Physical Maps of the Six Smallest Chromosomes of Saccharomyces Cerevisiae at a Resolution of 2.6 Kilobase Pairs

Physical maps of the six smallest chromosomes of Saccharomyces cerevisiae are presented. In order of increasing size, they are chromosomes I, VI, III, IX, V and VIII, comprising 2.49 megabase pairs of DNA. The maps are based on the analysis of an overlapping set of lambda and cosmid clones. Overlaps between adjacent clones were recognized by shared restriction fragments produced by the combined action of EcoRI and HindIII. The average spacing between mapped cleavage sites is 2.6 kb. Five of the six chromosomes were mapped from end to end without discontinuities; a single internal gap remains in the map of chromosome IX. The reported maps span an estimated 97% of the DNA on the six chromosomes; nearly all the missing segments are telomeric. The maps are fully cross-correlated with the previously published SfiI/NotI map of the yeast genome by A. J. Link and M. V. Olson. They have also been cross-correlated with the yeast genetic map at 51 loci.     

Conservation of water molecules in an antibody–antigen interaction

The solvation of the antibody–antigen Fv D1.3–lysozyme complex is investigated through a study of the conservation of water molecules in crystal structures of the wild-type Fv fragment of antibody D1.3, 5 free lysozyme, the wild-type Fv D1.3–lysozyme complex, 5 Fv D1.3 mutants complexed with lysozyme and the crystal structure of an idiotope (Fv D1.3)-abti-idiotope (Fv E5.2) complex. In all, there are 99 water molecules common to the wild-type and mutant antibody–lysozyme complexes. The antibody–lysozyme interface includes 25 well-ordered solvent molecules, conserved among the wild-type and mutant Fv D1.3–lysozyme complexes, which are bound directly or through other water molecules to both antibody and antigen. In addition to contributing hydrogen bonds to the antibody–antigen interaction the solvent molecules fill many interface cavities. Comparison with x-ray crystal structures of free Fv D1.3 and free lysozyme shows that 20 of these conserved interface waters in the complex were bound to one of the free proteins. Uo to 23 additional water molecules are also found in the antibody–antigen interface, however these waters do no bridge antibody and antigen and their temperature factors are much higher than those of the 25 well-ordered waters. Fifteen water molecules are displaced to form the complex, some of which are substituted by hydrophilic protein atoms, and 5 water molecules are added at the antibody–antigen interface with the formation of the complex. While the current crystal models of the D1.3–lysozyme complex do not demonstrate the increase in bound waters found in a physico-chemical study of the interaction at decreased water activities, the 25 well-ordered interface water contribute a net gain of 10 hydrogen bonds to complex stability.     

Functional and morphological characteristics of the first corpus luteum formed after parturition in ewes

The ability of sheep luteal cells from the first corpus luteum formed after parturition (Group F) to secrete progesterone in the presence or absence of LH was compared with that of luteal cells obtained from normal cyclic ewes (Group C). Luteal concentrations of receptors for LH and prostaglandins (PG) F-2 alpha (PGF-2 alpha) and the cellular composition of corpora lutea from Groups F and C were also compared. Luteal cells from Group F secreted less progesterone in either the presence or absence of LH (P less than 0.01). There was no difference in the number of receptors for LH or PGF-2 alpha per luteal cell between Groups F and C (P greater than 0.1), nor was there a difference in the number of large or small steroidogenic luteal cells (P greater than 0.1). It was concluded that, if short-lived corpora lutea are insensitive to gonadotrophins, this response is not mediated by decreased numbers of receptors for LH. In addition, if the first corpus luteum formed post partum in ewes is more sensitive to the luteolytic effects of PGF-2 alpha, this effect is not mediated by an increased number of receptors for PGF-2 alpha or an increased proportion of PGF-2 alpha-sensitive large luteal cells.     

The Structure of the Biofilm-controlling Response Regulator BfmR from Acinetobacter baumannii Reveals Details of Its DNA-binding Mechanism

The rise of drug-resistant bacterial infections coupled with decreasing antibiotic efficacy poses a significant challenge to global health care. Acinetobacter baumannii is an insidious, emerging bacterial pathogen responsible for severe nosocomial infections aided by its ability to form biofilms. The response regulator BfmR, from the BfmR/S two-component system, is the master regulator of biofilm initiation in A. baumannii and is a tractable therapeutic target. Here we present the structure of A. baumannii BfmR using a hybrid approach combining X-ray crystallography, nuclear magnetic resonance spectroscopy, chemical crosslinking mass spectrometry, and molecular modeling. We also show that BfmR binds the previously proposed bfmRS promoter sequence with moderate affinity. While BfmR shares many traits with other OmpR/PhoB family response regulators, some unusual properties were observed.Most importantly, we observe that when phosphorylated, BfmR binds this promoter sequence with a lower affinity than when not phosphorylated. All other OmpR/PhoB family members studied to date show an increase in DNA-binding affinity upon phosphorylation. Understanding the structural and biochemical mechanisms of BfmR will aid in the development of new antimicrobial therapies.     

Pseudosynapsis and Decreased Stringency of Meiotic Repair Pathway Choice on the Hemizygous Sex Chromosome of Caenorhabditis elegans Males

During meiosis, accurate chromosome segregation relies on homology to mediate chromosome pairing, synapsis, and crossover recombination. Crossovers are dependent upon formation and repair of double-strand breaks (DSBs) by homologous recombination (HR). In males of many species, sex chromosomes are largely hemizygous, yet DSBs are induced along nonhomologous regions. Here we analyzed the genetic requirements for meiotic DSB repair on the completely hemizygous X chromosome of Caenorhabditis elegans males. Our data reveal that the kinetics of DSB formation, chromosome pairing, and synapsis are tightly linked in the male germ line. Moreover, DSB induction on the X is concomitant with a brief period of pseudosynapsis that may allow X sister chromatids to masquerade as homologs. Consistent with this, neither meiotic kleisins nor the SMC-5/6 complex are essential for DSB repair on the X. Furthermore, early processing of X DSBs is dependent on the CtIP/Sae2 homolog COM-1, suggesting that as with paired chromosomes, HR is the preferred pathway. In contrast, the X chromosome is refractory to feedback mechanisms that ensure crossover formation on autosomes. Surprisingly, neither RAD-54 nor BRC-2 are essential for DSB repair on the X, suggesting that unlike autosomes, the X is competent for repair in the absence of HR. When both RAD-54 and the structure-specific nuclease XPF-1 are abrogated, X DSBs persist, suggesting that single-strand annealing is engaged in the absence of HR. Our findings indicate that alteration in sister chromatid interactions and flexibility in DSB repair pathway choice accommodate hemizygosity on sex chromosomes.     

Material properties of articular cartilage in the rabbit tibial plateau

The material properties of articular cartilage in the rabbit tibial plateau were determined using biphasic indentation creep tests. Cartilage specimens from matched-pair hind limbs of rabbits approximately 4 months of age and greater than 12 months of age were tested on two locations within each compartment using a custom built materials testing apparatus. A three-way ANOVA was used to determine the effect of leg, compartment, and test location on the material properties (aggregate modulus, permeability, and Poisson's ratio) and thickness of the cartilage for each set of specimens. While no differences were observed in cartilage properties between the left and right legs, differences between compartments were found in each set of specimens. For cartilage from the adolescent group, values for aggregate modulus were 40% less in the medial compartment compared to the lateral compartment, while values for permeability and thickness were greater in the medial compartment compared to the lateral compartment (57% and 30%, respectively). Values for Poisson's ratio were 19% less in the medial compartment compared to the lateral compartment. There was also a strong trend for thickness to differ between test locations. Similar findings were observed for cartilage from the mature group with values for permeability and thickness being greater in the medial compartment compared to the lateral compartment (66% and 34%, respectively). Values for Poisson's ratio were 22% less in the medial compartment compared to the lateral compartment.     

Effects of increased chronic loading on articular cartilage material properties in the Lapine tibio-femoral joint

Methods of producing relevant and quantifiable load alterations in vivo with which to study load-induced cartilage degeneration analogous to osteoarthritis are limited. An animal model was used to investigate the effects of increased chronic loads on articular cartilage. Mature rabbits were randomized into one of three experimentally loaded groups and a fourth unoperated control group. A mechanical-loading device was skeletally fixed to the hind limb of animals in the loaded groups. Engaging the device resulted in an additional load of 0%, +22% or +44% body weight to the medial compartment of the experimental knee, while allowing normal joint function. Following a 12-week loading protocol, a creep-indentation test and needle probe test were used to determine the biphasic material properties and thickness of the cartilage at four locations of each femoral and tibial condyle of the experimental and contralateral limbs. Analyses of covariance were performed to compare outcome measures across the treatment groups. The effect of increased load was site and load-level specific with alterations of material properties and thickness most prominent in the posterior region of the medial compartment of the tibia. At this site, permeability increased 128% and thickness increased 28% in the +44% body weight group relative to the 0% body weight group. This model of altered chronic loading initiated changes in the material properties to the articular cartilage at the sites of increased load over 12-weeks that were consistent with early degenerative changes suggesting that increased tibio-femoral loading may be responsible for the alterations. This work begins to elucidate the chronic-load threshold and the time course of cartilage degeneration at different levels of altered loading.     

Automatic determination of anatomical coordinate systems for three-dimensional bone models of the isolated human knee

The combination of three-dimensional (3-D) models with dual fluoroscopy is increasingly popular for evaluating joint function in vivo. Applying these modalities to study knee motion with high accuracy requires reliable anatomical coordinate systems (ACSs) for the femur and tibia. Therefore, a robust method for creating ACSs from 3-D models of the femur and tibia is required. We present and evaluate an automated method for constructing ACSs for the distal femur and proximal tibia based solely on 3-D bone models. The algorithm requires no observer interactions and uses model cross-sectional area, center of mass, principal axes of inertia, and cylindrical surface fitting to construct the ACSs. The algorithm was applied to the femur and tibia of 10 (unpaired) human cadaveric knees. Due to the automated nature of the algorithm, the within specimen variability is zero for a given bone model. The algorithm’s repeatability was evaluated by calculating variability in ACS location and orientation across specimens. Differences in ACS location and orientation between specimens were low (<1.5 mm and <2.5°). Variability arose primarily from natural anatomical and morphological differences between specimens. The presented algorithm provides an alternative method for automatically determining subject-specific ACSs from the distal femur and proximal tibia.