Detection of genes influencing economic traits in three French dairy cattle breeds

A project of QTL detection was carried out in the French Holstein, Normande, and Montbéliarde dairy cattle breeds. This granddaughter design included 1 548 artificial insemination bulls distributed in 14 sire families and evaluated after a progeny-test for 24 traits (production, milk composition, persistency, type, fertility, mastitis resistance, and milking ease). These bulls were also genotyped for 169 genetic markers, mostly microsatellites. The QTL were analysed by within-sire linear regression of daughter yield deviations or deregressed proofs on the probability that the son receives one or the other paternal QTL allele, given the marker information. QTL were detected for all traits, including those with a low heritability. One hundred and twenty QTL with a chromosome-wise significance lower than 3% were tabulated. This threshold corresponded to a 15% false discovery rate. Amongst them, 32 were genome-wise significant. Estimates of their contribution to genetic variance ranged from 6 to 40%. Most substitution effects ranged from 0.6 to 1.0 genetic standard deviation. For a given QTL, only 1 to 5 families out of 14 were informative. The confidence intervals of the QTL locations were large and always greater than 20 cM. This experiment confirmed several already published QTL but most of them were original, particularly for non-production traits.     

Elephant Genomes Reveal Accelerated Evolution in Mechanisms Underlying Disease Defenses

Disease susceptibility and resistance are important factors for the conservation of endangered species, including elephants. We analyzed pathology data from 26 zoos and report that Asian elephants have increased neoplasia and malignancy prevalence compared with African bush elephants. This is consistent with observed higher susceptibility to tuberculosis and elephant endotheliotropic herpesvirus (EEHV) in Asian elephants. To investigate genetic mechanisms underlying disease resistance, including differential responses between species, among other elephant traits, we sequenced multiple elephant genomes. We report a draft assembly for an Asian elephant, and defined 862 and 1,017 conserved potential regulatory elements in Asian and African bush elephants, respectively. In the genomes of both elephant species, conserved elements were significantly enriched with genes differentially expressed between the species. In Asian elephants, these putative regulatory regions were involved in immunity pathways including tumor-necrosis factor, which plays an important role in EEHV response. Genomic sequences of African bush, forest, and Asian elephant genomes revealed extensive sequence conservation at TP53 retrogene loci across three species, which may be related to TP53 functionality in elephant cancer resistance. Positive selection scans revealed outlier genes related to additional elephant traits. Our study suggests that gene regulation plays an important role in the differential inflammatory response of Asian and African elephants, leading to increased infectious disease and cancer susceptibility in Asian elephants. These genomic discoveries can inform future functional and translational studies aimed at identifying effective treatment approaches for ill elephants, which may improve conservation.     

Anchor side chains of short peptide fragments trigger ligand-exchange of class II MHC molecules

Class II MHC molecules display peptides on the cell surface for the surveillance by CD4+ T cells. To ensure that these ligands accurately reflect the content of the intracellular MHC loading compartment, a complex processing pathway has evolved that delivers only stable peptide/MHC complexes to the surface. As additional safeguard, MHC molecules quickly acquire a 'non-receptive' state once they have lost their ligand. Here we show now that amino acid side chains of short peptides can bypass these safety mechanisms by triggering the reversible ligand-exchange. The catalytic activity of dipeptides such as Tyr-Arg was stereo-specific and could be enhanced by modifications addressing the conserved H-bond network near the P1 pocket of the MHC molecule. It affected both antigen-loading and ligand-release and strictly correlated with reported anchor preferences of P1, the specific target site for the catalytic side chain of the dipeptide. The effect was evident also in CD4+ T cell assays, where the allele-selective influence of the dipeptides translated into increased sensitivities of the antigen-specific immune response. Molecular dynamic calculations support the hypothesis that occupation of P1 prevents the 'closure' of the empty peptide binding site into the non-receptive state. During antigen-processing and -presentation P1 may therefore function as important "sensor" for peptide-load. While it regulates maturation and trafficking of the complex, on the cell surface, short protein fragments present in blood or lymph could utilize this mechanism to alter the ligand composition on antigen presenting cells in a catalytic way.     

Dean vortex membrane microfiltration and diafiltration of rBDNF E. coli inclusion bodies

Cross-flow microfiltration (CMF) and diafiltration were used to concentrate and purify recombinant Brain-Derived Neutrophic Factor (rBDNF) inclusion bodies from an E. coli cell suspension and a homogenized E. coli cell suspension (homogenate/lysate). Although these processes have been tested industrially in pilot scale with conventional linear membrane microfiltration modules, their performances were severely limited due to membrane fouling. The purpose of this work was to determine whether Dean vortex microfiltration with controlled centrifugal instabilities (Dean vortices produced in helical flow) could be used to improve filtration performance over that observed with conventional linear cross-flow microfiltration (CMF). For the microfiltration experiments with the feeds containing cell and homogenate suspensions, improvements in flux of about 50 and 70%, respectively, were obtained with the helical module as compared with that obtained with the linear module. For diafiltration with the homogenate suspension as feed, solute transport (as measured by mass) was from 100 to 40% higher after 40 and 100 min, respectively, with the helical module as compared with that obtained with the linear module. In the presence of the neutral surfactant, Tween 20, solute transport for diafiltration was at least 25 times higher during the first 10 min of operation and 100% higher after 300 min with the helical module as compared with that obtained with the linear module. Clearly, improved filtration performance, a purer and more concentrated product, and substantial savings can be expected with the new Dean vortex filters.     

Apolipoprotein E4 forms a molten globule. A potential basis for its association with disease

The amino-terminal domain of apolipoprotein (apo) E4 is less susceptible to chemical and thermal denaturation than the apoE3 and apoE2 domains. We compared the urea denaturation curves of the 22-kDa amino-terminal domains of the apoE isoforms at pH 7.4 and 4.0. At pH 7.4, apoE3 and apoE4 reflected an apparent two-state denaturation. The midpoints of denaturation were 5.2 and 4.3 m urea, respectively. At pH 4.0, a pH value known to stabilize folding intermediates, apoE4 and apoE3 displayed the same order of denaturation but with distinct plateaus, suggesting the presence of a stable folding intermediate. In contrast, apoE2 proved the most stable and lacked the distinct plateau observed with the other two isoforms and could be fitted to a two-state unfolding model. Analysis of the curves with a three-state unfolding model (native, intermediate, and unfolded) showed that the apoE4 folding intermediate reached its maximal concentration ( approximately 90% of the mixture) at 3.75 m, whereas the apoE3 intermediate was maximal at 4.75 m ( approximately 80%). These results are consistent with apoE4 being more susceptible to unfolding than apoE3 and apoE2 and more prone to form a stable folding intermediate. The structure of the apoE4 folding intermediate at pH 4.0 in 3.75 m urea was characterized using pepsin proteolysis, Fourier transform infrared spectroscopy, and dynamic light scattering. From these studies, we conclude that the apoE4 folding intermediate is a single molecule with the characteristics of a molten globule. We propose a model of the apoE4 molten globule in which the four-helix bundle of the amino-terminal domain is partially opened, generating a slightly elongated structure and exposing the hydrophobic core. Since molten globules have been implicated in both normal and abnormal physiological function, the differential abilities of the apoE isoforms to form a molten globule may contribute to the isoform-specific effects of apoE in disease.     

Viscoelastic fluid description of bacterial biofilm material properties

A mathematical model describing the constitutive properties of biofilms is required for predicting biofilm deformation, failure, and detachment in response to mechanical forces. Laboratory observations indicate that biofilms are viscoelastic materials. Likewise, current knowledge of biofilm internal structure suggests modeling biofilms as associated polymer viscoelastic systems. Supporting experimental results and a system of viscoelastic fluid equations with a linear Jeffreys viscoelastic stress-strain law are presented here. This system of equations is based on elements of associated polymer physics and is also consistent with presented and previous experimental results. A number of predictions can be made. One particularly interesting result is the prediction of an elastic relaxation time on the order of a few minutes-biofilm disturbances on shorter time scales produce an elastic response, biofilm disturbances on longer time scales result in viscous flow, i.e., nonreversible biofilm deformation. Although not previously recognized, evidence of this phenomenon is in fact present in recent experimental results.     

TAFA: a novel secreted family with conserved cysteine residues and restricted expression in the brain

We have discovered a family of small secreted proteins in Homo sapiens and Mus musculus using a novel database searching strategy. The family is composed of five highly homologous genes referred to as TAFA-1 to -5. The TAFA genes encode proteins of approximately 100 amino acids that contain conserved cysteine residues at fixed positions. TAFA-1 to -4 are more closely related to each other than to TAFA-5, in which a conserved motif including CC in TAFA-1 to -4 is not present. In H. sapiens, TAFA-3 has two isoforms formed by alternative splicing. Sequence homology analyses reveal that TAFA proteins appear distantly related to MIP-1alpha, a member of the CC-chemokine family. TAFA mRNAs are highly expressed in specific brain regions, with little expression seen in other tissues.     

Laboratory scale structural genomics

At Lawrence Livermore National Laboratory, the development of the TB structural genomics consortium crystallization facility has paralleled several local proteomics research efforts that have grown out of gene expression microarray and comparative genomics studies. Collective experience gathered from TB consortium labs and other centers involved in the NIH-NIGMS protein structure initiative allows us to explore the possibilities and challenges of pursuing structural genomics on an academic laboratory scale. We discuss our procedures and protocols for genomic targeting approaches, primer design, cloning, small scale expression screening, scale-up and purification, through to automated crystallization screening and data collection. The procedures are carried out by a small group using a combination of traditional approaches, innovative molecular biochemistry approaches, software automation, and a modest investment in robotic equipment.     

Cloning, expression, and one-step purification of the minimal essential domain of the light chain of botulinum neurotoxin type A

A truncated but functional form of the botulinum neurotoxin A light chain (Tyr 9-Leu 415) has been cloned into the three bacterial expression vectors, pET 28, pET 30, and PGEX-2T, and produced as fusion proteins. This 406-amino-acid light chain was expressed with 1 six-histidine tag (LC-pET28), 2 six histidine tags and a S-tag (LC-pET30), or a six-histidine tag and a glutathione S-transferase tag (LC-pGEX-2T). The three fusion proteins have been overexpressed in Escherichia coli, purified in a soluble form, and tested for protease activity. All three recombinant proteins were found to have similar enzymatic activity, comparable to the light chain purified from the whole toxin. The LC-pET30 protein was the most soluble and stable of the three fusion proteins, and it could be purified using a one-step affinity chromatography protocol. The purified protein was determined to be 98% pure as assessed by SDS-polyacrylamide gel. This protein has been crystallized and initial X-ray data show that the crystals diffract to 1.8 A.