person differences in the relative attractiveness of humans to this important malaria vector.     

Laser light-scattering evidence for an altered association of beta B1-crystallin deamidated in the connecting peptide

Deamidation is a prevalent modification of crystallin proteins in the vertebrate lens. The effect of specific sites of deamidation on crystallin stability in vivo is not known. Using mass spectrometry, a previously unreported deamidation in beta B1-crystallin was identified at Gln146. Another deamidation was investigated at Asn157. It was determined that whole soluble beta B1 contained 13%-17% deamidation at Gln146 and Asn157. Static and quasi-elastic laser light scattering, circular dichroism, and heat aggregation studies were used to explore the structure and associative properties of recombinantly expressed wild-type (wt) beta B1 and the deamidated beta B1 mutants, Q146E and N157D. Dimer formation occurred for wt beta B1, Q146E, and N157D in a concentration-dependent manner, but only Q146E showed formation of higher ordered oligomers at the concentrations studied. Deamidation at Gln146, but not Asn157, led to an increased tendency of beta B1 to aggregate upon heating. We conclude that deamidation creates unique effects depending upon where the deamidation is introduced in the crystallin structure.