Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

What can we learn from noncoding regions of similarity between genomes?

Background  

In addition to known protein-coding genes, large amounts of apparently non-coding sequence are conserved between the human and mouse genomes. It seems reasonable to assume that these conserved regions are more likely to contain functional elements than less-conserved portions of the genome.     

Probing the proton channel and the retinal binding site of Natronobacterium pharaonis sensory rhodopsin II

The sensory rhodopsin II from Natronobacterium pharaonis (NpSRII) was mutated to try to create functional properties characteristic of bacteriorhodopsin (BR), the proton pump from Halobacterium salinarum. Key residues from the cytoplasmic and extracellular proton transfer channel of BR as well as from the retinal binding site were chosen. The single site mutants L40T, F86D, P183E, and T204A did not display altered function as determined by the kinetics of their photocycles. However, the photocycle of each of the subsequent multisite mutations L40T/F86D, L40T/F86D/P183E, and L40T/F86D/P183E/T204A was quite different from that of the wild-type protein. The reprotonation of the Schiff base could be accelerated approximately 300- to 400-fold, to approximately two to three times faster than the corresponding reaction in BR. The greatest effect is observed for the quadruple mutant in which Thr-204 is replaced by Ala. This result indicates that mutations affecting conformational changes of the protein might be of decisive importance for the creation of BR-like functional properties.     

An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains

To select a Saccharomyces cerevisiae reference strain amenable to experimental techniques used in (molecular) genetic, physiological and biochemical engineering research, a variety of properties were studied in four diploid, prototrophic laboratory strains. The following parameters were investigated: 1) maximum specific growth rate in shake-flask cultures; 2) biomass yields on glucose during growth on defined media in batch cultures and steady-state chemostat cultures under controlled conditions with respect to pH and dissolved oxygen concentration; 3) the critical specific growth rate above which aerobic fermentation becomes apparent in glucose-limited accelerostat cultures; 4) sporulation and mating efficiency; and 5) transformation efficiency via the lithium-acetate, bicine, and electroporation methods. On the basis of physiological as well as genetic properties, strains from the CEN.PK family were selected as a platform for cell-factory research on the stoichiometry and kinetics of growth and product formation.     

Pre-Use Susceptibility to Ceftaroline in Clinical Staphylococcus aureus Isolates from Germany: Is There a Non-Susceptible Pool to be Selected?

Ceftaroline is a new cephalosporin active against Methicillin-resistant Staphylococcus aureus (MRSA). Based on a representative collection of clinical S. aureus isolates from Germany, supplemented with isolates of clonal lineages ST228 and ST239, we demonstrate the in-vitro susceptibility towards ceftaroline prior to its introduction into clinical use for a total of 219 isolates. Susceptibility testing was performed by broth microdilution, disc diffusion and Etest, respectively. Results were interpreted according to EUCAST guidelines and showed considerable variance in dependence on clonal affiliation of the isolates tested. Among isolates of widespread hospital-associated lineages we found a high proportion of clinical isolates with MICs close to the EUCAST breakpoint (MIC_50/90 1.0/1.5 mg/L); currently, interpretation of these “borderline” MICs is complicated by a lack of concordant susceptibility testing methods and reasonable breakpoint determination. Isolates of clonal lineages ST228 and ST239 demonstrated increased MIC_50/90 values of 2.5/3.33 mg/L. Sequencing of mecA revealed no association of resistance to a specific mecA polymorphism, but rather reveals two regions in the non-penicillin-binding domain of PbP2a which displayed different combinations of mutations putatively involved in resistance development. This study provides national baseline data to (i) adjust susceptibility testing methods and current breakpoints to clinical and epidemiological requirements, (ii) evaluate current breakpoints with respect to therapeutic outcome and (iii) monitor further resistance evolution.     

Conformational changes of the histidine ATP-binding cassette transporter studied by double electron–electron resonance spectroscopy

The conformational dynamics of the histidine ABC transporter HisQMP₂ from Salmonella enterica serovar Typhimurium, reconstituted into liposomes, is studied by site-directed spin labeling and double electron–electron resonance spectroscopy in the absence of nucleotides, in the ATP-bound, and in the post-hydrolysis state. The results show that the inter-dimer distances as measured between the Q-loops of HisP₂ in the intact transporter resemble those determined for the maltose transporter in all three states of the hydrolysis cycle. Only in the presence of liganded HisJ the closed conformation of the nucleotide binding sites is achieved revealing the transmembrane communication of the presence of substrate. Two conformational states can be distinguished for the periplasmic moiety of HisQMP₂ as detected by differences in distributions of interspin distances between positions 86 and 96 or 104 and 197. The observed conformational changes are correlated to proposed open, semi-open and closed conformations of the nucleotide binding domains HisP₂. Our results are in line with a rearrangement of transmembrane helices 4 and 4′ of HisQM during the closed to the semi-open transition of HisP₂ driven by the reorientation of the coupled helices 3a and 3b to occur upon hydrolysis.