Biochemistry

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Some aspects of the role of rat liver ribosomes in protein biosynthesis.
(1969) Nourse, Leonard Donald.; Quicke, George Venn.
No abstract available.
The isolation of a toxic factor from a local cultivar of Phaseolus vulgaris, and an assessment of its relation to growth depression.
(1969) Stead, Robin Hugh.; Quicke, George Venn.
No abstract available.
Comparative immunochemical studies on normal and monoclonal immunoglobulin M.
(1973) Conradie, Jan Dirk.; Visser, Leon.
No abstract available.
A comparative study of three toxic legume glycoproteins.
(1973) Dennison, Clive.; Quicke, George Venn.; Visser, Leon.
No abstract available.
The effect of zinc on cell division.
(1975) Duncan, John Richard.; Dreosti, J. E.
No abstract available.
The ruminal metabolism of lactic acid.
(1977) Mackie, Roderick Ian.; Quicke, George Venn.; Gilchrist, F. M. C.
Abstract available in PDF file.
Studies on the structural and biological functions of the Cu3 and Cu4 domains of IgM.
(1977) Bubb, Martin Owen.; Quicke, George Venn.
No abstract available.
A carboxymethylcellulase and a xylanase from Sclerotium rolfsii.
(1983) Lindner, William Andrew.; Dennison, Clive.; Dekker, Robert F. H.
No abstract available.
Enzymatic conversion of sterigmatocystin to aflatoxin B1.
(1984) Jeenah, Mohamed Sayed.; Dutton, Michael Francis.
The age of Aspergillus parasiticus (1-11-105Wh1) mycelium was found to have an influence on the level of enzymes, responsible for the conversion of sterigmatocystin to aflatoxin B[1] and O-methylsterigmatocystin, present. These enzymes were active over a wide range of temperature and pH. Production of a cell free system by lyophiliization yielded the highest aflatoxin B[1] synthesising activity. Three other methods of preparing the cell free system capable of synthesising aflatoxin B[1] were also studied, ie,: french press, protoplast, and grinding, but with limited success. The lyophilized preparation had narrower temperature and pH optima for the conversion than whole mycelia. Initial purification of the aflatoxin B[1] synthesising enzyme was achieved by separating the crude cell free extract by gel filtration. The enzyme activity was located in a membrane fraction. The involvement of endoplasmic reticulum was indirectly concluded by the use of marker enzyme and chelating agents. This membrane fraction was ultracentrifuged and the released extrinsic proteins were separated by gel filtration. A fraction containing two proteins which were capable of converting sterigmatocystin to aflatoxin B[1] was isolated and characterised by isoelectric focusing and gel electrophoresis. The temperature and pH optima together with the cofactor requirements were studied. The Michaelis-Menten constant (Km) and the stoichiometry for the conversion of sterigmatocystin to aflatoxin B[1] was determined.
The occurrence of mycotoxins in feedstuffs in Natal and aspects of their metabolism in the rumen.
(1985) Westlake, Kenneth.; Quicke, George Venn.
The fluorodinitrobenzene (FONB), succinic anhydride (SA), dansyl chloride (DAN), dye-binding lysine (OBL), total lysine (TL), ninhydrin (NIN) and Tetrahymena lysine (TET) methods were compared for their ability to assess available lysine in soyaprotein heated in the absence or presence of glucose, lactose or xylose and in formaldehyde-treated lactalbumin. The reactive lysine methods showed comparable sensitivity to lysine damage in soyaprotein heated in the absence of sugar, the results indicating the presence of acid labile isopeptides and unidentified acid stable derivatives. Results for soyaprotein heated with glucose, lactose or xylose showed that the type of sugar and the extent of heat treatment has a strong influence on the progress of the Maillard reaction. Furthermore since fructoselysine (F-L) and lactulosyl-lysine (L-L) are colourless up to 30% loss of available lysine can occur without any change in product colour. The FONB method is the most sensitive for mildly damaged glucose-soya samples followed by DAN or OBL, SA and TL whereas for mildly damaged lactose-soya samples the order is OBL, FONB, SA, TL and DAN. For severely damaged samples the DAN or SA methods were the most sensitive followed by OBL, FONB and TL. Formylation of lactalbumin occurred more readily at higher formaldehyde concentrations. Exposure time had less effect while pH (5 and 9) had no effect. Methylene derivatives reached maximum levels sooner than the methylol compounds. Lysine and tyrosine but not histidine formed methylene bridges while tyrosine was found to condense with free formaldehyde during acid hydrolysis raising questions as to the interpretation of similar studies reported in the literature. The FONB, OBL and DAN methods were all very sensitive to this type of damage with the NIN and TL methods being less sensitive and the SA method being completely unsuitable. The TET assay is unsuitable for 'early' Maillard damage since at low sample-N levels growth is stimulated by its ability to utilise unavailable F-L and L-L while at higher N-levels growth is inhibited. No single method is most suitable for all types of damage. Furthermore, all except DAN and DBL are either too long, rather complicated, require expensive equipment or involve the use of dangerous chemicals. The DAN method appears promising but the problem of converting arbitrary fluorescence units to lysine values needs to be overcome. The DBL is recommended for routine analysis since it is simple, economical and highly sensitive to all lysine damage provided care is taken to optimise dye-binding for each type of material analysed.
Effect of nitrate upon the digestibility of kikuyu grass (Pennisetum clandestinum)
(1985) Marais, Johan Pieter.; Dennison, Clive.
The factors affecting the accumulation of nitrate in kikuyu grass pastures and the effect of elevated nitrate levels upon digestion in the ruminant were investigated. A high potassium level in the soil seems to be the major factor stimulating the accumulation of excessive amounts of nitrate in kikuyu grass, when the nitrate content of the soil is also high. The continuous elongation of kikuyu grass tillers allows constant exposure of high nitrate containing stem tissue to the grazing ruminant. Digestibility studies in vitro showed that nitrite, formed during the assimilatory reduction of nitrate to ammonia, reduces cellulose digestion, but the degree of reduction also depends upon the presence of readily available carbohydrates and protein in the digest. Studies in vivo showed that the microbial population can adapt to metabolise high concentrations of nitrate (500 mg% N, m/m) in fresh kikuyu grass, without the accumulation of nitrite in the rumen. However, introduction into the rumen of nitrite in excess of the capacity of the nitrite reducing microbes, causes nitrite accumulation. Nitrite has no direct effect upon rumen cellulase activity. Due to the affinity of rumen carbohydrases for the substrate, attempts to isolate these enzymes by means of isoelectric focusing and other separation techniques met with limited success. Nitrite strongly reduces the xylanolytic, total and cellulolytic microbial numbers with a concomitant decrease in xylanase and cellulase activity of the digest. Decreased microbial numbers could not be .attributed to a less negative redox potential of the digest in the presence of nitrite, nor could the effect upon the cellulolytic microbes be attributed to an effect of nitrite on branched chain fatty acid synthesis required for cellulolytic microbial growth. A study of the effect of nitrite upon the specific growth rate of pure cultures of the major cellulolytic bacteria, Ruminococcus flavefaciens strain FDI, Butyrivibrio fibrisolvens strain Ce 51, Bacteroides succinogenes strain S 85 and Ruminococcus albus strain 22.08.6A and the non-cellulolytic bacterium Selenomonas ruminantium strain ATCC 19205 revealed the extreme sensitivity to nitrite of some of these bacteria and the relative insensitivity of others. Growth inhibition seems to depend primarily upon the extent to which these microbes derive their energy from electron transport-mediated processes.
Assessment of lysine damage during food processing.
(1985) Anderson, Trevor Ryan.; Quicke, George Venn.
The fluorodinitrobenzene (FONB), succinic anhydride (SA), dansyl chloride (DAN), dye-binding lysine (OBL), total lysine (TL), ninhydrin (NIN) and Tetrahymena lysine (TET) methods were compared for their ability to assess available lysine in soyaprotein heated in the absence or presence of glucose, lactose or xylose and in formaldehyde-treated lactalbumin. The reactive lysine methods showed comparable sensitivity to lysine damage in soyaprotein heated in the absence of sugar, the results indicating the presence of acid labile isopeptides and unidentified acid stable derivatives. Results for soyaprotein heated with glucose, lactose or xylose showed that the type of sugar and the extent of heat treatment has a strong influence on the progress of the Maillard reaction. Furthermore since fructoselysine (F-L) and lactulosyl-lysine (L-L) are colourless up to 30% loss of available lysine can occur without any change in product colour. The FONB method is the most sensitive for mildly damaged glucose-soya samples followed by DAN or OBL, SA and TL whereas for mildly damaged lactose-soya samples the order is OBL, FONB, SA, TL and DAN. For severely damaged samples the DAN or SA methods were the most sensitive followed by OBL, FONB and TL. Formylation of lactalbumin occurred more readily at higher formaldehyde concentrations. Exposure time had less effect while pH (5 and 9) had no effect. Methylene derivatives reached maximum levels sooner than the methylol compounds. Lysine and tyrosine but not histidine formed methylene bridges while tyrosine was found to condense with free formaldehyde during acid hydrolysis raising questions as to the interpretation of similar studies reported in the literature. The FONB, OBL and DAN methods were all very sensitive to this type of damage with the NIN and TL methods being less sensitive and the SA method being completely unsuitable. The TET assay is unsuitable for 'early' Maillard damage since at low sample-N levels growth is stimulated by its ability to utilise unavailable F-L and L-L while at higher N-levels growth is inhibited. No single method is most suitable for all types of damage. Furthermore, all except DAN and DBL are either too long, rather complicated, require expensive equipment or involve the use of dangerous chemicals. The DAN method appears promising but the problem of converting arbitrary fluorescence units to lysine values needs to be overcome. The DBL is recommended for routine analysis since it is simple, economical and highly sensitive to all lysine damage provided care is taken to optimise dye-binding for each type of material analysed.
A contribution to the biochemistry of Erwinia chrysanthemi.
(1985) Gray, James Steward Sanders.; Dutton, Michael Francis.
No abstract available.
Studies on the preparation and interaction of modified transferrin-DNA complexes with HeLa cells.
(1986) Hawtrey, Richard William.; Ariatti, Mario.; Hawtrey, Arthur O.
The correction of human genetic disorders by transfer of genetic material to cells is under intensive investigation in a number of 1aboratories. One possible way of trying to achieve the transfer of nucleic acid is by attaching DNA to a protein which has specific receptors on cells and which undergoes receptor-mediated endocytosis. In order to make use of the ligand protein-receptor approach for DNA transfer, iron-loaded human serum transferrin has been modified with the water soluble carbodiimides N-ethy1-N I -(3-dilllethy1aminopropyl) carbodiimide (CDI) and its quaterary analogue (ECDI) to give modified N-acy1urea transferrins. N-Acy1urea CDI (Fe 3+) transferrin and N-acy1urea CDI (Fe ) transferrin have been found to interact with and bind DNA in a reversible manner which i! dependent on ionic strength. [1251] N-Acy1urea CDI+(Fe3+) transferrin binds to transferrin receptors on Hea cells in culture and undergoes internalization through receptor-mediated endocytosis. Binding of the modified transferrin in the presence of calf thymus DNA to transferrin receptors also takes place. However, although internalization in the presence of DNA doe! appear to take place, the results of the internalization are not fully understood. Transfection studies with N-acy1urea CDI (Fe ) transferrin and plasmid pBR322 DNA as well as plasmid ptkNEO DNA complexes in the HeLa cell system are reported. The results of a number of transfection experiments suggests that N-acy1urea transferrins are capable of transfecting DNA (ptkNEO DNA), carrying genes for resistance to the antibiotic Geneticin (G41S) in the HeLa cell system. However, further development of the transfection system is necessary in order that consistantly reproducible results may be achievd.
Preparation of chemically modified transferrin proteins and an investigation of their reactions with DNA and other nucleic acids.
(1986) Gordhan, Hasha.; Hawtrey, Arthur O.; Ariatti, Mario.
The molecular biology of human genetic disorders is under intensive investigation at present. In those cases where the disorder is clearly defined in terms of altered gene structure, possibilities may exist for the correction of the disorder by insertion of normal genes through the process of DNA transfection. A possible method for the transfer of genetic material is by attempting to attach DNA to a protein which has specific receptors on cells and which undergoes receptor-mediated endocytosis. By this means one might be able to get DNA into cells. This thesis deals with experimental work on the chemical modification of human serum transferrin by means of water-soluble carbodiimides. The resulting N-acylurea transferrins bind DNA in a reversible manner. Characteristics and properties of the binding interactions are dealt with in detail. N-acylurea derivatives of transferrin were prepared with the water-soluble carbodiimides, N-ethyl-N' -(3-dimethylaminopropyl) carbodiimide and N-ethyl-N' -(3-trimethylpropylammonium) carbodiimide iodide. Reactions were carried out under mild conditions at room temperature for 48-72 hours. [³ H] N-ethyl-N' -(3-trimethylpropylammonium)carbodiimide iodide was used for the determination of covalently attached N-acylurea groups in the protein. Changes in charge properties were determined by agarose gel electrophoresis. Carbodiimide modification of proteins is thought to occur at side chain carboxyl groups of glutamic and aspartic acid residues. This was confirmed by the use of Staphylococcus aureus V8 protease, which cleaves peptide bonds at the carboxyl side of glutamic and aspartic acid residues, but not in the case of substituted side chain carboxyl groups. Through the use of puromycin as a nucleophile it has been shown that other functional groups were not activated upon reaction of transferrin with carbodiimide. The carbodiimide-modified proteins bind various types of DNA and RNA in a reversible manner. Low concentrations of N-acylurea transferrin retarded the migration of pBR322 DNA, M 13 mp 8 single-stranded DNA and Pst 1 restricted lambda DNA on agarose gel electrophoresis, while at higher concentrations the DNA was unable to enter the gel. Nitrocellulose filter binding assays showed that binding of DNA to Nacylurea transferrins was rapid, dependent on concentration of the modified transferrin and sensitive to ionic conditions. Binding was found to occur mainly through electrostatic interactions between phosphate groups of DNA and N-acylurea groups. These conclusions were based on experiments which showed that protein-DNA complexes were dissociated by increasing salt concentrations and by heparin. Non-electrostatic interactions such as hydrophobic interactions and hydrogen bonding are also involved in binding, since half dissociation of complexes, induced by chaotropic salts, KSCN and NaC10₄occurs at lower concentrations of salt than in the case of NaCl. Also RNA polynucleotides inhibit binding of DNA to Nacylurea transferrins to varying extents. The N-acyl urea transferrins have been shown to bind certain specific restriction endonuclease cleavage sites on pBR322 DNA. The N-acylurea transferrin-DNA complexes would thus be suitable for experiments in cell transfections using cells which have transferrin receptors.
Canine anti-endotoxin immunotherapy in cranial mesenteric arterial occlusion shock and canine parvovirus disease endotoxaemia.
(1986) Wessels, Brian C.; Gaffin, Stephen L.
Endotoxin (LPS, lipopolysaccharide) forms an integral part of the outer cellular membrane of gram negative bacteria (GNB). The canines' intestine always contains large amounts of GNB, and hence LPS. If these GNB with their LPS, remain within the intestinal lumen, they are not harmful to the host. When GNB do gain entry into a hosts' circulation a bacteraemia will occur with a concurrent endotoxaemia. In the past, it had been accepted that GNB were, themselves, primarily responsible for the mortality and morbidity of bacteraemic and septicaemic patients. Evidence has emerged to indicate that this is not altogether true as isolated LPS, without the presence of GNB, can also lead to fatalities. Circulating LPS is exceptionally chemically stable and highly toxic to host cells. Antimicrobial chemotherapy can destroy GNB, but this therapy does not reduce the toxicity of LPS, nor does it clear LPS from the circulation. Destruction of the GNB by certain antibiotics can, in fact, increase the concentration of circulating plasma LPS in a host. The functional integrity of the intestinal wall is highly dependent upon an adequate blood supply, and the mucosal cells acts as the primary defence against the potentially pathogenic, endogenous and exogenous GNB and LPS. Once these pathogens become intravascular then the liver is the next most important organ of defence. Shock, irrespective of its aetiology, without adequate therapy, leads to reduced micro-vascular circulation, and thus a state of either localised or generalised hypoxia occurs. Partial or complete intestinal vascular ischaemia will produce a state of regional hypoxia, and lead to damage of the intestinal wall allowing GNB, with their LPS, or LPS by itself, to enter into the hosts' blood circulation. Therefore, an aetiology that gives rise to any type of "classified shock," may eventually give rise to concurrent endotoxaemia. In clinical practice there are numerous different diseases, physical onslaughts, and either acquired or congenital anatomical defects, that can give rise to intestinal vascular ischaemia, and hence, endotoxaemia. Many treatment regimens to combat the effects of an endotoxaemia have been advocated over the years, but this problem still has an unacceptably high mortality and morbidity index, probably because almost all such therapeutic regimens fail to destroy the LPS molecule. Recent clinical studies have shown that immunotherapy is effective in combating gram negative bacteraemia and septicaemia in humans and animals. Research workers have been able to produce a "broad- spectrum" or "polyvalent" equine, hyperimmune, anti-endotoxir, antibody-enriched plasma (ANTI- LPS), with favourab"^ responses recorded when this plasma was used to treat a variety of experimentally-induced endotoxin-shocked subjects. ANTI-LPS significantly reduced the mortality in experimentally produced superior mesenteric arterial occlusion endotoxaemia in rabbits, presumably by neutralizing and opsonizing the circulating plasma LPS. Equine practitioners have reported successful results when ANTI-LPS was incorporated into the treatment of certain medical and surgical equine endotoxic related problems. A ^/ery recent, independent, Canadian study showed the effectivness of ANTI-LPS, where this preparation was tested against other anti-LPS products, to treat experimentally-induced sepsis in rats. The polyvalent equine ANTI- LPS was the most effective, in that its use resulted in the longest survival. In order to establish the generality of the use of equine ANTI-LPS plasma, I have extended these studies to the canine, since an abdominal vascular ischaemia carries a serious, high-risk, surgical emergency with unsatisfactorily high mortality rates, despite successful surgical intervention with concurrent supportive medical therapy. Twenty healthy dogs were divided into four groups; a control group (n=5) and three experimentally treated groups (n=5 in each group). All twenty dogs were subjected to the well-documented cranial (superior) mesenteric arterial occlusion (CMAO) shock model. The three experimental groups received the polyvalent equine, ANTI-LPS at different times and by two different routes, with no side effects being observed in any of these dogs. One group (n=5)received ANTI-LPS s.c. before CMAO was performed, a second group (n= 5) received their dosage of ANTI-LPS i.v. during the three-hour occlusion period, and a third group (n=5) received their dose s.c, within three minutes after the CMAO was released. Survival was recorded when any dog lived for a minimum of 14 days after the occluded vessel was released. All 5/5 (100%) controls died within 17 hours after the release of the occluded vessel, whereas only one of the 15 (6,5%) experimentally ANTI-LPS treated dogs died (P
Synthesis of DNA - protein conjugates and a preliminary study of their interaction with eukaryotic cell receptors.
(1986) Weiler, Solly.; Hawtrey, Arthur O.; Ariatti, Mario.
Thymidine oligomers were chemically synthesised and linked to available amino functions of transferrin in alternative orientations: (a) A CMP residue attached to the 3' end of (pT)₁₀ with terminal deoxynucleotidyl transferase was oxidised with NaI0 and linked to transferrin via a Schiff base formation. (b) The 5' terminal phosphate group of (pT)₅ was activated with imidazole and reacted with transferrin to form a phosphoramide bond. The (pT)₅ thus attached to the protein was elongated to (pT)₃₀₀ using terminal deoxnucleotidyl transferase and TTP. The latter conjugate was capable of hybridising poly(A) tailed linear PBR322 DNA. The binding of this hybridisation complex to the transferrin receptor on various cell types was investigated.
A biochemical and immunological comparison of the Jaagsiekte and two related retroviruses.
(1987) York, Denis Francis.; Verwoerd, D. W.; Dennison, Clive.
Jaagsiekte is a contagious cancer affecting the lungs of sheep. Although the etiological agent is Jaagsiekte retrovirus (JSRV), two other retroviruses viz South African maedi visna virus ( SA - OMVV) and a novel Bovine retrovirus (BRV) have been associated with or implicated in the jaagsiekte disease complex. JSRV was sufficiently purified from lung rinse material using a Freon extraction, Percoll density gradient centrifugation and chranatography on a Sephacryl column, its polypeptide composition was studied by gel electrophoresis and its morphology observed electron microscopically. Monoclonal antibodies were made against purified preparations of the virus. Two hybridomas were isolated that produced MAbs which appear to be tumour cell specific. A third hybridoma, called 4A1O, produces antibodies considered to be viral specific. These MAbs have been used in the development of JS specific immunoassays. A cross reaction between JSRV and a polyclonal serum against Mason Pfizer monkey virus (MPMV) was confirmed and used in a Western blot technique to identify, monitor and differentiate JSRV from other viruses. During the study of JSRV it became apparent that another retrovirus was often present in JS infected lungs. This virus, referred to as SA - OM1V I, is a novel South African isolate of maedi visna virus (MVV). As SA - OM1V I has physicochemical characteristics similar to JSRV, it was often found in purified JSRV preparations. Being a retrovirus it is also detected by the reverse transcriptase assay which was the only method used to assay and monitor for JSRV during the early stages of our work. Using a Westen blot technique and sera against MVV and MPMV it was possible to simultaneously detect and differentiate JSRV from SA - OMVV I. A method was also developed whereby the two viruses could be separated from each other during purification. The information gained and techniques developed whilst studyiing JSRV were also used to isolate and characterize BRV. This novel virus originated from bovine cells that had been co-cultivated with white blood cells from an ox suffering from malignant catarrhal fever. Three out of four sheep inoculated with BRV developed JS. It therefore had to be· ascertained whether this virus was related to JSRV or not. The comparative study revealed that BRV was biochemically and morphologically quite different fran JSRV. Interestingly, it was shown that serum against MPMV cross reacted with a 32 kd protein of BRV indicating a serological relationship between JSRV, MPMV and BRV. The possible role of BRV in the etiology of jaagsiekte remains to be elucidated.
Studies on the coupling of DNA to low density lipoproteins (LDL) and the interaction of these complexes with eukaryotic cells.
(1987) Khan, Zainub.; Hawtrey, Arthur O.; Ariatti, Mario.
The application of Molecular Biochemistry for transfection studies in eukaryotic systems is well documented. Of the numerous methods employed for the introduction of foreign DNA into eukaryotic cells, the use of low density lipoproteins (LDL) as carriers of DNA into cells has not been reported. LDL was isolated, characterized with respect to its protein and lipid components, and then variously modified in an attempt to enhance its affinity for DNA. It was found that both unmodified and modified LDL could interact with DNA, at physiological pH. The carbodiimide modified LDL (ECDI - LDL) showed the greatest affinity for DNA. LDL and ECDI - LDL were used to study LDL receptor binding in skin fibroblasts. This was followed by a study of receptor binding activities of both unmodified LDL and ECDI - LDL complexed to DNA (pBR322). Although the extent of binding of ECDI - LDL and ECDI - LDL - DNA complexes to plasma membranes was greater, the internalization and degradation of both modified and unmodified LDL complexes were equivalent. This additional binding was attributed to non - receptor - specific affinity of the carbodiimide modified complexes for the plasma membrane. The transfection of foreign DNA into eukaryotic cells in culture was monitored by assaying for the expression of the cloning vector, pSV2cat, complexed to LDL or ECDI - LDL and introduced into the cells by LDL receptor - mediated endocytosis. Of the cell lines in which the expression of the pSV2cat recombinant DNA was monitored, the human lung fibroblasts showed the greatest activity of the expressed chloramphenicol acetyl transferase enzyme. Although transfection efficiency was lower than that of the calcium phosphate - DNA coprecipitation procedure, the LDL receptor - mediated transfection of eukaryotic cells was carried out under physiological conditions and may be applicable in vivo.
Gene transfer by receptor-mediated endocytosis : stable expression of NEO following insulin-directed entry into HepG2 cells.
(1989) Huckett, Barbara Isobel.; Hawtrey, Arthur O.; Ariatti, Mario.
Evidence is presented for DNA delivery to cultured HepG2 hepatoma cells via the endocytotic pathway, under the direction of insulin, in a soluble system of transfection leading to stable gene expression. Serum albumin treated at pH 5.5 and 20°C for 48-60h with the water-soluble carbodiimide N-ethyl-N'(3-dimethylaminopropyl) carbodiimide hydrochloride has been found to produce positively charged N-acylurea albumin capable of binding different types of DNA in a reaction which is at least partially electrostatic in nature (Huckett et al, 1986). N-Acylurea albumin, synthesised at an albumin to carbodiimide mole ratio of 1 : 500, resulting in the attachment of 27 Nacylurea moieties per albumin molecule, was covalently conjugated to insulin by glutaraldehyde cross-linkage in order to produce a macromolecule, insulin-[N-acylurea albumin], with the facilities f or both DNA transport and receptor binding. The resultant conjugate, purified by gel filtration through Sephadex G-100, was characterised in terms of molecular size, charge properties and insulin content by polyacrylamide gel electrophoresis, agarose gel electrophoresis and immuno-dotblotting respectively. The conjugated protein was shown by gel band shift and nitrocellulose filter binding assays to bind DNA non-specifically in a reversible reaction which occurs rapidly, is dependent upon protein concentration and the ionic strength of the medium, and involves at least two types of intermolecular interaction. Furthermore, the conjugate was shown by competitive displacement of [ 125I ]insulin to bind specifically and particularly avidly to the HepG2 insulin receptor. When the expression vectors ptkNEO and pAL-8 which incorporate the neo gene were complexed to the conjugate in an in vitro transfection procedure using HepG2 cells, G418 resistant clones developed at frequencies of 2.0 - 5.5 X 10-5, possibly dependent upon vector promoter. Subsequently, a 923bp PstI fragment within the neD sequence was identified by Southern transfer in genomic DNA extracted from transfected cell populations which had been grown on a G418 regime through several subculture passages over a period of 44 days.

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