Genetic construction and functional analysis of hybrid polyketide synthases containing heterologous acyl carrier proteins.

The gene that encodes the acyl carrier protein (ACP) of the actinorhodin polyketide synthase (PKS) of Streptomyces coelicolor A3(2) was replaced with homologs from the granaticin, oxytetracycline, tetracenomycin, and putative frenolicin polyketide synthase gene clusters. All of the replacements led to expression of functional synthases, and the recombinants synthesized aromatic polyketides similar in chromatographic properties to actinorhodin or to shunt products produced by mutants defective in the actinorhodin pathway. Some regions within the ACP were also shown to be interchangeable and allow production of a functional hybrid ACP. Structural analysis of the most abundant polyketide product of one of the recombinants by electrospray mass spectrometry suggested that it is identical to mutactin, a previously characterized shunt product of an actVII mutant (deficient in cyclase and dehydrase activities). Quantitative differences in the product profiles of strains that express the various hybrid synthases were observed. These can be explained, at least in part, by differences in ribosome-binding sites upstream of each ACP gene, implying either that the ACP concentration in some strains is rate limiting to overall PKS activity or that the level of ACP expression also influences the expression of another enzyme(s) encoded by a downstream gene(s) in the same operon as the actinorhodin ACP gene. These results reaffirm the idea that construction of hybrid polyketide synthases will be a useful approach for dissecting the molecular basis of the specificity of PKS-catalyzed reactions. However, they also point to the need for reducing the chemical complexity of the approach by minimizing the diversity of polyketide products synthesized in strains that produce recombinant polyketide synthases.     

Exploring the legacy of African and Indigenous Caribbean admixture in Puerto Rico

Objectives

From an anthropological genetic perspective, little is known about the ethnogenesis of African descendants in Puerto Rico. Furthermore, historical interactions between Indigenous Caribbean and African descendant peoples that may be reflected in the ancestry of contemporary populations are understudied. Given this dearth of genetic research and the precedence for Afro-Indigenous interactions documented by historical, archeological, and other lines of evidence, we sought to assess the biogeographic origins of African descendant Puerto Ricans and to query the potential for Indigenous ancestry within this community.

Materials and Methods

Saliva samples were collected from 58 self-identified African descendant Puerto Ricans residing in Puerto Rico. We sequenced whole mitochondrial genomes and genotyped Y chromosome haplogroups for each male individual (n = 25). Summary statistics, comparative analyses, and network analysis were used to assess diversity and variation in haplogroup distribution between the sample and comparative populations.

Results

As indicated by mitochondrial haplogroups, 66% had African, 5% had European, and 29% had Indigenous American matrilines. Along the Y chromosome, 52% had African, 28% had Western European, 16% had Eurasian, and, notably, 4% had Indigenous American patrilines. Both mitochondrial and Y chromosome haplogroup frequencies were significantly different from several comparative populations.

Discussion

Biogeographic origins are consistent with historical accounts of African, Indigenous American, and European ancestry. However, this first report of Indigenous American paternal ancestry in Puerto Rico suggests distinctive features within African descendant communities on the island. Future studies expanding sampling and incorporating higher resolution genetic markers are necessary to more fully understand African descendant history in Puerto Rico.     

Trophic structure in open waters of the marginal ice zone in the Scotia-Weddell confluence region during spring (1983)

The structure of the food web was investigated in open waters adjacent to the marginal ice zone in the southern Scotia Sea in spring 1983. Diets were defined for dominant zooplankton, micronekton, and flying seabird species and then aggregated by cluster analysis into feeding groups. Most zooplankton were omnivorous, feeding on phytoplankton, protozoans, and in some cases, small metazoans (copepods). Only two species were found to be exclusively herbivorous:Calanoides acutus andRhincalanus gigas. Micronekton were carnivores with copepods being the dominant prey in all their diets. The midwater fishElectrona antarctica was the dominant food item in seven of the nine seabird species examined. Cephalopods, midwater decapod shrimps and carrion were also important in the diets of a few seabird species. Comparison (cluster analysis) of diets in spring with other seasons (winter, fall) indicated that over half the species examined (18 of 31) had similar diets in all seasons tested. The significant intraspecific shifts in diet that did occur were attributable to regional, seasonal, and interannual effects. A scheme is presented that describes the major energetic pathways through the open water ecosystem from phytoplankton to apex predators. At the base are phytoplankton and protozoans which are the principal food resource for the biomass copepods and krill. Krill and the biomass copepods are the principal forage of the midwater fishElectrona antarctica which, in turn, is the central diet component of flying seabirds as well as important food for the Antarctic fur seal and cephalopods. Krill are a major diet element for the fur seal and cephalopods, and the principal food of the minke whale.     

A chromosome-level genome assembly enables the identification of the follicule stimulating hormone receptor as the master sex-determining gene in the flatfish Solea senegalensis

Sex determination (SD) shows huge variation among fish and a high evolutionary rate, as illustrated by the Pleuronectiformes (flatfishes). This order is characterized by its adaptation to demersal life, compact genomes and diversity of SD mechanisms. Here, we assembled the Solea senegalensis genome, a flatfish of great commercial value, into 82 contigs (614 Mb) combining long- and short-read sequencing, which were next scaffolded using a highly dense genetic map (28,838 markers, 21 linkage groups), representing 98.9% of the assembly. Further, we established the correspondence between the assembly and the 21 chromosomes by using BAC-FISH. Whole genome resequencing of six males and six females enabled the identification of 41 single nucleotide polymorphism variants in the follicle stimulating hormone receptor (fshr) consistent with an XX/XY SD system. The observed sex association was validated in a broader independent sample, providing a novel molecular sexing tool. The fshr gene displayed differential expression between male and female gonads from 86 days post-fertilization, when the gonad is still an undifferentiated primordium, concomitant with the activation of amh and cyp19a1a, testis and ovary marker genes, respectively, in males and females. The Y-linked fshr allele, which included 24 nonsynonymous variants and showed a highly divergent 3D protein structure, was overexpressed in males compared to the X-linked allele at all stages of gonadal differentiation. We hypothesize a mechanism hampering the action of the follicle stimulating hormone driving the undifferentiated gonad toward testis.     

Linking life history traits in successive phases of a complex life cycle: effects of larval biomass on early juvenile development in an estuarine crab, Chasmagnathus granulata

In marine benthic invertebrates with complex life cycles, recruitment success, juvenile survival, and growth may be affected by variation in both maternal factors and environmental conditions prevailing during preceding embryonic or larval development. In an estuarine crab, Chasmagnathus granulata, previous investigations have shown that initial larval biomass is positively correlated with the biomass of recently extruded eggs, and it depends also on the salinity experienced during embryogenesis. Biomass at hatching has consequences for the subsequent larval development which, in this species, comprises two alternative developmental pathways with four or five zoeal instars (short or long pathway) and a megalopa. Larvae hatching with a lower than average biomass tend to develop through the long pathway and metamorphose to megalopae with higher biomass. In the present study, we show experimentally that the long pathway produces also significantly larger juveniles (crab size measured as carapace width, biomass as dry mass, carbon and nitrogen contents). Compared with juveniles originating from the short pathway, those from the long pathway showed in successive instars longer moulting cycles and larger carapace width, but lower size increments at ecdysis. In consequence, differences in size or biomass of long pathway vs short pathway crabs tended to disappear in later instars (after stage V). Furthermore, we tested in juveniles the tolerance of starvation at three salinities (5‰, 15‰, 32‰). Tolerance of starvation was significantly higher in juveniles originating from the long pathway, indicating higher energy reserves. While salinity played only a minor role for survival, it exerted significant effects on the time of moulting to the second juvenile instar, regardless of the preceding developmental pathway. The biomass of first juveniles obtained from the short pathway showed a significant positive correlation with the biomass of the freshly hatched zoea I, but not in those from the long pathway. In conclusion, the fitness of juvenile C. granulata is linked with previous developmental processes and environmental conditions during the embryonic and larval phase. Hence, a better understanding and prediction of the recruitment success of marine benthic invertebrates with a complex life cycle may require more comprehensive life‐history investigations.     

Characterization of xanthans from selected Xanthomonas strains cultivated under constant dissolved oxygen

Seventeen wild-type Xanthomonas isolates were screened in terms of broth viscosity in shake-flasks. As culture conditions affect polymer characteristics, a fair comparison among isolates required their cultivation in a fermenter under controlled dissolved oxygen tension. Three isolates and a reference strain were studied. The mean molecular weights and molecular weight distributions of their xanthans were determined. Products showed different pyruvate (0.2–7%), acetate (5–10%) and proteinaceous nitrogen (1–3%) contents. The selected isolates exhibit properties which could improve xanthan gum production and some could be used to produce polymers with specific characteristics.     

Establishment of the Dorso-ventral Axis in Xenopus Embryos Is Presaged by Early Asymmetries in β-Catenin That Are Modulated by the Wnt Signaling Pathway

Eggs of Xenopus laevis undergo a postfertilization cortical rotation that specifies the position of the dorso-ventral axis and activates a transplantable dorsal-determining activity in dorsal blastomeres by the 32-cell stage. There have heretofore been no reported dorso-ventral asymmetries in endogenous signaling proteins that may be involved in this dorsal-determining activity during early cleavage stages. We focused on β-catenin as a candidate for an asymmetrically localized dorsal-determining factor since it is both necessary and sufficient for dorsal axis formation. We report that β-catenin displays greater cytoplasmic accumulation on the future dorsal side of the Xenopus embryo by the two-cell stage. This asymmetry persists and increases through early cleavage stages, with β-catenin accumulating in dorsal but not ventral nuclei by the 16- to 32cell stages. We then investigated which potential signaling factors and pathways are capable of modulating the steady-state levels of endogenous β-catenin. Steadystate levels and nuclear accumulation of β-catenin increased in response to ectopic Xenopus Wnt-8 (Xwnt-8) and to the inhibition of glycogen synthase kinase-3, whereas neither Xwnt-5A, BVg1, nor noggin increased β-catenin levels before the mid-blastula stage. As greater levels and nuclear accumulation of β-catenin on the future dorsal side of the embryo correlate with the induction of specific dorsal genes, our data suggest that early asymmetries in β-catenin presage and may specify dorso-ventral differences in gene expression and cell fate. Our data further support the hypothesis that these dorso-ventral differences in β-catenin arise in response to the postfertilization activation of a signaling pathway that involves Xenopus glycogen synthase kinase-3.     

Variation in heat shock proteins within tropical and desert species of poeciliid fishes

The 70-kilodalton heat shock protein (hsp70) family of molecular chaperones, which contains both stress-inducible and normally abundant constitutive members, is highly conserved across distantly related taxa. Analysis of this protein family in individuals from an outbred population of tropical topminnows, Poeciliopsis gracilis, showed that while constitutive hsp70 family members showed no variation in protein isoforms, inducibly synthesized hsp70 was polymorphic. Several species of Poeciliopsis adapted to desert environments exhibited lower levels of inducible hsp70 polymorphism than the tropical species, but constitutive forms were identical to those in P. gracilis, as they were in the confamilial species Gambusia affinis. These differences suggest that inducible and constitutive members of this family are under different evolutionary constraints and may indicate differences in their function within the cell. Also, northern desert species of Poeciliopsis synthesize a subset of the inducible hsp70 isoforms seen in tropical species. This distribution supports the theory that ancestral tropical fish migrated northward and colonized desert streams; the subsequent decrease in variation of inducible hsp70 may have been due to genetic drift or a consequence of adaptation to the desert environment. Higher levels of variability were found when the 30- kilodalton heat shock protein (hsp30) family was analyzed within different strains of two desert species of Poeciliopsis and also in wild-caught individuals of Gambusia affinis. In both cases the distribution of hsp30 isoform diversity was similar to that seen previously with allozyme polymorphisms.