This work presents the development of a highly sensitive and selective chemical assay for mono(ADP-ribose) residues covalently bound to proteins in vivo. An extensive review of the literature is presented in the introduction of this work. The physiological.functions of mono(ADP-ribosyl)transferase activities associated with certain bacterial toxins (e.g., diphtheria, cholera and pertussis toxins) are well established. However, the roles of endogenous vertebrate transferases are unknown. The elucidation of the roles of these cellular transferases will likely require identification of the physiologically relevant target proteins. Toward this end, it will also be important to identify the types of (ADP-ribose)-protein linkages present in vivo. ADP-ribosylation reactions catalyzed by the different bacterial and vertebrate transferases are specific for different amino acid acceptors in vitro. However, the vertebrate transferases that have been characterized thus far are NAD:arginine mono(ADP-ribosyl)transferases. The work presented here describes the development of a chemical assay for the detection of in vivo modified, ADP-ribosylated proteins containing N-glycosylic linkages to arginine. The assay was applied to the analysis of ADP-ribose residues in adult rat liver. The strategy employed for detection of protein-bound ADP-ribose residues eliminated potential artifacts arising from trapped nucleotides (or their degradation products), since the acid-insoluble material was completely dissolved in …

Two phage lambda clones encompassing human tRNA genes have been isolated from a human gene library harbored in bacteriophage lambda Charon-UA. One of the clones (designated as hLeuU) containing a 20-kb human DNA fragment was isolated and found to contain a cluster of four tRNA genes. An 8.2-kb Hindlll fragment encompassing the four tRNA genes was isolated from the 20-kb fragment and subcloned into pBR322 for restriction mapping and DNA sequence analysis. The four tRNA genes are arranged as two tandem pairs with the first pair containing a proline tRNAAGQ gene and a leucine tRNAAAQ gene and the second pair containing another proline tRNAAGG gene and a threonine tRNAuQU gene. The two pairs are separated about 3 kb from each other, and the leucine tRNAAAG gene is of opposite polarity from the other three tRNA genes. The tRNA transcription units were sequenced by a unidirectional deletion dideoxyribonucleotide chain-termination method in the M13mpl8 and 19 vectors. The coding regions of the four tRNA genes contain characteristic internal split promoter sequences and do not encode intervening sequences nor the CCA trinucleotide found in mature tRNAs. The proline t R N A A G G gene is separated from the leucine t R …

This study demonstrates the presence of protein kinase C activity in both cytosolic and membrane fractions of bovine lens epithelial cells in culture. Protein kinase C activity is similar in normal and hyperglycemic cells. Furthermore, the ability of the enzyme to translocate from the cytosol to the membrane following phorbol ester treatment is unimpeded by hyperglycemic conditions. Moreover, protein kinase C activation had no effect on myoinositol uptake either in normal cells or in cells exposed to hyperglycemic conditions.

Two plaque-pure phage lambda clones designated as λhtX-l and λhtX-2 that hybridized to unfractionated bovine liver tRNA were isolated from a human X chromosome-specific library. The λDNAs were characterized by restriction mapping and Southern blot hybridization techniques. The human DNA segment in λhtX-l contains five or more presumptive tRNA genes and at least one Alu family member. The 19-kilobase human DNA insert in λhtX-2 contains two or more presumptive tRNA genes and at least three Alu family members. Another human genomic clone designated λhVKV7 hybridized to mammalian valine tRNA IAC. The clone was characterized by physical mapping and Southern blot hybridization techniques. The 18.5-kilobase human DNA fragment in λhVKV7 contains a cluster of three tRNA genes and at least nine Alu family members.

Two different cloned human DNA segments encompassing transfer RNA gene and pseudogene clusters have been isolated from a human gene library harbored in bacteriophage lambda Charon 4-A. One clone (designated as λhVal7) encompassing a 20.5-kilobase (Kb) human DNA insert was found to contain a valine transfer RNA_AAC gene and several Alu-like elements by Southern blot hybridization analysis and DNA sequencing with the dideoxyribonucleotide chain-termination method in the bacteriophage M13mp19 vector. Another lambda clone (designated as λhLeu8) encompassing a 14.3-Kb segment of human DNA was found to contain a methionine elongator transfer RNA_CAT pseudogene and other as yet unidentified transfer RNA pseudogenes.

Glucose phosphate isomerase (GPI) was purified from human placenta utilizing cross-linked spherical particle phosphocellulose. In three steps, GPI could be purified approximately 5500 fold with greater than 50% recovery. The purified enzyme exhibited four bands upon non-denaturing PAGE and isoelectric focusing (IEF) when stained with GPI specific activity stain. The four isozymes were isolated by preparative IEF. The isoelectric points of the isozymes were determined. Sodium dodecyl sulfate (SDS) gel electrophoresis showed two types of subunits with different molecular weights. Structural analyses showed both types of subunits had blocked amino termini. Other properties of the isozymes and subunits, including immunological reactivity, pH stability, peptide mapping and amino acid composition, were also established.

Methodology was developed which allowed the rapid and routine quantitation of subpicomole quantities of diadenosine-5ʹ,5ʹʹʹ-P¹,P⁴-tetraphosphate (Ap₄A) in cultured mammalian cells. This methodology includes the rapid extraction of cellular nucleotides in cold alkali, resolution of Ap₄A from the bulk of cellular materials on a highly specific boronate affinity resin, and quantitation of the dinucleotide in a coupled bioluminescence assay utilizing venom phosphodiesterase and firefly luciferase. The sensitivity and selectivity of this assay is demonstrated and contrasted with previously developed techniques. This assay was used to examine the role of Ap₄A in DNA replication and the cellular stress response.