Expression of N-linked sialyl Le(x) determinants and O-glycans in the carbohydrate moiety of human amniotic fluid transferrin during pregnancy

Transferrin, a glycoprotein involved in iron transport in body fluids, was isolated from amniotic fluid of a hydramniospatient by sequential anion-exchange chromatography and gel filtration. The N-glycans of human amniotic fluid transferrin (hAFT) were enzymatically liberated by PNGase-F digestion, isolated by gel filtration and fractionated by (high-pH) anion-exchange chromatography. After alkaline borohydride treatment of native hAFT, the released O-glycans were isolated by gel filtration and fractionated by anion-exchange chroma-tography. Structure elucidation of 14 N- and 2 O-glycans was performed by 500 or 600 MHz1H-NMR spectroscopy. Besides conventional N-glycans established earlier for human serum transferrin (hST), new (alpha1-3)-fucosylated N- glycans were found, representing sialyl Le(x) elements. Furthermore, as compared to hST, a higher degree of (alpha1-6)-fucosylation and an increase in branching from di- to triantennary compounds has been detected. The presence of O-glycans is demonstrated for the first time in transferrin.      

Vegetal cover estimated by digital photographs related to biomass in a grassland site in northern Mexico

A regression model was used to determine the relationship between aerial herbaceous biomass and vegetation coverage estimated by digital images. Four samplings (n=36 each date) of vegetation cover and herbaceous biomass were performed during the growing season in 2011 in a grassland dominated by Bouteloua gracilis in La Cieneguilla, Municipality of Villa Hidalgo, Durango. Average production of dry biomass was 37.36 ± 9.66 g/m², and mean vegetation cover 30.02%. Dry biomass data were tested for normality using the test of Kolmogorov Smirnov, finding a lack of fit. The data were subjected to a logarithmic transformation and the model Ln(y) = 1.637926 + 0.08501X - 0.000586X² with an adjusted R² = 0.89 was found. In order to validate this model, another five samplings were carried out in 2013 at the same site during summer and autumn, using the same sampling size for each date as in 2011. Data collected in 2013 were analyzed with the model Ln (y) = β₀ + β₁X + β₂X². A comparison of regression coefficients was carried out between the 2011 and 2013 models with t (180+144-9-11-2=302, p<0.05) = 1.967. The results indicated that it is possible to use the 2011 regression model to estimate herbaceous aerial biomass from vegetation cover measurements with aerial photographs in La Cieneguilla site during summer and fall.     

Duplicate gene evolution and expression in the wake of vertebrate allopolyploidization

Background  

The mechanism by which duplicate genes originate – whether by duplication of a whole genome or of a genomic segment – influences their genetic fates. To study events that trigger duplicate gene persistence after whole genome duplication in vertebrates, we have analyzed molecular evolution and expression of hundreds of persistent duplicate gene pairs in allopolyploid clawed frogs (Xenopus and Silurana). We collected comparative data that allowed us to tease apart the molecular events that occurred soon after duplication from those that occurred later on. We also quantified expression profile divergence of hundreds of paralogs during development and in different tissues.     

RecQ and RecG helicases have distinct roles in maintaining the stability of polypurine.polypyrimidine sequences

DNA triplex structures can block the replication fork and result in double-stranded DNA breaks (DSBs). RecQ and RecG helicases may be important for replication of such sequences as RecQ resolves synthetic triplex DNA structures and RecG mediates replication restart by fork regression. Primer extension on an 88bp triplex-forming polypurine.polypyrimidine (Pu.Py) tract from the PKD1 gene demonstrated that RecQ, but not RecG, facilitated primer extension by T7 DNA polymerase. A high-throughput, dual plasmid screening system using isogenic bacterial lines deficient in RecG, RecQ, or both, revealed that RecQ deficiency increased mutation to sequence flanking this 88bp tract by eight to ten-fold. Although RecG facilitated small deletions in an 88bp mirror repeat-containing sequence, it was absolutely required to maintain a 2.5kb Pu.Py tract containing multiple mirror repeats. These results support a two-tiered model where RecQ facilitates fork progression through triplex-forming tracts and, failing processivity, RecG is critical for replication fork restart.     

Analysis of PKD1 for genomic deletion by multiplex ligation-dependent probe assay: absence of hot spots

Autosomal dominant polycystic kidney disease is largely due to mutations in PKD1. PKD1 has an unusual genomic structure, including a 2.5-kb polypyrimidine sequence in intron 21, which has been postulated to lead to a high rate of spontaneous genomic mutation events. In addition, the majority of the gene is duplicated three to six times at 97-99% identity elsewhere in the genome. To identify genomic mutations in PKD1, we developed a multiplex ligation-dependent probe assay (MLPA) in which sites of variation between PKD1 and its copies were positioned at the ligation sites of the MLPA probe sets. Thirteen probe sets covered PKD1 exons 2 through 46, at an average spacing of 2.5 kb. Analysis of 27 independent PKD patient samples showed no evidence for genomic deletions confined to PKD1. Analysis of 15 tuberous sclerosis patient samples in which deletions in TSC2 extended into PKD1 showed no evidence of clustering of breakpoints near the polypyrimidine tract.