Coagulation factor XIII: A useful polymorphic genetic marker

Summary The plasmas of two groups of subjects were examined for blood coagulation Factor XIII-A (FXIII-A, F13A) by electrophoresis in agarose using a Tris-EDTA-borate buffer to separate the common variants, F13A^*1, F13A^*2, and F13A^*3. Dimeric subunits were visualized in UV light as monodansyl cadaverine bound to casein at the position of the transglutaminase activity representing F13A. One test group consisted of 307 members of three large Caucasian families. The other consisted of 148 consecutive patients whose plasmas had been sent to the clinical laboratory for determination of prothrombin time. Segregation analysis and father-to-son transmission confirmed that F13A is inherited as an autosomal co-dominant trait. The allelic frequencies in the random sample were F13A^*1=0.82 and F13A^*2=0.18. This sample included both blacks and whites, and the gene frequencies were not significantly different in the two races. The gene frequencies among the unrelated spouses of the three white families were A^*1=0.75, A^*2=0.24, A^*3=0.01. Genetic equilibrium was present in both groups.The degree of polymorphism, the availability of blood, the ease of assessment, the absence of selective pressure, and the uniformity of gene frequencies in two major American ethnic groups make F13A a very useful marker for linkage studies and paternity testing. F13A has been provisionally assigned to chromosome 6. Linkage analysis of our family data did not provide evidence of linkage to two chromosome 6 markers, properdin factor B (BF) and glyoxalase 1 (GLO). The highest lod score (Z) was between F13A and the Kidd (Jk) blood group (Z=0.68 at -0.24).     

Insights into correlated motions and long-range interactions in CheY derived from molecular dynamics simulations

CheY is a response regulator protein involved in bacterial chemotaxis. Much is known about its active and inactive conformations, but little is known about the mechanisms underlying long-range interactions or correlated motions. To investigate these events, molecular dynamics simulations were performed on the unphosphorylated, inactive structure from Salmonella typhimurium and the CheY-BeF(3)(-) active mimic structure (with BeF(3)(-) removed) from Escherichia coli. Simulations utilized both sequences in each conformation to discriminate sequence- and structure-specific behavior. The previously identified conformational differences between the inactive and active conformations of the strand-4-helix-4 loop, which are present in these simulations, arise from the structural, and not the sequence, differences. The simulations identify previously unreported structure-specific flexibility features in this loop and sequence-specific flexibility features in other regions of the protein. Both structure- and sequence-specific long-range interactions are observed in the active and inactive ensembles. In the inactive ensemble, two distinct mechanisms based on Thr-87 or Ile-95 rotameric forms, are observed for the previously identified g+ and g- rotamer sampling by Tyr-106. These molecular dynamics simulations have thus identified both sequence- and structure-specific differences in flexibility, long-range interactions, and rotameric form of key residues. Potential biological consequences of differential flexibility and long-range correlated motion are discussed.     

WISE 2005: vascular responses to 60-day bed rest in women

WISE-2005 studied 24 women during a 60-day head down bed rest (HDBR) who look part in an exercise countermeasure (LBNP-treadmill plus flywheel, EX) and no-exercise (No-EX). We conducted a series of experiments to explore changes in cardiovascular function and the ability of EX to prevent these changes. Resting arterial diameter in the arm was not affected but the leg arteries (femoral and popliteal) were significantly reduced in Np-EX, but was increased in EX. In this study we report on drug stimulated responses with sublingual nitroglycerin and infused isoproterenol. Heart rate increased in response to nitroglycerin with larger increases in No-EX after HDBR. Likewise during isoproterenol infusion the HR increase was greater after HDBR in the No-EX group. In all cases, the higher HR was associated with lower stroke volume in No-EX while stroke volume was protected in EX. These data do not support a change in sensitivity of beta-adrenergic receptors after HDBR. The leg vascular resistance decreased in response to isoproterenol and it decreased to a greater extent in No-EX than EX. These data were consistent with observations of lower leg vascular resistance during orthostatic challenge tests after HDBR. We conclude that consistent changes in cardiovascular function in the No-EX were detected by different methods that point to mechanisms contributing to orthostatic intolerance after HDBR.     

Mapping of New Escherichia coli K and 15 Restriction Sites on Specific Fragments of Bacteriophage φX174 DNA

We have isolated several new phiX174 mutants which contain sites sensitive to restriction by Escherichia coli. One contains an E. coli 15 restriction site and three are double mutants containing an E. coli K site as well as the E. coli 15 site. The replicative form (RF) DNA of one of the mutants containing a K site has been shown to be restricted in spheroplasts of a K-12 strain. The infectivity of this RF, but not wild-type RF, has also been shown to be inactivated by an E. coli K extract and by purified K restriction enzyme in vitro. The product of the RF treated with purified K restriction enzyme in vitro is a full length linear molecule. The mutant sites have also been localized to specific regions of the phiX174 genome by a fragment mapping technique, making use of specific fragments of phiX174 RF DNA obtained by digestion with a specific endonuclease.     

Learning to live together: mutualism between self-splicing introns and their hosts

Group I and II introns can be considered as molecular parasites that interrupt protein-coding and structural RNA genes in all domains of life. They function as self-splicing ribozymes and thereby limit the phenotypic costs associated with disruption of a host gene while they act as mobile DNA elements to promote their spread within and between genomes. Once considered purely selfish DNA elements, they now seem, in the light of recent work on the molecular mechanisms regulating bacterial and phage group I and II intron dynamics, to show evidence of co-evolution with their hosts. These previously underappreciated relationships serve the co-evolving entities particularly well in times of environmental stress.     

Assembly of a fragmented ribonucleotide reductase by protein interaction domains derived from a mobile genetic element

Ribonucleotide reductase (RNR) is a critical enzyme of nucleotide metabolism, synthesizing precursors for DNA replication and repair. In prokaryotic genomes, RNR genes are commonly targeted by mobile genetic elements, including free standing and intron-encoded homing endonucleases and inteins. Here, we describe a unique molecular solution to assemble a functional product from the RNR large subunit gene, nrdA that has been fragmented into two smaller genes by the insertion of mobE, a mobile endonuclease. We show that unique sequences that originated during the mobE insertion and that are present as C- and N-terminal tails on the split NrdA-a and NrdA-b polypeptides, are absolutely essential for enzymatic activity. Our data are consistent with the tails functioning as protein interaction domains to assemble the tetrameric (NrdA-a/NrdA-b)(2) large subunit necessary for a functional RNR holoenzyme. The tails represent a solution distinct from RNA and protein splicing or programmed DNA rearrangements to restore function from a fragmented coding region and may represent a general mechanism to neutralize fragmentation of essential genes by mobile genetic elements.