Robert C. Benjamin

Identification of enzymatic activities which process protein bound mono(ADP-ribose)

Enzymatic activities have been identified in extracts of cultured mouse cells which catalyze the removal of intact mono(ADP-ribosyl) residues linked to proteins at arginine. Activities that sequentially remove AMP and ribose 5-phosphate have also been identified. These results suggest that mono(ADP-ribosylation) of proteins is a reversible post translational modification.

Characterization of recognition sites on bacteriophage HP1c1 DNA which interact with the DNA uptake system of Haemophilus influenzae Rd

The 32.4-kb genome of the Haemophilus influenzae bacteriophage HP1c1 contains at least twelve sites, each conferring high affinity for the DNA uptake system of transformable H. influenzae Rd. Five of these high-affinity sites have been located and their nucleotide sequences determined. Three sites contained a contiguous 9-bp sequence identical to the first nine residues of the 11-bp site previously identified as conferring high affinity for the H. influenzae transformation receptor to DNA fragments. The remaining two sites contained complete 11-bp sequences. In contrast, an HP1c1 restriction fragment containing a sequence identical to the final nine residues of the 11-bp uptake site exhibits only a low affinity for the DNA uptake system. An 8-bp sequence consisting of the first eight residues of the 11-bp site was 1% as active as the longer, high-affinity sites. Thus the first 9-bp of the 11-bp site are sufficient to direct high-affinity uptake, while the first 8-bp or the distal 9-bp are not. These results provide an initial assessment of the relative contributions of the individual residues constituting the 11-bp site to the apparent affinity of DNA fragments for the receptor of Haemophilus transformation.

Assessment of the role of DNA repair in damaged forensic samples

Previous studies on DNA damage and repair have involved in vitro laboratory procedures that induce a single type of lesion in naked templates. Although repair of singular, sequestered types of DNA damage has shown some success, forensic and ancient specimens likely contain a number of different types of lesions. This study sought to (1) develop protocols to damage DNA in its native state, (2) generate a pool of candidate samples for repair that more likely emulate authentic forensic samples, and (3) assess the ability of the PreCRTM Repair Mix to repair the resultant lesions. Complexed, native DNA is more difficult to damage than naked DNA. Modified procedures included the use of higher concentrations and longer exposure times. Three types of samples, those that demonstrated damage based on short tandem repeat (STR) profile signals, were selected for repair experiments: environmentally damaged bloodstains, bleach-damaged whole blood, and human skeletal remains. Results showed trends of improved performance of STR profiling of bleach-damaged DNA. However, the repair assay did not improve DNA profiles from environmentally damaged bloodstains or bone, and in some cases resulted in lower RFU values for STR alleles. The extensive spectrum of DNA damage and myriad combinations of lesions that can be present in forensic samples appears to pose a challenge for the in vitro PreCRTM assay. The data suggest that the use of PreCR in casework should be considered with caution due to the assay’s varied results.

Parentage and relatedness in captive and natural populations of the Roseate Spoonbill (Aves: Ciconiiformes) based on microsatellite data

This study constitutes a first approach to evaluate the use of genetic information and relatedness estimators in the investigation of questions related to mating system and parentage in ex situ and in situ populations of the Roseate Spoonbill. We assessed the parentage assignments in 17 supposed families from US captive populations and investigated the genetic relationships among 67 nestlings sampled within 28 nests in Brazilian natural breeding colonies. Estimations of genetic relatedness values, hypothesis testing methods, simulations and maximum likelihood approaches were performed on data from four microsatellite loci. Parentage was confirmed in 61.5% of the registered parent-offspring relationships at zoo parks. Inconsistencies in assignments were investigated and the likely parents were identified for most of the hatchlings. Matings among relatives, not previously noticed based on behavioral observations, were identified by the use of genetic analyses. In natural populations, 33% of the sampled dyads were confidently classified as full-sibs. Above 25% of the analyzed dyads were unrelated, indicating that more than one parent-pair may have been responsible for the progeny. Our results demonstrate that genetic information can augment the precision in parentage assignment in captive Roseate Spoonbill populations, and this approach can contribute to their management and conservation. Results obtained using three different methodologies are concordant and point to the existence of a mating system other than monogamy for this species in the wild. The approaches implemented in this study can be applied to other waterbird species in which capture of adults is difficult.

Alteration of poly(adenosine diphosphoribose) metabolism by ethanol: Mechanism of action☆

We present evidence that ethanol alters intracellular poly(adenosine diphosphoribose) metabolism and we further describe the mechanism by which ethanol exerts its effect on polymer synthesis. One percent ethanol stimulates polymer accumulation as much as 2.5-fold but does not alter polymer degradation in intact cells following DNA damage. Ethanol directly stimulates polymer synthesis following low doses of DNA damage induce by deoxyribonuclease I in a nucleotide-permeable cell system that does not possess a functional polymer turnover system. Ethanol has no measurable effect on polymer synthesis in undamaged nucleotide-permeable cells or in permeable cells treated with high doses of deoxyribonuclease I. Ethanol concentrations that stimulate poly(adenosine diphosphoribose) polymerase activity in vitro specifically lower KDNA without affecting KNAD or Vmax. The results clearly show that ethanol alters the binding of this enzyme to the DNA component of chromatin and that this altered binding is responsible for the activation of the enzyme. Altered affinity of poly(adenosine diphosphoribose) polymerase and perhaps other regulatory proteins for chromatin may play an important role in the pathology of alcohol.

Kinetic Mechanism of Poly(ADP-Ribose) Polymerase

Poly(ADP-ribose) polymerase is a chromatin-associated enzyme which, in the presence of fragmented DNA, assembles branched homopolymers from the ADP-ribose moiety of NAD (reviewed in [1]). Fragmented DNA is an essential activator of the polymerase and is not modified by the reaction. This report presents data related to the kinetic mechanism of DNA activation of the poly(ADP-ribose) polymerase.

Modification of an arginine residue essential for the activity of NAD-malic enzyme from Ascaris suum

Purified NAD-malic enzyme from Ascaris suum is rapidly inactivated by the arginine reagent, 2,3-butanedione, and this inactivation is facilitated by 30 mM borate. Determination of the inactivation rate as a function of butanedione concentration suggests a second-order process overall, which is first order in butanedione. A second-order rate constant of 0.6 M-1 s-1 at pH 9 is obtained for the butanedione reaction. The inactivation is reversed by removal of the excess reagent upon dialysis. The enzyme is protected against inactivation by saturating amounts of malate in the presence and absence of borate. The divalent metal Mg2+ affords protection in the presence of borate but has no effect in its absence. The nucleotide reactant NAD+ has no effect on the inactivation rate in either the presence or absence of borate. A dissociation constant of 24 mM is obtained for E:malate from the decrease in the inactivation rate as a function of malate concentration. An apparent Ki of 0.5 mM is obtained for oxalate (an inhibitor competitive vs malate) from E:Mg:oxalate while no significant binding is observed for oxalate using the butanedione modified enzyme. The pH dependence of the first-order rate of inactivation by butanedione gives a pKa of 9.4 +/- 0.1 for the residue(s) modified, and this pK is increased when NAD is bound. The arginine(s) modified is implicated in the binding of malate.

A POSSIBLE ROLE FOR POLY ADP-RIBOSE IN DNA REPAIR

ABSTRACT The synthesis of poly ADP-ribose by BSC or HeLa cell ghosts is dramatically increased by X-irradiation or endonuclease treatment. In a cell-free system it is stimulated by restriction nuclease-digested plasmid DNA according to the type and number of cuts. Thus it probably occurs at or near cut ends of double-stranded DNA and may well occur there in vivo after X-irradiation.

Nucleotide sequence of cloned DNA segments of the Haemophilus influenzae bacteriophage HP1c1

Restriction fragments obtained by digestion of Haemophilus influenzae phage HP1c1 DNA with HaeIII have been cloned by insertion into the HindIII site of pBR322 using synthetic linkers. The nucleotide sequences have been determined for three adjacent fragments, HaeIII-E, HaeIII-C and HaeIII-K, which comprise and 8.2-kb segment of the HP1c1 genome. The distribution and location of restriction sites in the sequenced region were determined. Restriction sites containing the dinucleotides -GG- and -CC- occurred infrequently in the sequence. The region contains numerous sequences which are subsets of the high-affinity recognition sequence of Haemophilus transformation, including five sites which direct high-affinity uptake of fragments containing them. Shorter subsets of the uptake sequence, including those with little or no measurable affinity for the DNA transport system, are considerably over-represented in HP1c1 DNA. Nine open reading frames (ORFs) corresponding to presumed polypeptides longer than 90 amino acid residues were identified; all of these shared a common orientation, suggesting the probable direction of transcription in this segment of the phage genome. These ORFs were preceded by appropriately spaced polypurine stretches which might function as ribosome-binding sites. One ORF coincides with the site of a mutation affecting the production of phage tails.

Validation of alternative capillary electrophoresis detection of STRs using POP-6 polymer and a 22 cm array on a 3130xl genetic analyzer

The goal of this project was to reduce capillary electrophoresis detection time on a 3130xl Genetic Analyzer for amplification product obtained from 4-dye and 5-dye STR amplification kits while still generating high quality STR profiles. This was accomplished by utilizing a more viscous polymer (POP-6™) and a shorter array (22 cm) than that which are typically used (POP-4(®) polymer and a 36 cm array) for human identification purposes. Spatial calibration and detection run modules were modified in response to the use of this polymer/array combination and to reduce detection time. Alternative detection resulted in 24-28 min run times, as compared to ∼45 min using traditional POP-4(®)/36 cm detection methods. POP-6™/22 cm detection run modules were validated for use with 4-dye Promega STR kits (e.g., PowerPlex(®) 16 and PowerPlex(®) 16HS) and 5-dye Life Technologies kits (e.g., Identifiler(®) and Identifiler(®) Plus). Three hundred ninety-five samples, controls and allelic ladders were used for the validation studies, which consisted of a comparison of alternative POP-6™/22 cm detection to traditional POP-4(®)/36 cm (including reproducibility/concordance of allele calls, resolution, ILS sizing quality, peak height and pass rates), a sizing study (precision and accuracy) and a sensitivity study to obtain a usable range of injection times. Compared to traditional POP-4(®)/36 cm detection, alternative detection resulted in 100% reproducible and concordant alleles, the ability to achieve one base resolution, slightly reduced ILS sizing quality, slightly reduced peak height and statistically similar pass rates (α=0.05). It should be noted that alternative detection offered improved resolution over that of traditional for amplicons less than ∼200 b, but had reduced resolution for products greater than ∼200 b. Additionally, alternative detection yielded acceptable precision and accuracy of sizing using Life Technologies criteria (<0.15 standard deviation of allele sizing and ±0.5b sizing differences for the same allele) and usable injection parameters of 2 kV 4-15s (compared to 3 kV 10s for traditional). The run modules developed and validated for 4-dye and 5-dye STR kits using POP-6™ polymer on a 22 cm array offer a tremendous reduction in detection time (∼40%) while still generating high quality STR profiles.