The equilibrium constant and the reversibility of the reaction catalysed by nicotinamide-adenine dinucleotide kinase from pigeon liver.

The reversibility of the NAD+ kinase reaction was established, and the kinetic parameters of the rate equation in the reverse direction were determined. The equilibrium constant of the reaction was determined by using the purified pigeon liver enzyme and radioactively labelled nicotinamide nucleotides. The relationship between kinetic parameters of the forward and reverse reactions is in reasonable agreement with the measured equilibrium constant. As expected from the proposed mechanism of action, the enzyme does not catalyse isotope exchange between NAD+ and NADP+ in the absence of ADP and ATP. Although homogeneous as judged by polyacrylamide-gel electrophoresis, the enzyme preparation exhibits ADP/ATP isotope-exchange activity which could not be separated from NAD+ kinase activity, but kinetic evidence suggests that this is probably due to a contaminant.     

Early changes in concanavalin A-stimulated lymphocytes detected by the fluorescent probe N-phenyl-1-naphthylamine

Summary The hydrophobic fluorescent cell-membrane probe N-phenyl-1-naphthylamine (NPN) is a useful investigative tool for studies of early lymphocyte activation. NPN-labelled mouse thymus cells incubated with 5 g/ml concanavalin A (Con A) for 30 min at 37° C gave a reproducible increase in mean cell-fluorescence intensity measured by microfluorimetry on 100 single cells. The dose-response curve was similar to that obtained by ³H-thymidine assay.Increased fluorescence was not observed in the presence of 10 mM -methyl mannoside, 5mM sodium azide, 10^–5 M cytochalasin B, or Ca²⁺-free culture medium.However, incubation with 10^–5 M colchicine did not alter the probe response. Fluorescence change was also shown by spleen cells from a normal mouse but not from an athymic mouse, indicating T cell dependence of the response.Comparison with other lectins showed that increased fluorescence followed incubation with phytohaemagglutinin, and the non-mitogenic wheat germ lectin, but there was no change with succinyl-Con A, and decreased fluorescence with pokeweed mitogen. Use of fluorescent-labelled lectins showed that the NPN fluorescence change did not correlate with surface receptor patching and capping. Increased phospholipid-fatty acid turnover and subsequent increased membrane fluidity with alteration of molecular polarity are suggested as likely explanations of increased NPN fluorescence.Supported by a grant from the Anti-Cancer Council of VictoriaWe are grateful to Miss R. Jenkins and Mr. R. McGready for preparations of succinyl-Con A, to Dr. H.A. Ward for helpful discussion, and to Dr. M. Hohnes of the Walter and Eliza Hall Institute for providing BALB/c.nu mice     

Unexpected complexity of the dm1 mutation revealed in the structure of three H-2D/L-related antigens

The H-2L^dm1 and H-2D^dm1 MHC antigens of the B10.D2 (H-2 ^dm1 ) mutant mouse strain (formerly known as M504 or H-2 ^da ) have been compared to the H-2L^d and H-2D^d antigens of the B10.D2 (H-2 ^d ) mouse strain. L^dm1 and L^d are 45 000 M_r antigens and both are reactive with anti-H-2.28 (k/r anti-h2) serum and unreactive with anti-H-2.4 (k/b anti-a) serum which detects private determinants of the D^dm1 and D^d antigens. However, the tryptic peptide compositions of these two antigens are different and, based on the number of major tryptic peptides which coelute during ion-exchange chromatography, the estimated peptide homology between L^dm1 and L^d is 80 percent. A newly defined antigen (M_r = 39 000), designated gp39^dm1, was found in glycoprotein extracts of the ^dm1 strain but not of the d strain. This antigen coprecipitates with L^dm1 but does not coprecipitate with D^dm1 indicating that it lacks the H-2.4 determinant. In comparison with L^dm1, gp39^dm1 appears to contain far fewer Arg and Lys residues and is most likely not a simple proteolytic fragment of L^dm1. Finally, peptide maps of the D^dm1 antigen show that the majority of its Arg peptides are identical to D^d Arg peptides, whereas at least five of its Lys peptides and three of its Arg peptides correspond not to D^d peptides but to L^d and L^dm1 peptides. These data raise the possibility that the D^dm1 antigen is a hybrid molecule and they have also revealed an unexpected level of complexity in the dm1 mutant phenotype.     

DNA-mediated gene transfer in Chinese hamster ovary cells: Clonal variation in transfer efficiency

Summary Thymidine kinase-deficient Chinese hamster ovary (CHO) cells were genetically transformed with the BamHI restriction fragment encoding the thymidine kinase gene of herpes simplex virus (HSV-tk). We have observed considerable clonal variation among independent CHO sublines with respect to transformation competence for the DNA-mediated gene transfer of HSV-tk. Transformation frequencies 3×10^-4 were observed consistently in one subline, with a transformation efficiency of approximately 1 transformant per ng viral gene. The frequency and efficiency of transformation we observed in this system are at least 10-fold greater than those previously reported for DNA-mediated transformation of CHO cells by HSV-tk. All of the CHO HSV-tk⁺ transformants examined were stable for the transferred genotype in the absence of selection, and all showed evidence of co-transformation by unselected plasmid pBR322 sequences.A preliminary account of these results was given at the ICN-UCLA Symposia, March 21–28, 1982     

Role of the nucleotide excision repair gene ERCC1 in formation of recombination-dependent rearrangements in mammalian cells

Spontaneous recombination between direct repeats at the adenine phosphoribosyltransferase (APRT) locus in ERCC1-deficient cells generates a high frequency of rearrangements that are dependent on the process of homologous recombination, suggesting that rearrangements are formed by misprocessing of recombination intermediates. Given the specificity of the structure-specific Ercc1/Xpf endonuclease, two potential recombination intermediates are substrates for misprocessing in ERCC1^– cells: heteroduplex loops and heteroduplex intermediates with non-homologous 3′ tails. To investigate the roles of each, we constructed repeats that would yield no heteroduplex loops during spontaneous recombination or that would yield two non-homologous 3′ tails after treatment with the rare-cutting endonuclease I-SceI. Our results indicate that misprocessing of heteroduplex loops is not the major source of recombination-dependent rearrangements in ERCC1-deficient cells. Our results also suggest that the Ercc1/Xpf endonuclease is required for efficient removal of non-homologous 3′ tails, like its Rad1/Rad10 counterpart in yeast. Thus, it is likely that misprocessing of non-homologous 3′ tails is the primary source of recombination-dependent rearrangements in mammalian cells. We also find an unexpected effect of ERCC1 deficiency on I-SceI-stimulated rearrangements, which are not dependent on homologous recombination, suggesting that the ERCC1 gene product may play a role in generating the rearrangements that arise after I-SceI-induced double-strand breaks.     

Neuron-specific phosphorylation of Alzheimer's beta-amyloid precursor protein by cyclin-dependent kinase 5

The mature form of Alzheimer's beta-amyloid precursor protein (APP) is phosphorylated specifically at Thr(668) in neurons. In mature neurons, phosphorylated APP is detected in neurites, with dephosphorylated APP being found mostly in the cell body. In vitro, active cyclin-dependent kinase 5 (Cdk5) phosphorylated the cytoplasmic domain of APP at Thr(668). Treatment of mature neurons with an antisense oligonucleotide to Cdk5 suppressed Cdk5 expression and significantly diminished the level of phosphorylated APP. The expression of APP was unaffected in antisense-treated neurons. These results indicate that in neurons APP is phosphorylated by Cdk5, and that this may play a role in its localization.     

Role of ERCC1 in removal of long non-homologous tails during targeted homologous recombination

The XpF/Ercc1 structure-specific endonuclease performs the 5' incision in nucleotide excision repair and is the apparent mammalian counterpart of the Rad1/Rad10 endonuclease from Saccharomyces cerevisiae. In yeast, Rad1/Rad10 endonuclease also functions in mitotic recombination. To determine whether XpF/Ercc1 endonuclease has a similar role in mitotic recombination, we targeted the APRT locus in Chinese hamster ovary ERCC1(+) and ERCC1(-) cell lines with insertion vectors having long or short terminal non-homologies flanking each side of a double-strand break. No substantial differences were evident in overall recombination frequencies, in contrast to results from targeting experiments in yeast. However, profound differences were observed in types of APRT(+) recombinants recovered from ERCC1(-) cells using targeting vectors with long terminal non-homologies-almost complete ablation of gap repair and single-reciprocal exchange events, and generation of a new class of aberrant insertion/deletion recombinants absent in ERCC1(+) cells. These results represent the first demonstration of a requirement for ERCC1 in targeted homologous recombination in mammalian cells, specifically in removal of long non-homologous tails from invading homologous strands.     

Use of green fluorescent protein as A non-destructive marker for peanut genetic transformation

Summary The ability to non-destructively visualize transient and stable gene expression has made green fluorescent protein (GFP) a most efficient reporter gene for routine plant transformation studies. We have assessed two fluorescent protein mutants, enhanced GFP (EGFP) and enhanced yellow fluorescent protein (EYFP), under the control of the CaMV35S promoter, for their transient expression efficiencies after particle bombardment of embryogenic cultures of the peanut cultivar, Georgia Green. A third construct (p524EGFP.1) that expressed EGFP from a double 35S promoter with an AMV enhancer sequence also was compared. The brightest and most dense fluorescent signals observed during transient expression were from p524EGFP. 1 and EYFP. Optimized bombardment conditions consisted of 0.6 μm diameter gold particles, 12410 kPa bombardment pressure, 95 kPa vacuum pressure, and pretreatment with 0.4 M mannitol. Bombardments with p524EGFP.1 produced tissue sectors expressing GFP that could be visually selected under the fluorescence microscope over multiple subcultures. Embryogenic lines selected for GFP expression initially may have been chimeric since quantitative analysis of expression sometimes showed an increase when GFP-expressing lines, that also contained a hygromycin-resistance gene, subsequently were cultured on hygromycin. Transformed peanut plants expressing GFP were obtained from lines selected either visually or on hygromycin. Integration of the gfp gene in the genomic DNA of regenerated plants was confirmed by Southern blot hybridization and transmission to progeny.     

Functional roles of nonconserved structural segments in CFTR's NH2-terminal nucleotide binding domain

The cystic fibrosis transmembrane conductance regulator (CFTR), encoded by the gene mutated in cystic fibrosis patients, belongs to the family of ATP-binding cassette (ABC) proteins, but, unlike other members, functions as a chloride channel. CFTR is activated by protein kinase A (PKA)-mediated phosphorylation of multiple sites in its regulatory domain, and gated by binding and hydrolysis of ATP at its two nucleotide binding domains (NBD1, NBD2). The recent crystal structure of NBD1 from mouse CFTR (Lewis, H.A., S.G. Buchanan, S.K. Burley, K. Conners, M. Dickey, M. Dorwart, R. Fowler, X. Gao, W.B. Guggino, W.A. Hendrickson, et al. 2004. EMBO J. 23:282-293) identified two regions absent from structures of all other NBDs determined so far, a "regulatory insertion" (residues 404-435) and a "regulatory extension" (residues 639-670), both positioned to impede formation of the putative NBD1-NBD2 dimer anticipated to occur during channel gating; as both segments appeared highly mobile and both contained consensus PKA sites (serine 422, and serines 660 and 670, respectively), it was suggested that their phosphorylation-linked conformational changes might underlie CFTR channel regulation. To test that suggestion, we coexpressed in Xenopus oocytes CFTR residues 1-414 with residues 433-1480, or residues 1-633 with 668-1480, to yield split CFTR channels (called 414+433 and 633+668) that lack most of the insertion, or extension, respectively. In excised patches, regulation of the resulting CFTR channels by PKA and by ATP was largely normal. Both 414+433 channels and 633+668 channels, as well as 633(S422A)+668 channels (lacking both the extension and the sole PKA consensus site in the insertion), were all shut during exposure to MgATP before addition of PKA, but activated like wild type (WT) upon phosphorylation; this indicates that inhibitory regulation of nonphosphorylated WT channels depends upon neither segment. Detailed kinetic analysis of 414+433 channels revealed intact ATP dependence of single-channel gating kinetics, but slightly shortened open bursts and faster closing from the locked-open state (elicited by ATP plus pyrophosphate or ATP plus AMPPNP). In contrast, 633+668 channel function was indistinguishable from WT at both macroscopic and microscopic levels. We conclude that neither nonconserved segment is an essential element of PKA- or nucleotide-dependent regulation.