Fusions between green fluorescent protein and β-glucuronidase as sensitive and vital bifunctional reporters in plants

By fusing the genes encoding green fluorescent protein (GFP) and -glucuronidase (GUS) we have created a set of bifunctional reporter constructs which are optimized for use in transient and stable expression studies in plants. This approach makes it possible to combine the advantage of GUS, its high sensitivity in histochemical staining, with the advantages of GFP as a vital marker. The fusion proteins were functional in transient expression studies in tobacco using either DNA bombardment or potato virus X as a vector, and in stably transformed Arabidopsis thaliana and Lotus japonicus plants. The results show that high level of expression does not interfere with efficient stable transformation in A. thaliana and L. japonicus. Using confocal laser scanning microscopy we show that the fusion constructs are very suitable for promoter expression studies in all organs of living plants, including root nodules. The use of these reporter constructs in the model legume L. japonicus offers exciting new possibilities for the study of the root nodulation process.     

Deletion of the pseudoautosomal region and lack of sex-chromosome pairing at pachytene in two infertile men carrying an X;Y translocation

Two males with a 46,Y,der(X),t(X;Y)(p22.3;q11) complement were referred independently for evaluation of sterility with azoospermia. Both patients exhibited minimal symptomatology, characterized only by psychological disturbances. Study of X-chromosome breakpoints with pseudoautosomal probes 68B (DXYZ2 elements), 113D (locus DXYS15), and 19B (locus MIC2) indicated in both patients that at least 97% of the X pseudoautosomal sequences are lost. Hybridization with Xp22.3-specific probes DXS283, DXS284, and DXS31 shows that these loci are retained on the rearranged chromosome. Thus, the X-chromosome breakpoints are located close to the proximal boundary of the pseudoautosomal region, between MIC2 and DXS284.     

Acetylcholine-gated current translates wake neuronal firing rate information into a spike timing-based code in Non-REM sleep,stabilizing neural network dynamics during memory consolidation

Sleep is critical for memory consolidation, although the exact mechanisms mediating this process are unknown. Combining reduced network models and analysis of in vivo recordings, we tested the hypothesis that neuromodulatory changes in acetylcholine (ACh) levels during non-rapid eye movement (NREM) sleep mediate stabilization of network-wide firing patterns, with temporal order of neurons’ firing dependent on their mean firing rate during wake. In both reduced models and in vivo recordings from mouse hippocampus, we find that the relative order of firing among neurons during NREM sleep reflects their relative firing rates during prior wake. Our modeling results show that this remapping of wake-associated, firing frequency-based representations is based on NREM-associated changes in neuronal excitability mediated by ACh-gated potassium current. We also show that learning-dependent reordering of sequential firing during NREM sleep, together with spike timing-dependent plasticity (STDP), reconfigures neuronal firing rates across the network. This rescaling of firing rates has been reported in multiple brain circuits across periods of sleep. Our model and experimental data both suggest that this effect is amplified in neural circuits following learning. Together our data suggest that sleep may bias neural networks from firing rate-based towards phase-based information encoding to consolidate memories.     

The role of protein in the estrogen-stimulated in vitro RNA synthesis of isolated rat uterine nucleoli

Immature rat uterine nucleoli were isolated and their ability to synthesize RNA in vitro was determined. Estradiol-17β injected intraperitoneally 2 hr prior to killing stimulated rat uterine nucleolar in vitro RNA synthesis both quantitatively and qualitatively. The intraperitoneal administration of cycloheximide as late as 10 min prior to the end of a 2-hr estrogen exposure prevented both the quantitative and qualitative changes stimulated by estrogen. The data suggest that estrogen-stimulated rat uterine nucleolar RNA synthesis requires the continuous synthesis of protein.     

Expression, purification, kinetic, and structural characterization of an alpha-class carbonic anhydrase from Aedes aegypti (AaCA1)

Carbonic anhydrases (CAs) are zinc-containing metalloenzymes that catalyze the interconversion of carbon dioxide and bicarbonate. The alpha-class CAs are found predominantly in vertebrates, but they are also expressed in insects like mosquitoes. Recently, an alpha-CA from the midgut of Aedes aegypti larvae (AaCA1) was identified, cloned, and subsequently shown to share high sequence homologous to human CA I (HCA I). This paper presents the bacterial expression, purification, and kinetic characterization of the soluble CA domain of AaCA1. The data show AaCA1 is a highly active CA that displays inhibition by methazolamide and ethoxzolamide with nM affinity. Additionally, a homology model of AaCA1, based on the crystal structure of HCA I, is presented and the overall structure, active site, and surface charge properties are compared to those of HCA I and II. Measurements of catalysis show that AaCA1 is more like HCA II in terms of proton transfer, but more similar to HCA I in terms of conversion of carbon dioxide to bicarbonate, and these differences are rationalized in terms of structure. These results also indicate that amino acid differences in the active site of AaCA1 compared to human CAs could be used to design specific CA inhibitors for the management of mosquito populations.