Implications of intragenic marker homozygosity and haplotype sharing in a rare autosomal recessive disorder: the example of the collagen type XVII (COL17A1) locus in generalised atrophic benign epidermolysis bullosa

Generalised atrophic benign epidermolysis bullosa (GABEB) is a form of junctional epidermolysis bullosa with a recessive mode of inheritance. The gene considered likely to be involved in this disease is COL17A1, since in the majority of GABEB patients the product of that gene, the 180-kD bullous pemphigoid antigen (BP180), is undetectable in skin. We have identified an intragenic COL17A1 microsatellite marker for which 83% of randomly selected control individuals are heterozygous. We observed homozygosity for different alleles of this marker in five out of six collagen type XVII-negative GABEB patients of different European descent. Five of the six COL17A1 alleles of three patients originating from the eastern part of The Netherlands were identical, as were the haplotypes including flanking markers. The 2342delG mutation was identified in all these five alleles. This confirms the expectation that due to genetic drift and hidden inbreeding for an autosomal recessive disorder with low gene frequency, such as collagen type XVII-negative GABEB, most disease alleles from a restricted geographical area will be “identical by descent”. Our results demonstrate that involvement of a candidate gene can be confirmed by looking for identity by descent of highly informative intragenic markers. Received: 25 October 1996 / Accepted: 6 March 1997     

The role of packing interactions in stabilizing folded proteins.

In order to investigate the role of nonpolar side chains in determining protein stability, we have carried out a molecular dynamics simulation study of the thermodynamics of interconverting isoleucine and valine side chains in the core of ribonuclease T1. The free energy change in the unfolded state, which we take to be fully solvated, was small and agrees qualitatively with experimental studies of alkane solvation. In the two Ile----Val mutations studied, the protein was able to relax around the smaller side chains, while in the case of the two Val----Ile mutations, the ability of the core to accommodate the extra methylene group depended on where the mutation took place. We argue that the experimentally observed decrease in stability for mutating isoleucine into valine results from a loss of favorable packing interactions of the side chain in the folded form of the protein. This supports the view that packing interactions in the folded state are an important contributor to the overall stability of the folded protein and that the core of the native protein is packed efficiently and almost completely.     

Electrostatic effects in short superhelical DNA

We present Monte Carlo simulations of the equilibrium configurations of short closed circular DNA that obeys a combined elastic, hard-sphere, and electrostatic energy potential. We employ a B-spline representation to model chain configuration and simulate the effects of salt on chain folding by varying the Debye screening parameter. We obtain global equilibrium configurations of closed circular DNA, with several imposed linking number differences, at two salt concentrations (specifically at the extremes of no added salt and the high salt regime), and for different chain lengths. Minimization of the composite elastic/long-range potential energy under the constraints of ring closure and fixed chain length is found to produce structures that are consistent with the configurations of short supercoiled DNA observed experimentally. The structures generated under the constraints of an electrostatic potential are less compact than those subjected only to an elastic term and a hard-sphere constraint. For a fixed linking number difference greater than a critical value, the interwound structures obtained under the condition of high salt are more compact than those obtained under the condition of no added salt. In the case of no added salt, the electrostatic energy plays a dominant role over the elastic energy in dictating the shape of the closed circular DNA. The DNA supercoil opens up with increasing chain length at low salt concentration. A branched three-leaf rose structure with a fixed linking number difference is higher in energy than the interwound form at both salt concentrations employed here.     

The differential cardio-respiratory responses to ambient hypoxia and systemic hypoxaemia in the South American lungfish, Lepidosiren paradoxa

Lungfishes (Dipnoi) occupy an evolutionary transition between water and air breathing and possess well-developed lungs and reduced gills. The South American species, Lepidosiren paradoxa, is an obligate air-breather and has the lowest aquatic respiration of the three extant genera. To study the relative importance, location and modality of reflexogenic sites sensitive to oxygen in the generation of cardio-respiratory responses, we measured ventilatory responses to changes in ambient oxygen and to reductions in blood oxygen content. Animals were exposed to aquatic and aerial hypoxia, both separately and in combination. While aerial hypoxia elicited brisk ventilatory responses, aquatic hypoxia had no effect, indicating a primary role for internal rather than branchial receptors. Reducing haematocrit and blood oxygen content by approximately 50% did not affect ventilation during normoxia, showing that the specific modality of the internal oxygen sensitive chemoreceptors is blood PO(2) per se and not oxygen concentration. In light of previous studies, it appears that the heart rate responses and the changes in pulmonary ventilation during oxygen shortage are similar in lungfish and tetrapods. Furthermore, the modality of the oxygen receptors controlling these responses is similar to tetrapods. Because the cardio-respiratory responses and the modality of the oxygen receptors differ from typical water-breathing teleosts, it appears that many of the changes in the mechanisms exerting reflex control over cardio-respiratory functions occurred at an early stage in vertebrate evolution.     

Population genomics reveals evolution and variation of Saccharomyces cerevisiae in the human and insects gut

The quest to discover the variety of ecological niches inhabited by Saccharomyces cerevisiae has led to research in areas as diverse as wineries, oak trees and insect guts. The discovery of fungal communities in the human gastrointestinal tract suggested the host's gut as a potential reservoir for yeast adaptation. Here, we report the existence of yeast populations associated with the human gut (HG) that differ from those isolated from other human body sites. Phylogenetic analysis on 12 microsatellite loci and 1715 combined CDSs from whole-genome sequencing revealed three subclusters of HG strains with further evidence of clonal colonization within the host's gut. The presence of such subclusters was supported by other genomic features, such as copy number variation, absence/introgressions of CDSs and relative polymorphism frequency. Functional analysis of CDSs specific of the different subclusters suggested possible alterations in cell wall composition and sporulation features. The phenotypic analysis combined with immunological profiling of these strains further showed that sporulation was related with strain-specific genomic characteristics in the immune recognition pattern. We conclude that both genetic and environmental factors involved in cell wall remodelling and sporulation are the main drivers of adaptation in S. cerevisiae populations in the human gut.     

Application of dynamic metabolic flux analysis for process modeling: Robust flux estimation with regularization,confidence bounds,and selection of elementary modes

In macroscopic dynamic models of fermentation processes, elementary modes (EM) derived from metabolic networks are often used to describe the reaction stoichiometry in a simplified manner and to build predictive models by parameterizing kinetic rate equations for the EM. In this procedure, the selection of a set of EM is a key step which is followed by an estimation of their reaction rates and of the associated confidence bounds. In this paper, we present a method for the computation of reaction rates of cellular reactions and EM as well as an algorithm for the selection of EM for process modeling. The method is based on the dynamic metabolic flux analysis (DMFA) proposed by Leighty and Antoniewicz (2011, Metab Eng, 13(6), 745–755) with additional constraints, regularization and analysis of uncertainty. Instead of using estimated uptake or secretion rates, concentration measurements are used directly to avoid an amplification of measurement errors by numerical differentiation. It is shown that the regularized DMFA for EM method is significantly more robust against measurement noise than methods using estimated rates. The confidence intervals for the estimated reaction rates are obtained by bootstrapping. For the selection of a set of EM for a given st oichiometric model, the DMFA for EM method is combined with a multiobjective genetic algorithm. The method is applied to real data from a CHO fed-batch process. From measurements of six fed-batch experiments, 10 EM were identified as the smallest subset of EM based upon which the data can be described sufficiently accurately by a dynamic model. The estimated EM reaction rates and their confidence intervals at different process conditions provide useful information for the kinetic modeling and subsequent process optimization.     

Lytic transglycosylases RlpA and MltC assist in Vibrio cholerae daughter cell separation

The cell wall is a crucial structural feature in the vast majority of bacteria and comprises a covalently closed network of peptidoglycan (PG) strands. While PG synthesis is important for survival under many conditions, the cell wall is also a dynamic structure, undergoing degradation and remodeling by ‘autolysins’, enzymes that break down PG. Cell division, for example, requires extensive PG remodeling, especially during separation of daughter cells, which depends heavily upon the activity of amidases. However, in Vibrio cholerae, we demonstrate that amidase activity alone is insufficient for daughter cell separation and that lytic transglycosylases RlpA and MltC both contribute to this process. MltC and RlpA both localize to the septum and are functionally redundant under normal laboratory conditions; however, only RlpA can support normal cell separation in low‐salt media. The division‐specific activity of lytic transglycosylases has implications for the local structure of septal PG, suggesting that there may be glycan bridges between daughter cells that cannot be resolved by amidases. We propose that lytic transglycosylases at the septum cleave PG strands that are crosslinked beyond the reach of the highly regulated activity of the amidase and clear PG debris that may block the completion of outer membrane invagination.