Prostaglandin‐endoperoxide synthase 2 is not required for preimplantation ovine conceptus development in sheep

Conceptus development and elongation is required for successful pregnancy establishment in ruminants and is coincident with the production of interferon τ (IFNT) and prostaglandins (PGs). In both the conceptus trophectoderm and endometrium, PGs are primarily synthesized through a prostaglandin‐endoperoxide synthase 2 (PTGS2) pathway and modify endometrial gene expression and thus histotroph composition in the uterine lumen to promote conceptus growth and survival. Chemical inhibition of PG production by both the endometrium and the conceptus prevented elongation in sheep. However, the contributions of conceptus‐derived PGs to preimplantation conceptus development remain unclear. In this study, CRISPR‐Cas9 genome editing was used to inactivate PTGS2 in ovine embryos to determine the role of PTGS2‐derived PGs in conceptus development and elongation. PTGS2 edited conceptuses produced fewer PGs, but secreted similar amounts of IFNT to their Cas9 control counterparts and elongated normally. Expression of PTGS1 was lower in PTGS2 edited conceptuses, but PPARG expression and IFNT secretion were unaffected. Content of PGs in the uterine lumen was similar as was gene expression in the endometrium of ewes who received either Cas9 control or PTGS2 edited conceptuses. These results support the idea that intrinsic PTGS2‐derived PGs are not required for preimplantation embryo or conceptus survival and development in sheep.     

COLINEARITY OF CHLOROPLAST GENOMES IN DIVERGENT ECOTYYES OF THE MARINE DIATOM SKELETONEMA COSTATUM (BACILLARIOPHYTA)1

The chloroplast genomes of three isolates of the marine diatom Skeletonema costatum (Grev.) Cleve were mapped and found to be 131 ± 2 kb with inverted repeats (IRs) of approximately 20 kb. In contrast to higher plants, the psbA gene mapped to the IR, and rbcS mapped to the same fragment as the rbcL gene in the large single-copy region. The maps of the three isolates were colinear and revealed as many as 20 site mutations out of a total of 47 sites. The number of site mutations among isolates was consistent with previous data on their genetic diversity and physiology. Comparisons of gene order among our maps and those of three other diatom species showed that closely related genera retained similar gene orders but that more distantly related taxa exhibited extensive rearrangements. We conclude that simple restriction fragment analysis of chloroplast DNA is useful in comparative studies of diatom populations and species but that other analytical methods are more appropriate for phylogenetic studies at higher levels.     

Tyrosine and tyrosinate fluorescence of pig intestinal Ca2+-binding protein.

The single tyrosine residue in both pig and cow intestinal Ca2+-binding proteins fluoresces at 303 nm although the crystal structure of the cow protein shows a hydrogen bond between the hydroxy group of the tyrosine and glutamate-38 [Szebenyi & Moffat (1986) J. Biol. Chem. 261, 8761-8777]. The latter interaction suggests that tyrosinate fluorescence should dominate the emission spectra of these proteins. A fluorescence difference spectrum, produced by subtracting the spectrum of free tyrosine from the spectrum of the protein, gives a peak at 334 nm due to ionized tyrosine. That this component of the emission spectrum is not due to a tryptophan-containing contaminant is shown by its elimination when the protein is denatured by guanidine and when glutamate-38 is protonated. We conclude that, in solution, the tyrosine residue in this protein interacts occasionally with glutamate-38 but that a permanent hydrogen bond is not formed.     

Interspecific Comparison of the Transformer Gene of Drosophila Reveals an Unusually High Degree of Evolutionary Divergence

The transformer (tra) gene of Drosophila melanogaster occupies an intermediate position in the regulatory pathway controlling all aspects of somatic sexual differentiation. The female-specific expression of this gene's function is regulated by the Sex lethal (Sxl) gene, through a mechanism involving sex-specific alternative splicing of tra pre-mRNA. The tra gene encodes a protein that is thought to act in conjunction with the transformer-2 (tra-2) gene product to control the sex-specific processing of doublesex (dsx) pre-mRNA. The bifunctional dsx gene carries out opposite functions in the two sexes, repressing female differentiation in males and repressing male differentiation in females. Here we report the results from an evolutionary approach to investigate tra regulation and function, by isolating the tra-homologous genes from selected Drosophila species, and then using the interspecific DNA sequence comparisons to help identify regions of functional significance. The tra-homologous genes from two Sophophoran subgenus species, Drosophila simulans and Drosophila erecta, and two Drosophila subgenus species, Drosophila hydei and Drosophila virilis, were cloned, sequenced and compared to the D. melanogaster tra gene. This comparison reveals an unusually high degree of evolutionary divergence among the tra coding sequences. These studies also highlight a highly conserved sequence within intron one that probably defines a cis-acting regulator of the sex-specific alternative splicing event.     

Maize Y9 encodes a product essential for 15-cis-zeta-carotene isomerization

Carotenoids are a diverse group of pigments found in plants, fungi, and bacteria. They serve essential functions in plants and provide health benefits for humans and animals. In plants, it was thought that conversion of the C40 carotenoid backbone, 15-cis-phytoene, to all-trans-lycopene, the geometrical isomer required by downstream enzymes, required two desaturases (phytoene desaturase and zeta-carotene desaturase [ZDS]) plus a carotene isomerase (CRTISO), in addition to light-mediated photoisomerization of the 15-cis-double bond; bacteria employ only a single enzyme, CRTI. Characterization of the maize (Zea mays) pale yellow9 (y9) locus has brought to light a new isomerase required in plant carotenoid biosynthesis. We report that maize Y9 encodes a factor required for isomerase activity upstream of CRTISO, which we term Z-ISO, an activity that catalyzes the cis- to trans-conversion of the 15-cis-bond in 9,15,9'-tri-cis-zeta-carotene, the product of phytoene desaturase, to form 9,9'-di-cis-zeta-carotene, the substrate of ZDS. We show that recessive y9 alleles condition accumulation of 9,15,9'-tri-cis-zeta-carotene in dark tissues, such as roots and etiolated leaves, in contrast to accumulation of 9,9'-di-cis-zeta-carotene in a ZDS mutant, viviparous9. We also identify a locus in Euglena gracilis, which is similarly required for Z-ISO activity. These data, taken together with the geometrical isomer substrate requirement of ZDS in evolutionarily distant plants, suggest that Z-ISO activity is not unique to maize, but will be found in all higher plants. Further analysis of this new gene-controlled step is critical to understanding regulation of this essential biosynthetic pathway.     

Role of DNA flexibility in sequence-dependent activity of uracil DNA glycosylase

Uracil DNA glycosylase (UDG) is a base excision repair enzyme that specifically recognizes and removes uracil from double- or single-stranded DNA. The efficiency of the enzyme depends on the DNA sequence surrounding the uracil. Crystal structures of UDG in complex with DNA reveal that the DNA is severely bent and distorted in the region of the uracil. This suggests that the sequence-dependent efficiency of the enzyme may be related to the energetic cost of DNA distortion in the process of specific damage recognition. To test this hypothesis, molecular dynamics simulations were performed on two sequences representing extreme cases of UDG efficiency, AUA/TAT (high efficiency) and GUG/CAC (low efficiency). Analysis of the simulations shows that the effective bending force constants are lower for the AUA/TAT sequence, indicating that this sequence is more flexible than the GUG/CAC sequence. Fluorescence lifetimes of the adenine analogue 2-aminopurine (2AP), replacing adenine opposite the uracil, are shorter in the context of the AUA/TAT sequence, indicating more dynamic base-base interaction and greater local flexibility than in the GUG/CAC sequence. Furthermore, the K(M) of Escherichia coli UDG for the AUA/TAT sequence is 10-fold smaller than that for the GUG/CAC sequence, while the k(cat) is only 2-fold smaller. This indicates that differences in UDG efficiency largely arise from differences in binding and not catalysis. These results link directly flexibility near the damaged DNA site with the efficiency of DNA repair.