Mechanism of sequence-specific template binding by the DNA primase of bacteriophage T7

DNA primases catalyze the synthesis of the oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Biochemical studies have elucidated the mechanism for the sequence-specific synthesis of primers. However, the physical interactions of the primase with the DNA template to explain the basis of specificity have not been demonstrated. Using a combination of surface plasmon resonance and biochemical assays, we show that T7 DNA primase has only a slightly higher affinity for DNA containing the primase recognition sequence (5′-TGGTC-3′) than for DNA lacking the recognition site. However, this binding is drastically enhanced by the presence of the cognate Nucleoside triphosphates (NTPs), Adenosine triphosphate (ATP) and Cytosine triphosphate (CTP) that are incorporated into the primer, pppACCA. Formation of the dimer, pppAC, the initial step of sequence-specific primer synthesis, is not sufficient for the stable binding. Preformed primers exhibit significantly less selective binding than that observed with ATP and CTP. Alterations in subdomains of the primase result in loss of selective DNA binding. We present a model in which conformational changes induced during primer synthesis facilitate contact between the zinc-binding domain and the polymerase domain.     

Peptide ligands specific to the oxidized form of Escherichia coli thioredoxin

Thioredoxin (Trx) is a highly conserved redox protein involved in several essential cellular processes. In this study, our goal was to isolate peptide ligands to Escherichia coli Trx that mimic protein-protein interactions, specifically the T7 polymerase-Trx interaction. To do this, we subjected Trx to affinity selection against a panel of linear and cysteine-constrained peptides using M13 phage display. A novel cyclized conserved peptide sequence, with a motif of C(D/N/S/T/G)D(S/T)-hydrophobic-C-X-hydrophobic-P, was isolated to Trx. These peptides bound specifically to the E. coli Trx when compared to the human and spirulina homologs. An alanine substitution of the active site cysteines (CGPC) resulted in a significant loss of peptide binding affinity to the Cys-32 mutant. The peptides were also characterized in the context of Trx's role as a processivity factor of the T7 DNA polymerase (gp5). As the interaction between gp5 and Trx normally takes place under reducing conditions, which might interfere with the conformation of the disulfide-bridged peptides, we made use of a 22 residue deletion mutant of gp5 in the thioredoxin binding domain (gp5Delta22) that bypassed the requirements of reducing conditions to interact with Trx. A competition study revealed that the peptide selectively inhibits the interaction of gp5Delta22 with Trx, under oxidizing conditions, with an IC50 of approximately 10 microM.     

First report of paralytic shellfish poisoning (PSP) caused by Alexandrium tamiyavanichii in Kuantan Port,Pahang, East Coast of Malaysia

Harmful algal bloom (HAB) is a proliferation of algae, which naturally produce biotoxins and cause harmful effects to humans, the environment and organisms associated with it. Paralytic shellfish poisoning (PSP) was reported for the first time in Kuantan Port, Pahang, Malaysia, in November 2013, followed by a second episode in August 2014. The toxicity level reported during the second event was as high as 3500 μg of STX equiv./100 g shellfish. Ten people were hospitalized with PSP symptoms after consuming contaminated shellfish. This study was conducted at Kuantan Port to identify the organisms responsible for these events. Water samples were collected monthly for a period of 12 months beginning in September 2014. HAB species were identified based on their morphology using light and fluorescence microscopes, and their classification was supported by molecular evidence based on internal transcribed spacer (ITS) sequences. Monthly cell abundance of Alexandrium tamiyavanichii was measured at four sampling stations. Toxin production by three strains isolated from the area was determined using HPLC. Our results revealed the presence of several HAB species, including the PSP‐producing species A . tamiyavanichii . The highest cell density of A . tamiyavanichii was 840 cells L^?1. The presence of GTX components was detected in these strains. However, other toxin components could not be determined. This study reported, for the first time, the presence of PSP‐producing A . tamiyavanichii on the Pahang coast of east Peninsular Malaysia and confirmed that the PSP events in Kuantan Port were attributable to this species. The presence of this species further indicates that several safety measures need to be considered to safeguard public health, particularly in Pahang coastal waters.     

Chemostat optimization of biomass production of a mixed bacterial culture utilizing methanol

Summary A continuous culture technique was used to optimize the medium composition and growth conditions of a mixed bacterial culture utilizing methanol. The improved medium resulted in satisfactory growth, high-yield coefficients and gave a product containing reduced polysaccharide concentrations. Optimal growth and biomass yields occurred at pH 6.8 a temperature of 37° C and dissolved oxygen at >20% saturation. The maximum growth rate was 0.58 h^–1 and maximum biomass yield 0.48 g g^–1. The protein content of the product ranged between 81%–83%, and nucleic acid content between 10%–12%, increasing with growth rate. The amino acid profile of the mixed culture product met and, in some cases, exceeded the UN Food and Agricultural Organization standard, indicating a good source of feed protein.Offprint requests to: A. S. Abu-Ruwaida     

Yield depression in methanol-utilizing batch cultures

Summary Yield depression, as opposed to growth inhibition, in batch cultures of methanol-utilizing microorganisms is discussed. Under conditions where the yield coefficient varies, the effect on oxygen demand has been predicted for exponentionally growing cultures.     

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness¹ and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them². This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia³. The male-to-female ratio of mutation rate is estimated at about 4–6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males⁴. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.     

Reduction of nucleic acid content in cell biomass of methanol-utilizing mixed bacterial culture by heat treatment

Summary A heat treatment method to reduce nucleic acid content in cell biomass of a mixed methanol-utilizing bacterial culture was studied. Maximum nucleic acid reduction in the bacterial cells was achieved by using heat shock at 65°C for 5–10 min followed by 2 h incubation at 55°C and 7.2±0.2 pH. In this treatment, 81–85% nucleic acid content was removed from the cells without affecting their true protein content and essential amino acids profile.