Purification and characterization of ribulose-5-phosphate kinase from spinach

An efficient purification procedure utilizing affinity chromatography is described for spinach ribulose-5-phosphate kinase, a light-regulated chloroplastic enzyme. Gel filtration and polyacrylamide gel electrophoresis of the purified enzyme reveal a dimeric structure of 44,000 Mr subunits. Chemical crosslinking with dimethyl suberimidate confirms the presence of two subunits per molecule of native kinase, which are shown to be identical by partial NH2-terminal sequencing. Based on sulfhydryl titrations and on amino acid analyses, each subunit contains four to five cysteinyl residues. The observed slow loss of activity during spontaneous oxidation in air-saturated buffer correlates with the intramolecular oxidation of two sulfhydryl groups, presumably those involved in thioredoxin-mediated regulation.     

Ribosomal subunit antiassociation activity in rabbit reticulocyte lysates. Evidence for a low molecular weight ribosomal subunit antiassociation protein factor (Mr = 25,000)

A ribosomal subunit antiassociation activity has been purified from both the postribosomal supernatant and ribosomal salt-wash protein fractions of rabbit reticulocyte lysates. A majority (greater than 90%) of the activity is associated with a low molecular weight protein of Mr of approximately 25,000. A small but significant level of antiassociation activity (less than 10%) was found to be associated with higher molecular weight protein fractions. The purified 25,000-dalton antiassociation factor interacts with 60 S ribosomal subunits to prevent them from reassociating with 40 S ribosomal subunits. The factor does not seem to interact directly with 40 S subunits nor does it dissociate 80 S monosomes. The properties of this factor are thus similar to the eukaryotic initiation factor 6 isolated from both wheat germ and calf liver extracts.     

Reparative growth in the rat thyroid

This study was designed to investigate the mitotic response to wounding in the rat thyroid. The spatial distribution of mitotic activity 48 hr after incision of the thyroid isthmus, or mere exposure of the gland (sham-operation), was assessed using a stathmokinetic technique. Incision resulted in a 66-fold increase over normal in metaphase index adjacent to the wound, falling over 2 mm to a stable 13-fold elevation. Sham-operation produced a smaller response with a complete return to normal levels over 1-1 X 5 mm. The results demonstrate that there is a dramatic localized mitotic response to wounding in the thyroid together with a smaller generalized response. Further, the response to sham-operation indicates that thyroid follicular cells respond to a diffusible 'wound hormone'. We suggest that this may be a major mechanism mediating reparative growth in this gland.     

Refixation of xylem sap CO2 in Populus deltoides

Vascular plants have respiring tissues which are perfused by the transpiration stream, allowing solubilization of respiratory CO₂ in the xylem sap. The transpiration stream could provide a conduit for the internal delivery of respiratory CO₂ to leaves. Trees have large amounts of respiring tissues in the root systems and stems, and may have elevated levels of CO₂ in the xylem sap which could be delivered to and refixed by the leaves. Xylem sap from the shoots of three Populus deltoides trees had mean dissolved inorganic carbon concentrations (CO₂+H₂CO₃+HCO^?₃) ranging from 0. 5 to 0. 9 mM. When excised leaves were allowed to transpire 1 mM[¹⁴C]NaHCO₃, 99. 6% of the label was fixed in the light. Seventy-seven percent of the label was fixed in major veins and the remainder was fixed in the minor veins. Autoradiography confirmed that label was confined to the vasculature. In the dark, approximately 80% of the transpired label escaped the leaf, the remainder was fixed in the major veins, slightly elevating dark respiration measurements. This indicates that the vascular tissue in P. deltoides leaves is supplied with a carbon source distinct from the atmospheric source fixed by interveinal lamina. However, the contribution of CO₂ delivered to the leaves in the transpiration stream and fixed in the veins was only 0. 5% of atmospheric CO₂ uptake. In the light 90% of the label was found in sugar, starch and protein, a pattern similar to that found for atmospheric uptake of[¹⁴C]CO₂. Compared with leaves labelled in the light, leaves labelled in the dark had more label in organic acid, amino acid and protein and less label in sugar and starch. After a 5-s pulse the majority of the label fed to petioles in both the light and the dark was found in malate. The majority of the label was found in malate at 120 s in the dark; only 2% of the label was found in phosphorylated compounds at 120 s. The proportion of label found in phosphorylated compounds increased from 17% at 5 s to 80% at 120 s in the light. This suggests that CO₂ delivered to leaves in the light via the transpiration stream is fixed in the veins, a small portion through dark fixation into malate, the remainder by C-3 photosynthesis.     

Mugharet el'Aliya: Affinities of an enigmatic north African Aterian maxillary fragment

Objectives

This study uses a virtual framework to examine the left maxillary fragment of the juvenile fossil from Mugharet el'Aliya, Morocco, found in association with an Aterian lithic industry. Previously, this fossil had been ascribed to modern humans or the Neanderthal lineage based on its “archaic”/“Neanderthal-like” features and apparent large size. Here, we conducted a novel 3D shape comparative analysis of the maxillary fragment to clarify its taxonomic affinities with regard to its size and ontogeny.

Materials and Methods

Eighty Computed Tomography and surface scans representing ontogenetic samples of Homo sapiens and Homo neanderthalensis were used to capture species-specific differences. The toolkit of geometric morphometrics in combination with surface registration and an elastic iterative closest point algorithm were used to create a dataset of meshes with an identical number of corresponding vertices for the maxillae. Multivariate statistics were applied to Procrustes superimposed coordinates derived from the vertices of this dataset.

Results

Our analysis showed affinities of the Mugharet el'Aliya individual with our H. sapiens sample, especially with a subadult individual from Qafzeh. No size-independent affinities with Neanderthals of comparable dental age could be identified.

Discussion

Our results add to the evidence connecting fossils from western Asia, especially Qafzeh and Skhul, and the North African Aterian. Furthermore, Mugharet el'Aliya adds to our knowledge of the ontogenetic development of adult morphology that is frequently used to characterize hominin groups, for example, Neanderthals and modern humans.     

FLP-mediated site-specific recombination in microinjected murine zygotes

The FLP recombinase of yeast catalyses site-specific recombination between repeated FLP recombinase target (FRT) elements in yeast and in heterologous system (Escherichia coli, Drosophila, mosquito and cultured mammalian cells). In this report, it is shown that transient FLP recombinase expression can recombine and activate an extrachromosomal silent reporter gene following coinjection into fertilized one-cell mouse eggs. Furthermore, it is demonstrated that introduction of a FLP-recombinase expression vector into transgenic one-cell fertilized mouse eggs induces a recombination event at a chromosomal FRT target locus. The resulting event occured at the one-cell stage and deleted a chromosomal tandem array of a FRT containinglacZ expression cassette down to one or two copies. These results demonstrate that the FLP recombinase can be utilized to manipulate the genome of transgenic animals and suggest that FLP recombinase-mediated plasmid-to-chromosome targeting is feasible in microinjected eggs.     

Sequence variation at the major histocompatibility complex locus DQ beta in beluga whales (Delphinapterus leucas) [published erratum appears in Mol Biol Evol 1995 Nov;12(6):1174]

Genetic variation at the Major Histocompatibility Complex locus DQ beta was analyzed in 233 beluga whales (Delphinapterus leucas) from seven populations: St. Lawrence Estuary, eastern Beaufort Sea, eastern Chukchi Sea, western Hudson Bay, eastern Hudson Bay, southeastern Baffin Island, and High Arctic and in 12 narwhals (Monodon monoceros) sympatric with the High Arctic beluga population. Variation was assessed by amplification of the exon coding for the peptide binding region via the polymerase chain reaction, followed by either cloning and DNA sequencing or single-stranded conformation polymorphism analysis. Five alleles were found across the beluga populations and one in the narwhal. Pairwise comparisons of these alleles showed a 5:1 ratio of nonsynonymous to synonymous substitutions per site leading to eight amino acid differences, five of which were nonconservative substitutions, centered around positions previously shown to be important for peptide binding. Although the amount of allelic variation is low when compared with terrestrial mammals, the nature of the substitutions in the peptide binding sites indicates an important role for the DQ beta locus in the cellular immune response of beluga whales. Comparisons of allele frequencies among populations show the High Arctic population to be different (P < or = .005) from the other beluga populations surveyed. In these other populations an allele, Dele-DQ beta*0101-2, was found in 98% of the animals, while in the High Arctic it was found in only 52% of the animals. Two other alleles were found at high frequencies in the High Arctic population, one being very similar to the single allele found in narwhal.