Fusion protein-based epitope mapping of phytochrome. Precise identification of an evolutionarily conserved domain

Fusion proteins are used to define with precision an evolutionarily conserved domain on the carboxyl-terminal portion of the chromoprotein phytochrome. Simultaneously, assignments of two other epitopes are made with significantly greater precision, while the location of a fourth is confirmed. The epitope-mapping method that is described here is systematic, using complementary, overlapping nested sets of fusion proteins of predefined sequence rather than randomly generated peptides. Moreover, in contrast to previous methods, this approach yields rigorous and unambiguous assignments because it relies solely upon the ability of an antibody to detect a given polypeptide. A cDNA fragment encoding phytochrome amino acids 464-1129, which is its carboxyl terminus, was identified in lambda gt11 and subcloned in frame into the lacZ alpha sequence of pUC18. Four nested sets of subclones in pUC18 were created by digestion with selected restriction endonucleases and with the exonuclease Bal31. Fusion proteins were analyzed by immunoblotting following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The epitope for monoclonal antibody Oat-13 was confirmed to be between residues 551 and 617, while the epitopes for Oat-8 and Oat-28 were narrowed to 624-686 and 624-747, respectively. The epitope recognized by Pea-25, Pea-2, and Oat-15 was resolved unequivocally to a sequence of only seven residues (residues 765-771): N-Pro-Ile-Phe-Gly-Ala-Asp-Glu-C.     

Multiple mechanisms of membrane anchoring of Escherichia coli penicillin-binding proteins

Abstract: The major penicillin-binding proteins (PBPs) of Escherichia coli play vital roles in cell wall biosynthesis and are located in the inner membrane. The high M _r PBPs 1A, 1B, 2 and 3 are essential bifunctional transglycosylases/transpeptidases which are thought to be type II integral inner membrane proteins with their C-terminal enzymatic domains projecting into the periplasm. The low M _r PBP4 is a DD-carboxypeptidase/endopeptidase, whereas PBPs 5 and are DD-carboxypeptidases. All three low M _r , PBPs act in the modification of peptidoglycan to allow expansion of the sacculus and are thought to be periplasmic proteins attached with varying affinities to the inner membrane via C-terminal amphiphilic α-helices. It is possible that the PBPs and other inner membrane proteins form a peptidoglycan synthesizing complex to coordinate their activities.     

Linkage of a new mutation in the proteolipid protein (PLP) gene to Pelizaeus-Merzbacher disease (PMD) in a large Finnish kindred.

The purpose of this study was to confirm linkage of the proteolipid protein gene (PLP) and Pelizaeus-Merzbacher disease (PMD). A T-->A transversion in nucleotide pair 35 of exon 4 of PLP was found in a large Finnish kindred with PMD. This mutation results in the substitution Val165-->Glu165. We used a combination of single-strand conformational polymorphism and PCR primer extension to determine the presence or absence of the point mutation in family members. A lod score of 2.6 (theta = 0) was found for linkage of the gene and the disease. We examined 101 unrelated X chromosomes and found none with the transversion. This is the second report of linkage of PMD to a missense mutation in PLP. These findings support the hypothesis that PMD in this family is a result of the missense mutation present in exon 4 of PLP.     

Synthesis,characterization, and electrocatalytic behaviour of hydrothermally grown nanostructured La2O3 and La2O3/K-complex

Rare earth metals play a conspicuous role in magnetic resonance imaging (MRI) for detecting cancerous cells. The alkali metal potassium is a neurotransmitter in the sodium–potassium pump in biomedical sciences. This unique property of rare earth metals and potassium drew our attention to carry forward this study. Therefore, in this work, previously synthesized potassium (K) complexes formed by the reflux of 4-N,N-dimethylaminobenzoic acid (DBA) and potassium hydroxide in methanol, and named [(μ2–4-N,N-dimethylaminobenzoate-κO)(μ2–4-N,N-dimethylaminobenzoic acid-κO)(4-N,N-dimethylaminobenzoic acid-κO) potassium(I) coordination polymer)] were treated hydrothermally with La₂O₃ nanomaterials to obtain a nanohybrid La₂O₃/K-complex. After that, the K-complex was analyzed using single-crystal X-ray diffraction and ¹H and ¹³C NMR spectroscopy. In addition, the structural and morphological properties of the as-prepared nanostructured La₂O₃/K-complex were also characterized, which involved an investigation using X-ray diffraction (XRD)spectroscopy, Fourier transform infrared (FTIR) spectroscopy, atomic force spectroscopy (AFM), transmission electron microscopy (TEM), and energy dispersive X-ray (EDX) analysis. After this, the electrochemical redox behaviour of the synthesized nanohybrid material was studied using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Therefore, the results from these studies revealed that the as-prepared material was a La₂O₃/K-complex that has a promising future role in sensing various analytes, as it showed effective electrocatalytic behaviour.     

Habitat selection and space use overlap between feral horses,pronghorn, and greater sage-grouse in cold arid steppe

Populations of feral horses (Equus ferus caballus) in the western United States have increased during the past decade, consequently affecting co-occurring wildlife habitat. Feral horses may influence 2 native wildlife species, greater sage-grouse (Centrocercus urophasianus; sage-grouse) and pronghorn (Antilocapra americana) through mechanisms of habitat alteration and competition. Wyoming, USA, contains the largest populations of pronghorn and sage-grouse of any state and also has the highest degree of range overlap between feral horses and these species. Consequently, the effects that horses may have on pronghorn and sage-grouse populations in Wyoming have implications at local, state, and population-wide levels. Managers need information concerning habitat selection and space use overlap among these species to develop appropriate management strategies; yet this information is absent for most feral horse management areas. To address this knowledge need, we attached global positioning system (GPS) transmitters to horses, pronghorn, and sage-grouse within the greater Bureau of Land Management–Adobe Town Herd Management Area in southern Wyoming and northern Colorado, USA, between 2017 and 2021 to evaluate habitat selection and space use of all species during 3 biologically relevant seasons: spring (Apr–Jun; sage-grouse breeding, nesting, and early-brood rearing; pronghorn late gestation and early parturition), summer (Jul–Oct; sage-grouse summer and late-brood rearing; pronghorn late parturition and breeding), and winter (Nov–Mar; non-breeding season). Feral horses selected flatter slopes and shorter mean shrub height across all seasons and were closer to water in spring and summer. Pronghorn habitat selection was similar to horses, but they also avoided oil and gas well pads year-round. During spring, sage-grouse selected greater herbaceous cover, flatter slopes, and areas farther from well pads. In summer, sage-grouse selected greater mean shrub height, flatter slopes, and were closer to water. In winter, sage-grouse selected flatter slopes and areas with greater vegetation production during the preceding summer. Our results indicate strong year-round overlap in space use between horses and pronghorn, whereas overlap between horses and sage-grouse is greatest during the summer in this region. Consequently, managers should recognize the potential for horses to influence habitat quality of pronghorn and sage-grouse in the region.     

Recruitment and Other Communication Behavior in the Ponerine Ant Ectatomma ruidum

The ponerine ant Ectatomma ruidum, though previously reported to possess only rudimentary recruitment ability, was found to lay chemical trails for mass recruitment to rich or difficult food sources. The pheromone originates from the Dufour's gland, a new source of trail pheromones in the primitive ant subfamily Ponerinae. During nest emigrations, E. ruidum practices stereotyped social carrying in the myrmicine mode. The discovery of this form of social carrying and of a recruitment pheromone in the Dufour's gland secretions support the hypothesis that the subfamily Myrmicinae is derived from an ectatommine ancestor. Other communication behaviors exhibited by E. ruidum include exchange of liquid food carried between the mandibles, chemical alarm communication, nest entrance marking, and an additional social carrying posture previously unknown in ants.     

Tomato contains two differentially expressed genes encoding B-type phytochromes,neither of which can be considered an ortholog of Arabidopsis phytochrome B

Tomato (Solanum lycopersicon L.) contains two B-type phytochrome genes (PHYB1 and PHYB2). Fragments of these two PHYB were cloned following amplification by the polymerase chain reaction of a portion of their relatively well conserved 5 coding regions. Polypeptides encoded by these gene fragments exhibit 90% sequence identity. These two PHYB are independently expressed in organ-specific fashion. In mature plants, PHYB2 mRNA is most abundant in fruit and PHYB1 mRNA in expanded leaves. A phylogenetic analysis fails to establish which tomato PHYB is orthologous to either Arabidopsis PHYB or PHYD, the latter being a second B-type phytochrome. Instead, this analysis indicates that following the divergence of the Solanaceae and Brassicaceae from one another, a PHYB gene duplicated independently in each lineage. Consequently, Arabidopsis PHYB mutants cannot be considered strictly equivalent to the tomato tri mutants, which appear to be mutated at the PHYB1 locus. Similarly, other putative PHYB mutants might not be equivalent to those described for Arabidopsis and tomato. This situation complicates efforts to determine PHYB function because there might be no one answer to this question.Abbreviations PCR polymerase chain reaction - PHY undesignated phytochrome gene - PHYA, PHYB, etc phytochrome gene(s) of the A, B, etc. type This research was supported by USDA NRICGP grant 93-00939 and by NATO travel grant CRG 931183. It was initiated when two of us (L.H.P., M.-M.C.-P.) spent a sabbatical year at the Institut National de la Recherche Agronomique in Versailles, France. L.H.P. gratefully acknowledges support provided by a senior guest fellowship from the Ministère de l'enseignement superieur et de la recherche during his stay in Versailles. L.H.P. and M.-M.C.-P thank all of their colleagues in Versailles for their warm hospitality and their willingness to share their expertise with us. We also thank Russell Malmberg, Richard Meagher and Robert Price for helpful discussions concerning the interpretation of molecular phylogenies.     

Substrate requirements for ErmC' methyltransferase activity.

ErmC' is a methyltransferase that confers resistance to the macrolide-lincosamide-streptogramin B group of antibiotics by catalyzing the methylation of 23S rRNA at a specific adenine residue (A-2085 in Bacillus subtilis; A-2058 in Escherichia coli). The gene for ErmC' was cloned and expressed to a high level in E. coli, and the protein was purified to virtual homogeneity. Studies of substrate requirements of ErmC' have shown that a 262-nucleotide RNA fragment within domain V of B. subtilis 23S rRNA can be utilized efficiently as a substrate for methylation at A-2085. Kinetic studies of the monomethylation reaction showed that the apparent Km of this 262-nucleotide RNA oligonucleotide was 26-fold greater than the value determined for full-size and domain V 23S rRNA. In addition, the Vmax for this fragment also rose sevenfold. A model of RNA-ErmC' interaction involving multiple binding sites is proposed from the kinetic data presented.     

The MUC5AC gene: RFLP analysis with the Jer58 probe

We have recently obtained evidence that the locus corresponding to three groups of partial tracheobronchial cDNAs (A=Jer47, B=Jer57, C=Jer58) which mapped to chromosome 11p15 and was given the symbol MUC5 corresponds to two distinct genes which we have provisionally called MUC5B and MUC5AC. Here we describe the detection, using the Jer58 probe, which contains a 24-bp tandem repeat, of polymorphism in the MUC5AC gene with seven different restriction enzymes.