Jeff Hewitt

Biomining with bacteriophage: Selectivity of displayed peptides for naturally occurring sphalerite and chalcopyrite

During mineral processing, concentrates of sulfide minerals of economic interest are formed by froth flotation of fine ore particles. The method works well but recovery and selectivity can be poor for ores with complex mineralogy. There is considerable interest in methods that improve the selectivity of this process while avoiding the high costs of using flotation chemicals. Here we show the first application of phage biotechnology to the processing of economically important minerals in ore slurries. A random heptapeptide library was screened for peptide sequences that bind selectively to the minerals sphalerite (ZnS) and chalcopyrite (CuFeS2). After several rounds of enrichment, cloned phage containing the surface peptide loops KPLLMGS and QPKGPKQ bound specifically to sphalerite. Phage containing the peptide loop TPTTYKV bound to both sphalerite and chalcopyrite. By using an enzyme-linked immunosorbant assay (ELISA), the phage was characterized as strong binders compared to wild-type phage. Specificity of binding was confirmed by immunochemical visualization of phage bound to mineral particles but not to silica (a waste mineral) or pyrite. The current study focused primarily on the isolation of ZnS-specific phage that could be utilized in the separation of sphalerite from silica. At mining sites where sphalerite and chalcopyrite are not found together in natural ores, the separation of sphalerite from silica would be an appropriate enrichment step. At mining sites where sphalerite and chalcopyrite do occur together, more specific phage would be required. This bacteriophage has the potential to be used in a more selective method of mineral separation and to be the basis for advanced methods of mineral processing.

Nucleotide sequence of the phosphotransacetylase gene of Escherichia coli strain K12

The phosphotransacetylase gene (pta) from Escherichia coli strain K-12 1100 was identified in a cloned fragment of chromosomal DNA (Yamamoto-Otake, H., Matsuyama, A. and Nakano, A. (1990) Appl. Microbiol. Biotechnol. 33, 680-682). Overexpression in E. coli confirmed the presence of the pta gene within the cloned fragment. DNA sequence analysis of the cloned pta gene indicates that the predicted phosphotransacetylase polypeptide chain is 713 amino acids in length. The carboxyterminal region of the E. coli phosphotransacetylase shows 42.6% sequence identity with the corresponding enzyme from Methanosarcina thermophila (142 out of 333 residues in corresponding positions are identical). Several short regions of high sequence identity may be structurally or functionally important for enzymic activity.

Identification of the epitope of a monoclonal antibody that disrupts binding of human transferrin to the human transferrin receptor

The molecular basis of the transferrin (TF)–transferrin receptor (TFR) interaction is not known. The C‐lobe of TF is required to facilitate binding to the TFR and both the N‐ and C‐lobes are necessary for maximal binding. Several mAb have been raised against human transferrin (hTF). One of these, designated F11, is specific to the C‐lobe of hTF and does not recognize mouse or pig TF. Furthermore, mAb F11 inhibits the binding of TF to TFR on HeLa cells. To map the epitope for mAb F11, constructs spanning various regions of hTF were expressed as glutathione S‐transferase (GST) fusion proteins in Escherichia coli. The recombinant fusion proteins were analysed in an iterative fashion by immunoblotting using mAb F11 as the probe. This process resulted in the localization of the F11 epitope to the C1 domain (residues 365–401) of hTF. Subsequent computer modelling suggested that the epitope is probably restricted to a surface patch of hTF consisting of residues 365–385. Mutagenesis of the F11 epitope of hTF to the sequence of either mouse or pig TF confirmed the identity of the epitope as immunoreactivity was diminished or lost. In agreement with other studies, these epitope mapping studies support a role for residues in the C1 domain of hTF in receptor binding.

Isolation of a murine glucose-dependent insulinotropic polypeptide (GIP) cDNA from a tumor cell line (STC6–14) and quantification of glucose-induced increases in GIP mRNA

A 537 base pair cDNA clone for murine GIP has been isolated. The elucidated sequence encodes an open reading frame of 432 base pairs which codes for a 144 amino acid precursor. Murine GIP is predicted to differ from the human hormone by three amino acid substitutions: arginine for histidine at position 18, arginine for lysine at position 30 and serine for lysine at position 34. GIP mRNA levels in STC6-14 cells incubated in the presence of varying glucose concentrations was investigated using a competitive-PCR method. In the presence of a 5-mM glucose stimulus, 1 x 10(5) GIP cells were found to contain 3.9 +/- 0.59 amol of GIP mRNA while the same number of cells contained 11.6 +/- 1.4 amol when subjected to a high (25 mM) glucose stimulus.

A novel compensating mechanism for homozygous coagulation factor V deficiency suggested by enhanced activated partial thromboplastin time after reconstitution with normal factor V.

Factor V (FV) is an essential coagulation cofactor and is critical for production of thrombin, the effector of fibrin clot formation (Krishnaswamy et al, 1993). FV deficiency is a rare autosomal recessive bleeding disorder with an incidence of one per million. However, whilst FV knock-out mice die before or soon after birth, severe FV deficiency (<1%) is usually not fatal in humans (Cui et al, 1996) implying the presence of compensatory mechanisms that minimise the clinical severity of the deficiency (Duckers et al, 2009). In the current study, an 83-year-old male of Scottish heritage who had no spontaneous bleeding or abnormal blood loss except upon surgery or trauma was investigated. Clinical laboratory tests initially identified the FV deficiency (<2%). Other haemostatic plasma proteins were found to be within the normal range by routine assays conducted at a validated hospital clinical laboratory including; prothrombin (>65%), factor VII (123%), factor VIII (108%), factor IX (100%), factor X (90%), factor XI (67%), factor XII (95%), von Willebrand factor antigen (>0Æ50 iu), high molecular weight kininogen (81%), prekallikrein (109%) and antithrombin III (91%). Platelet count and liver and kidney function tests were also normal. Our aim was to determine the patient’s FV genotype, and to investigate the potential contribution of his platelet FV pool and possible hypercoagulant mechanisms that may attenuate the severity of the disease. DNA sequence analysis of the patient’s FV gene (F5) revealed a homozygous mis-sense mutation in exon 15 (A5279G) changing the codon for Tyr1702 to Cys. The remainder of the F5 sequence was identical with the wild type F5 (Jenny et al, 1987) suggesting that the homozygosity of the A5279G nucleotide was responsible for the FV deficiency. This homozygous mutation has been previously identified (Castoldi et al, 2001; Montefusco et al, 2003) and is thought to expose a cysteine residue that causes incorrect protein folding, reduced stability and/or secretion resulting in low plasma FV activity (Castoldi et al, 2001).

Molecular characterization of a 4 409 480 bp deletion of the human X chromosome in a patient with haemophilia B

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Molecular determination of the breakpoints of a 161 556 bp deletion at chromosome 13q34 that presented as severe factor VII deficiency in a neonate.

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Germline mutation identified after amplification of nuclear DNA from citrated plasma.

We read with interest the recent correspondence in this journal concerning the use of plasma as a source of mRNA for determining mutation status and clonality in patients with chronic lymphocytic leukaemia (Ma et al, 2006). We recently used a similar strategy involving a sample of citrated plasma as the source of DNA for genetic mutation analysis. Since its inception in 1984, the polymerase chain reaction (PCR) has been recognised for its sensitivity, specificity and speed of analysis. Several previous reports have demonstrated the presence of DNA in both serum (Ishizawa et al, 1991) and plasma (Fournie et al, 1993). Although the PCR is able to detect viral DNA load (Pardoe & Michalak, 1995) and fetal DNA (Lo et al, 1997) in blood plasma, the common protocol for genetic analysis of haemostatic disorders in clinical laboratories remains the enrichment or purification of DNA from leucocyte cells or buccal cells prior to PCR amplification. Recently, we analysed the molecular basis of prothrombin deficiency in a family from Alberta (Wong et al, 2006). Using a molecular genetic approach, we determined that the proband was a compound heterozygote for two prothrombin gene mutations – a point mutation in one allele and a small gene deletion in the other allele. We confirmed that the point mutation had been inherited from the maternal DNA, and wished to confirm the paternal origin of the deletion allele but we did not have any purified leucocyte DNA from the father. However, we did have a sample of plasma from the father; this plasma sample had been isolated from a tube of citrated blood and frozen and stored during the baseline coagulation factor work-up of the family. We hypothesised that the plasma sample would contain either soluble DNA or DNA-containing leucocyte cell debris that would serve as a template for PCR amplification. To test this hypothesis, we used a tube of paternal citrated plasma as the template for the PCR amplification of the exon 11 region of the human prothrombin gene using specific primers. A sample of plasma was placed into an Eppendorf tube and was centrifuged at 19 000 g for 10 min. A portion of the supernatant (10 ll) was placed into a clean tube, and the pellet was re-suspended in 10 ll of water. The samples were heated to 90 C for 10 min, cooled to room temperature and centrifuged as before. The supernatants were removed, transferred to clean tubes and then subjected to PCR analysis using primers that were specific to the region surrounding exon 11 where the prothrombin gene deletion was located. The results are highlighted in Fig 1. As shown in lane 3, the sample of maternal leucocyte DNA produced the amplification of a 600 bp fragment of DNA corresponding to the wild type prothrombin gene. PCR amplification of the paternal plasma sample (lane 2) resulted in the presence of a 600 bp fragment (corresponding to the wild type gene sequence) and a 250 bp fragment [corresponding to the prothrombin gene deletion in this family as determined by Wong et al, (2006)]. The cell debris pellet did not give any PCR fragments suggesting that the DNA in lane 2 was amplified from soluble plasma DNA. We conclude that blood plasma samples prepared in a clinical laboratory for coagulation factor testing contain sufficient soluble DNA for the identification of germline mutations in inherited single-gene disorders.

Confirmation of paternity suggests a new mutation in the factor VII gene: ‘Pater certus quouque est’ ‐ Response to Girolami et al

Thank you for giving us the opportunity to respond to the comments made by Dr Girolami and colleagues concerning our paper describing a patient with severe factor VII deficiency (Hewitt et al, 2005). In our paper, we proposed that the severe deficiency arose from a previously undetected maternal deletion of a region of chromosome 13 (including the factor VII and factor X genes) together with a new paternal point mutation. Dr Girolami and colleagues suggested that (i) germinal mosaicism in the father and (ii) different paternity could also explain the results in our paper. Concerning the paternity, we did state in our paper that paternity was confirmed using the commercially available AmpFlSTR Identifiler polymerase chain reaction Amplification Kit (Applied Biosystems). In addition, this kit was used to verify relationships between all familial samples and rule out any possibility of sample mis-identification. However, Dr Girolami and colleagues are correct in pointing out that we did not give details of the results in our paper. In fact, the short tandem repeat (STR) analysis confirmed paternity by using 16 different STR alleles including the amelogenin allele that distinguishes between the X- and Y-chromosomes. The proband’s family is Caucasian, so using the statistics supplied with the kit (for the US Caucasian population), the STR