Sequence comparisons of non-allelic late histone genes and their early stage counterparts. Evidence for gene conversion within the sea urchin late stage gene family

We have determined the nucleotide sequence of sea urchin (Lytechinus pictus) late stage H3 and H4 histone genes contained on the clone pLpH3H4 -21 and of the early stage H3 gene contained on the plasmid pLpA . Comparison of these differentially regulated histone genes with each other and with other L. pictus late and early stage histone H3 and H4 genes previously sequenced confirms that members of each histone gene family (early and late) are more homologous to each other than they are to members of other histone gene families. The spacer regions between two late H3-H4 gene pairs on the clones pLpH3H4 -19 and pLpH3H4 -21 have diverged to the point where they are no longer homologous. However, comparative analysis of the 5' flanking DNA has identified a sequence 5'C-T-C-A-T-G-T-A-T-T3' upstream of both late H4 genes and another, 5'A-G-A-T-T-C-A3', upstream of both H3 genes. Except for a short conserved sequence near the initiation codon, the transcribed 5' leaders of the late mRNAs differ in length and sequence in the two non-allelic late histone gene pairs. This divergence contrasts with the 95 to 96% conservation found between late histone gene coding sequences. The results suggest that there is intergenic exchange in the germline among members of the late histone gene family and that the unit of exchange is the individual gene rather than the heterotypic dimer which includes the common spacer DNA.     

Bovine serum conglutinin is a lectin which binds non-reducing terminal N-acetylglucosamine, mannose and fucose residues.

Carbohydrate recognition by bovine serum conglutinin has been investigated by inhibition and direct binding assays using glycoproteins and polysaccharides from Saccharomyces cerevisiae (baker's yeast), and neoglycolipids derived from N-acetylglucosamine oligomers, mannobiose and human milk oligosaccharides. The results clearly show that conglutinin is a lectin which binds terminal N-acetylglucosamine, mannose and fucose residues as found in chitobiose (GlcNAc beta 1-4GlcNAc), mannobiose (Man alpha 1-3Man) and lacto-N-fucopentaose II [Fuc alpha 1-4(Gal beta 1-3)GlcNAc beta 1-3Gal beta 1-4Glc] respectively.     

A new family of tandem repetitive early histone genes in the sea urchin Lytechinus pictus: evidence for concerted evolution within tandem arrays.

We have isolated and characterized a third nonallelic tandemly arrayed histone cluster (LpE) from the sea urchin Lytechinus pictus. Although this tandem array is not intermingled with the other two early histone gene families also found in the L. pictus genome, the order and polarity of the five histone coding sequences in this family are the same as every other well characterized sea urchin early histone gene family. Heteroduplex analysis and restriction endonuclease mapping experiments indicate that the LpE family is more closely related to the B-C than the A-D family of early histone genes. Examination of several individual sperm DNA samples has revealed considerable polymorphism in each of the three tandem repeat families. Within an individual, however, each family is remarkably homogeneous. Thus, our results indicate that rapid fixation of variants acts to homogenize the members of a single tandem array at a considerably faster rate within a family than between families. However, at least some exchange of sequences between families is evident based on the conservation of many restriction endonuclease recognition sites and from analysis of a a cosmid clone in which the A-D and E tandem repeats are found adjacent to one another. These differences in the rate of fixation of variants within and between these families are likely to be responsible for the maintenance of diversity between the different families.     

Multiple blood feeding in mosquitoes shortens the Plasmodium falciparum incubation period and increases malaria transmission potential

Many mosquito species, including the major malaria vector Anopheles gambiae, naturally undergo multiple reproductive cycles of blood feeding, egg development and egg laying in their lifespan. Such complex mosquito behavior is regularly overlooked when mosquitoes are experimentally infected with malaria parasites, limiting our ability to accurately describe potential effects on transmission. Here, we examine how Plasmodium falciparum development and transmission potential is impacted when infected mosquitoes feed an additional time. We measured P. falciparum oocyst size and performed sporozoite time course analyses to determine the parasite’s extrinsic incubation period (EIP), i.e. the time required by parasites to reach infectious sporozoite stages, in An. gambiae females blood fed either once or twice. An additional blood feed at 3 days post infection drastically accelerates oocyst growth rates, causing earlier sporozoite accumulation in the salivary glands, thereby shortening the EIP (reduction of 2.3 ± 0.4 days). Moreover, parasite growth is further accelerated in transgenic mosquitoes with reduced reproductive capacity, which mimic genetic modifications currently proposed in population suppression gene drives. We incorporate our shortened EIP values into a measure of transmission potential, the basic reproduction number R₀, and find the average R₀ is higher (range: 10.1%–12.1% increase) across sub-Saharan Africa than when using traditional EIP measurements. These data suggest that malaria elimination may be substantially more challenging and that younger mosquitoes or those with reduced reproductive ability may provide a larger contribution to infection than currently believed. Our findings have profound implications for current and future mosquito control interventions.     

An electrophoretic method for detecting hypoxanthine-guanine phosphoribosyl transferase variants

An electrophoretic method that detects hypoxanthine-guanine phosphoribosyl transferase variants is described.Aided by National Institutes of Health Grants HD 00486, HD 00004, GM 14155, and AM 10813.     

Unravelling Glucan Recognition Systems by Glycome Microarrays Using the Designer Approach and Mass Spectrometry

Glucans are polymers of d-glucose with differing linkages in linear or branched sequences. They are constituents of microbial and plant cell-walls and involved in important bio-recognition processes, including immunomodulation, anticancer activities, pathogen virulence, and plant cell-wall biodegradation. Translational possibilities for these activities in medicine and biotechnology are considerable. High-throughput micro-methods are needed to screen proteins for recognition of specific glucan sequences as a lead to structure–function studies and their exploitation. We describe construction of a “glucome” microarray, the first sequence-defined glycome-scale microarray, using a “designer” approach from targeted ligand-bearing glucans in conjunction with a novel high-sensitivity mass spectrometric sequencing method, as a screening tool to assign glucan recognition motifs. The glucome microarray comprises 153 oligosaccharide probes with high purity, representing major sequences in glucans. Negative-ion electrospray tandem mass spectrometry with collision-induced dissociation was used for complete linkage analysis of gluco-oligosaccharides in linear “homo” and “hetero” and branched sequences. The system is validated using antibodies and carbohydrate-binding modules known to target α- or β-glucans in different biological contexts, extending knowledge on their specificities, and applied to reveal new information on glucan recognition by two signaling molecules of the immune system against pathogens: Dectin-1 and DC-SIGN. The sequencing of the glucan oligosaccharides by the MS method and their interrogation on the microarrays provides detailed information on linkage, sequence and chain length requirements of glucan-recognizing proteins, and are a sensitive means of revealing unsuspected sequences in the polysaccharides.Glucan polysaccharides are polymers of d-glucose with differing linkages in linear or branched sequences. They occur as storage materials in animals, secreted virulence factors of bacteria, and conserved structural components of cell walls of yeasts, fungi, some bacteria, and plants. Polysaccharides of this type are of considerable interest in biology, medicine, and biotechnology and are acknowledged for their immunostimulatory, anticancer, and health-promoting activities (1, 2); for their elicitor activities in defense responses and signaling in plants (3); and for acting as functional ingredients in human nutrition (4). Unraveling recognition systems that mediate these activities is highly desirable as a lead to effective translational applications.Recognition systems involving glucan polysaccharides include those in mammals, such as recognition of fungal β-glucans by Dectin-1, the major receptor of the innate immune system against fungal pathogens (5), and by natural or vaccine-induced protective antifungal antibodies (6, 7); also recognition of mycobacterial α-glucan by the innate immune receptor DC-SIGN (dendritic cell-specific ICAM-3-grabbing nonintegrin) (8); those in insects, such as the Drosophila Gram-negative binding protein 3 (GNBP3) sensor protein, which binds β-glucans (9); and those in bacteria, such as Brucella abortus, where cyclic β-glucans can serve as virulence factors (10).Another important class of glucan-recognizing proteins comprises noncatalytic carbohydrate-binding modules (CBMs)¹ of bacterial glycoside hydrolases that mediate association with substrate and increase catalytic activity, likely through a targeting mechanism or by driving enzyme specificity (11, 12). Notable examples are CBMs of bacterial cellulolytic enzymes that promote enzymatic deconstruction of intact plant cell walls and that are of industrial significance in the biofuel and bioprocessing sectors (13, 14) and CBMs of rumen or commensal human microbiota with roles in animal and human health (14, 15). CBMs also have roles in other systems: for example, CBM-containing enzymes as virulence factors of bacterial pathogens (16) and CBM-containing human laforin that regulates glycogen metabolism and for which mutations can lead to neurodegenerative disease (17). The number of putative glucan-binding CBMs that have been identified and classified in the Carbohydrate-Active enZyme (CAZy) database (http://www.cazy.org) is expanding, but relatively few have been experimentally investigated for details of carbohydrate binding and fine specificity (11).Searching for and assigning the specificities of glucan-recognizing proteins has thus become increasingly important. It is desirable to have high-throughput and sensitive micro-methods to screen for and characterize ligands for structure–function studies toward effective exploitation in modern therapeutic, nutritional, agricultural, and biofuel-related technologies. Carbohydrate microarrays have served to advance knowledge on specificities of diverse carbohydrate-recognition systems (18–22). Where the desired oligosaccharide probes are unavailable, microarrays need to be generated from ligand-bearing glycomes (23). Using a prototype of such designer microarrays of neoglycolipid (NGL)-probes (23) derived from oligosaccharide fragments of glucans rich in β1,3- or β1,6-linked sequences, we showed that linear β1,3-linked glucose sequences with degree of polymerization (DP) 10 or longer are bound by Dectin-1 (24). Recognition of other types of glucan sequences by Dectin-1 and the applicability of microarrays of diverse gluco-oligosaccharide sequences to other glucan-recognizing proteins required investigation. Cummings, Smith, and colleagues have developed the shotgun strategy (20) to create glycome-scale “gangliome” and “human milk glycome” microarrays. In the shotgun microarrays, the printed probes may not be sequence-defined before array construction and require metadata-assisted glycan sequencing (MAGS), which combines MS analysis (25), binding data with glycan-binding proteins or antibodies, and exoglycosidase treatment after printing (26, 27).Mass spectrometry has become a primary technique in carbohydrate structural analysis (28), and electrospray mass spectrometry (ESI-MS) has been used to provide sequence and partial linkage information on various types of oligosaccharides (29–33). For neutral oligosaccharides, we have found that tandem MS with collision-induced dissociation (CID-MS/MS) in the negative-ion mode is particularly useful and have successfully applied for oligosaccharide chain and blood-group typing (34, 35) and for branching pattern analysis (36).This is because that some important linkages at certain monosaccharide residues can be unambiguously determined with high sensitivity without the need for derivatization and anion complexation as previously recognized, e.g. in the area of gluco-oligosaccharides, Cl⁻-anion adduction has been used to determine sequences of tetrasaccharides of dextran (37).Here, we describe a strategy using the designer approach combined with negative-ion ESI-CID-MS/MS for constructing a microarray of sequence-defined gluco-oligosaccharides representing major sequences in glucans (glucome microarray) as a tool for screening glucan-recognizing proteins and assigning their recognition motifs (Fig. 1). We selected a comprehensive panel of glucan polysaccharides isolated from plants, fungi, and bacteria with different sequences to represent the glucome. We used finely tuned chemical and enzymatic methods to partially depolymerize the polysaccharides and prepare gluco-oligosaccharide fragments with different chain lengths (up to DP-13 or DP-16). We developed a ESI-CID-MS/MS method that enables linkage and sequence determination of linear or branched gluco-oligosaccharides at high-sensitivity and applied this to the sequencing of oligosaccharide fragments prepared. These sequence-defined gluco-oligosaccharides were then converted into NGL probes and used for construction of the microarray. The oligosaccharides encompassed linear sequences with homo (single) linkages: 1,2-, 1,3-, 1,4-, or 1,6- with α or β configurations; and hetero (multiple) linkages: 1,3-, 1,4, or 1,6-; also branched oligosaccharide sequences with 1,3 and 1,6-linkages.Open in a separate windowFig. 1.Neoglycolipid (NGL)-based designer glucome microarray with mass spectrometry as a tool to assign carbohydrate ligands in glucan recognition. Ligand-bearing glucan polysaccharides, described in supplemental Fig. S1 and Table S1, were selected as sources of gluco-oligosaccharides for construction of the microarray. A total of 121 gluco-oligosaccharide fractions were obtained with different DP after partial depolymerization of polysaccharides and fractionation. ESI-CID-MS/MS method was developed using gluco-oligosaccharides with known sequences and applied to determination of sequences of oligosaccharide fragments from polysaccharides. Gluco-oligosaccharides were converted to NGL probes for microarray construction and interrogation with the glucan-recognizing proteins described in supplemental Table S2.To our knowledge, this is the first sequence-defined glycome-scale microarray constructed. We used 12 selected proteins (antibodies and CBMs) known to target α- or β-glucans to validate the approach. We then applied the microarray analysis to Dectin-1 and DC-SIGN, which revealed new insights into the specificities of these signaling molecules of the innate immune system.     

Multiscale model of CRISPR-induced coevolutionary dynamics: diversification at the interface of Lamarck and Darwin

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system is a recently discovered type of adaptive immune defense in bacteria and archaea that functions via directed incorporation of viral and plasmid DNA into host genomes. Here, we introduce a multiscale model of dynamic coevolution between hosts and viruses in an ecological context that incorporates CRISPR immunity principles. We analyze the model to test whether and how CRISPR immunity induces host and viral diversification and the maintenance of many coexisting strains. We show that hosts and viruses coevolve to form highly diverse communities. We observe the punctuated replacement of existent strains, such that populations have very low similarity compared over the long term. However, in the short term, we observe evolutionary dynamics consistent with both incomplete selective sweeps of novel strains (as single strains and coalitions) and the recurrence of previously rare strains. Coalitions of multiple dominant host strains are predicted to arise because host strains can have nearly identical immune phenotypes mediated by CRISPR defense albeit with different genotypes. We close by discussing how our explicit eco-evolutionary model of CRISPR immunity can help guide efforts to understand the drivers of diversity seen in microbial communities where CRISPR systems are active.