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Antiretroviral protease inhibitors were assessed in vitro for their activity against Giardia duodenalis and Trichomonas vaginalis. Kaletra (a co-formulation of ritonavir and lopinavir) was the most effective overall, with 50% effective drug concentrations (EC(50)) of 1.1-2.7 microM (ritonavir concentration) against G. duodenalis and 6.8-8 microM against metronidazole-sensitive and clinically metronidazole-resistant T. vaginalis. Minimal inhibitory concentrations were 2-2.5 microM and 10-50 microM for G. duodenalis and T. vaginalis, respectively. Within the range of human plasma concentrations for ritonavir, only G. duodenalis was inhibited. Lopinavir alone was less inhibitory than ritonavir but was associated with a blockage in cytokinesis of G. duodenalis trophozoites. Saquinavir was not effective. These findings are significant considering the association between human immunodeficiency virus and T. vaginalis, and between G. duodenalis and homosexual behaviour.

Primary vaccine strategies against group A streptococci (GAS) have focused on the M protein-the target of opsonic antibodies important for protective immunity. We have previously reported protection of mice against GAS infection following parenteral delivery of a multi-epitope vaccine construct, referred to as a heteropolymer. This current report has assessed mucosal (intranasal (i.n.) and oral) delivery of the heteropolymer in mice with regard to the induction and specificity of mucosal and systemic antibody responses, and compared this to parenteral delivery. GAS-specific IgA responses were detected in saliva and gut upon i.n. and oral delivery of the heteropolymer co-administered with cholera toxin B subunit, respectively. High titre serum IgG responses were elicited to the heteropolymer following all routes of delivery when administered with adjuvant. Moreover, as with parenteral delivery, serum IgG antibodies were detected to the individual heteropolymer peptides following i.n. but not oral delivery. These data support the potential of the i.n. route in the mucosal delivery of a GAS vaccine. (C) 2002 Elsevier Science Ltd. All rights reserved.

The intestinal, parasitic protozoa comprising the genus Giardia are major aetiological agents of diarrhoeal disease in numerous vertebrates, including humans, livestock and domestic pets. In previous investigations, morphological characteristics, in particular caudal flagellar length and asymmetry, have been used to distinguish avian from mammalian Giardia species, with scanning electron microscopy (SEM) often the technique of choice for observation. In this study, however, we observed considerable variation in caudal flagellar length and asymmetry, irrespective of avian or mammalian host origin. In addition, SEM sample preparation and the age of trophozoite cultures caused increases in the number of trophozoites with caudal flagellar length anomalies. Our findings, therefore, suggest that caudal flagellar characteristics are not reliable morphological markers for distinguishing Giardia species. In addition, the novel use of micromanipulation in establishing sub-clonal cultures of Giardia from single trophozoites was a highly accurate and successful microscopic technique.

Numerous studies have demonstrated various strain differences between Giardia isolates, but little is known about the immunology and pathogenesis of infections. This study aimed to compare host responses to strains of Giardi duodenalis differing in levels of virulence and pathogenicity and, by doing so, elucidate the mechanisms via which pathogenic strains establish infections. Marked differences were found in the infection dynamics, histopathological responses and serum antibody responses of neonatal mice infected with either G. duodenalis strain BRIS/83/HEPU/106 (isolated from a human) or BRIS/95/HEPU/2041 (isolated from a sulphur-crested cockatoo, Cacatua galerita ). Infections with the bird strain were more intense (6.7-times greater) and persisted longer (by 14 days) than infections with the human strain. The bird strain was more pathogenic and caused greater pathophysiological alteration to the gut mucosa, including increased villous atrophy, hyperplasia of goblet cells and vacuolated epithelial cells. Mice infected with the bird strain produced less serum anti- Giardia IgA and IgM, but more total (non-specific) serum IgA than those infected with the human strain of Giardia . This suggests that avian G. duodenalis strains are infective for mammalian hosts and may contribute to zoonotic infections. Furthermore, infection of mice with BRIS/95/HEPU/2041 serves as a good experimental model to provide further insight into the mechanisms via which G. duodenalis causes disease.

We have induced high levels of resistance to metronidazole (1 mM or 170 microg ml(-1)) in two different strains of Trichomonas vaginalis (BRIS/92/STDL/F1623 and BRIS/92/STDL/B7708) and have used one strain to identify two alternative T. vaginalis 2-keto acid oxidoreductases (KOR) both of which are distinct from the already characterised pyruvate:ferredoxin oxidoreductase (PFOR). Unlike the characterised PFOR which is severely down-regulated in metronidazole-resistant parasites, both of the alternative KORs are fully active in metronidazole-resistant T. vaginalis. The first, KORI, localized in all membrane fractions but predominantly in the hydrogenosome fraction, is soluble in Triton X-100 and the second, KOR2, is extractable in 1 M acetate from membrane fractions of metronidazole-resistant parasites. PFOR and both KORI and KOR2 use a broad range of 2-keto acids as substrates (pyruvate, alpha-ketobutyrate, alpha-ketomalonate), including the deaminated forms of aromatic amino acids (indolepyruvate and phenylpyruvate). However, unlike PFOR neither KORI or KOR2 was able to use oz-ketoglutarate. Deaminated forms of branched chain amino acids (alpha-ketoisovalerate) were not substrates for T. vaginalis KORs. Since KOR I and KOR2 do not apparently donate electrons to ferredoxin, and are not down-regulated in metronidazole-resistant parasites, we propose that KORI and KOR2 provide metronidazole-resistant parasites with an alternative energy production pathway(s) which circumvents metronidazole activation.

Early in 1995, a sulphur-crested cockatoo captured in the wild died along with several other cage mates, apparently of an overwhelming, acute infection of Giardia. Trophozoites isolated from the dead bird and established in traditional Giardia axenic medium were infective to mice and established chronic infections associated with weight gain impairment. Genetically and morphologically, the Giardia isolated from the bird belonged to the duodenalis group. Here, Jacqui Upcroft, Ann McDonnell and Peter Upcroft present data on pathogenic avian Giardia with he potential to contaminate watersheds and discuss the implications.

A gene encoding an unusually small dynein heavy chain homologue, hDYHH, was cloned from the genome of a free-living diplomonad, Hexamita inflata (Hi). The open reading frame (ORF) of hDYHH is 867bp and encodes a polypeptide of 289 amino acids (aa), hDYHH. hDYHH is homologous to the region around the third P-loop ATP-binding site of several dynein heavy chain polypeptides that are around 4000aa. Northern blot analysis showed that hDYHH is expressed in vivo and that the mRNA length (approximately 1.8kb) is consistent with the gene length (1.67kb). Southern blot analysis indicated that there are hDYHH homologues within the Hi genome, possibly including a longer dynein heavy chain gene. An hDYHH homologue was also identified in Hexamita pusilla (Hp). hDYHH is the first full-length protein-encoding gene cloned from Hexamita.

The protozoan parasite, Giardia duodenalis, shares many metabolic and genetic attributes of the bacteria, including fermentative energy metabolism which relies heavily on pyrophosphate rather than adenosine triphosphate and as a result contains two typically bacterial glycolytic enzymes which are pyrophosphate dependent. Pyruvate decarboxylation and subsequent electron transport to as yet unidentified anaerobic electron acceptors relies on a eubacterial-like pyruvate:ferredoxin oxidoreductase and an archaebacterial/eubacterial-like ferredoxin. The presence of another 2-ketoacid oxidoreductase (with a preference for alpha-ketobutyrate) and multiple ferredoxins in Giardia is also a trait shared with the anaerobic bacteria. Giardia pyruvate:ferredoxin oxidoreductase is distinct from the pyruvate dehydrogenase multienzyme complex invariably found in mitochondria. This is consistent with a lack of mitochondria, citric acid cycle, oxidative phosphorylation and glutathione in Giardia. Giardia duodenalis actively consumes oxygen and yet lacks the conventional mechanisms of oxidative stress management, including superoxide dismutase, catalase, peroxidase, and glutathione cycling, which are present in most eukaryotes. In their place Giardia contains a prokaryotic H2O-producing NADH oxidase, a membrane-associated NADH peroxidase, a broad-range prokaryotic thioredoxin reductase-like disulphide reductase and the low molecular weight thiols, cysteine, thioglycolate, sulphite and coenzyme A. NADH oxidase is a major component of the electron transport pathway of Giardia which, in conjunction with disulphide reductase, protects oxygen-labile proteins such as ferredoxin and pyruvate:ferredoxin oxidoreductase against oxidative stress by maintaining a reduced intracellular environment. As the terminal oxidase, NADH oxidase provides a means of removing excess H+, thereby enabling continued pyruvate decarboxylation and the resultant production of acetate and adenosine triphosphate. A further example of the bacterial-like metabolism of Giardia is the utilisation of the amino acid arginine as an energy source. Giardia contain the arginine dihydrolase pathway, which occurs in a number of anaerobic prokaryotes, but not in other eukaryotes apart from trichomonads and Chlamydomonas reinhardtii. The pathway includes substrate level phosphorylation and is sufficiently active to make a major contribution to adenosine triphosphate production. Two enzymes of the pathway, arginine deiminase and carbamate kinase, are rare in eukaryotes and do not occur in higher animals. Arginine is transported into the trophozoite via a bacterial-like arginine:ornithine antiport. Together these metabolic pathways in Giardia provide a wide range of potential drug targets for future consideration.

The major 2-oxoacid oxidoreductase (2-OR), pyruvate:ferredoxin oxidoreductase (PFOR) from Giardia duodenalis has been purified to apparent homogeneity. A second 2-OR with a preference for alpha-ketobutyrate as substrate was identified and was removed from PFOR containing fractions during purification. Only PFOR and the second 2-OR were identified in gels of crude Giardia extracts assayed for 2-OR activity. The native form of PFOR which is membrane associated, is a homodimer of 138 kDa subunits. Pyruvate is the preferred substrate: alpha-ketobutyrate and oxaloacetate, but not phenyl-pyruvate or alpha-ketoglutarate, are decarboxylated. PFOR from Giardia is more stable than PFOR from most other organisms and purified PFOR can be stored without deterioration at -70 degrees C. Purified PFOR donates electrons to Giardia ferredoxin (Fd I) with concomitant reduction of metronidazole. However, two other Giardia ferredoxins did not accept electrons from PFOR. Consistent with the involvement of PFOR in metronidazole activation, the activity of pyruvate dependent 2-OR activity was decreased in all metronidazole-resistant lines tested but not in furazolidone-resistant lines. The presence of three different ferredoxins and two 2-ORs in Giardia suggests that a number of different electron transport pathways operate in this organism providing unusual metabolic flexibility for a eukaryote.

A landmark physical map of the 2-Mb chromosome of the Giardia duodenalis cloned line WB-1B, constructed using randomly cloned, chromosome specific markers, was used to compare the organisation and map order of the equivalent chromosome in other strains. A representative marker from each of the 13 NotI segments of the 2-Mb chromosome was hybridized to NotI cleavages of whole chromosomes of the other strains. Two strains, one isolated from a human, and one from a cat, had the same chromosome hybridization patterns as WB-1B. A strain isolated from a sheep, had one NotI chromosome 5 segment larger than WB-1B. Two additional strains isolated from a calf and a human had significantly different NotI cleavage patterns from the previous strains and shared no similar-sized chromosome NotI segment from their 2Mb chromosome homologues and only one in common with WB-1B. In one strain, two markers from the same WB-1B NotI segment did not hybridize suggesting deletion events have occurred. The order of some NotI segments within the 2Mb chromosome homologue was maintained, as determined from partial NotI chromosome cleavages, while in the most divergent of strains internal chromosome rearrangements and deletions were evident. All but one of the 2Mb WB-1B chromosome markers examined hybridized to a single chromosome band in all strains. Thus, while Giardia chromosomes vary in size, copy number and organisation, some linkage of markers is apparently maintained in isolates from disparate hosts and localities. We have therefore generated a genetic analysis system for Giardia with landmark maps using representative markers to replace the paucity of classical genetic markers and mutants. This approach is being extended to the complete genome.

We have cloned a gene, CRP65, from genomic DNA of Giardia duodenalis (Gd) which contains four 228-bp tandem repeat units between a short (48 bp) 5' and long (942 bp) 3′ non-repeat region. CRP65 encodes a Cys-rich protein (CRP) with the typical transmembrane domain and CXXC amino acid (aa) motif of Gd CRP. Comparison of the nucleotide (nt) and deduced aa sequences of CRP65 and a gene we cloned previously, CRP136, indicates that the genes are highly homologous in the entire non-repeat regions, but not in the repeat regions. The repeat unit of CRP65 was found to be homologous to epidermal growth factor (EGF)-like domains from different proteins. Analysis of Gd genomic DNA showed that there are multiple copies of CRP65 and each copy varies in the number of repeat units, as well as in certain restriction sites in the units. In Gd strain WB-1B, a 2.0-kb transcript encoded by the gene was expressed, while in a metronidazole-resistant line (WB1B-M3) induced from WB-1B, two longer transcripts (5.5 and 7 kb) were expressed. Based on our results, we suggest that there is a unique CRP family in the Gd genome, whose members, including CRP65 and CRP136, carry various repeat units within a highly conserved ‘cassette’. CRP65 may be involved in EGF-like interactions with the host proteins

Duodenal aspirates from children investigated for diarrhoea have been examined for the presence of Giardia over an eleven year period, and where possible, in vitro or in vivo Giardia cultures in mice were established. Based on biochemical characteristics of electrophoretic karyotype, RFLP analysis and rDNA hybridization studies of 40 stocks at least two major varieties, or demes, of Giardia have infected the population of South East Queensland and environs during this period. These demes carried different rDNA repeat units and differed markedly in both the electrophoretic karyotype pattern and the molar representation of chromosome bands. From 1983 to 1991 only one deme was documented. The first evidence of a new deme seen in local children occurred in 1991 and was followed by a predominance of this deme in 1993. These 40 stocks do not represent all positive samples. Other stocks established in vivo were not able to be cultured in vitro, and these probably represent other demes. Since all of the stocks established in vivo were not able to be cultured in vitro, and these probably represent other demes. Since all of the stocks were derived from children with similar chronic symptoms it appears that at least two demes of Giardia are pathogenic.

Non-denaturing polyacrylamide gels were used to analyse superoxide dismutase (SOD), catalase, peroxidase, NADH oxidase and NADH peroxidase in the microaerophilic protozoan parasite Giardia duodenalis. A cytosolic H2O-producing NADH oxidase and membrane-associated NADH peroxidase were readily detected from G. duodenalis. In all Giardia strains investigated the NADH oxidase was present in high levels (1.2-2 U (mg protein)-1). Using the same technique, NADH oxidase activity was also detected in the microaerophilic protozoan parasites Tritrichomonas foetus, Trichomonas vaginalis and Entamoeba histolytica and in the bacterium Escherichia coli. The conventional enzymes of oxidative stress management (superoxide dismutase, catalase and peroxidase) were not detected in particulate or cytosolic extracts from recent and established strains of Giardia assayed in situ. Spectrophotometric assays also yielded negative results. The same methodology readily detected one or more of these enzyme activities in T. foetus, T. vaginalis and E. coli. Superoxide dismutase activity was not detected in lines of Giardia resistant to high levels of metronidazole or furazolidone. Furthermore, the agents 1,10 phenanthroline, diamide, MnCl2 and KNO3, which induce SOD in anaerobically cultured E. coli, did not induce SOD in Giardia. 1,10 phenanthroline has also been shown to induce iron-containing (Fe-) SOD in Entamoeba. Neither peroxidase nor catalase activities were detected in a peroxide-resistant line of Giardia. Viable trophozoites from parent lines were able to decompose H2O2 at a significant rate. It appears that the conventional SOD, catalase and peroxidase utilised in aerobic metabolism have been substituted in Giardia by NADH oxidase and NADH peroxidase, similar to anaerobic bacteria. The O2-scavenging NADH oxidase explains the previously observed futile 'respiration' in Giardia.

Aerobic metabolism can result in the production of reduced molecular oxygen intermediates, i.e., superoxide (O2-), hydroxyl radical ( .OH), and hydrogen peroxide (H202) [1]. Defence mechanisms specifically employed for removal of these toxic radicals include superoxide dismutase [2], catalase [3], and the glutathione (GSH) cycling enzymes, GSH peroxidase [4] and GSH reductase [5]. GSH cycling has been regarded as one of the primary mechanisms of detoxification in organisms ranging from Escherichia coli to man. All organisms contain high levels of at least one thiol whether it be GSH, GSH analogues or another low-molecular weight thiol. The haemoflagellate Trypanosoma, for example, conjugates the majority (>70%) of its free intracellular GSH with spermidine to form trypanothione, which acts as a cofactor with low levels of GSH in redox cycling [6,7]. During stationary phase under anaerobic conditions, E. coli, which exhibits GSH metabolism aerobically and during log phase, converts the majority of its free GSH to glutathionylspermidine [8]. Anaerobes must detoxify both oxygen and

Giardiasis is a worldwide disease that can cause serious morbidity. Metronidozole is the current recommended drug for treatment, and is mostly still effective. However, Giardia duodenalis , the causative agent, is capable of developing resistance to high levels of metronidozole and other drugs, in vitro , via a number of mechanisms. Resistance, in vivo , has been reported and many cases of treatment failure have been variously attributed to a number of causes, including resistance. Here, Jacqueline and Peter Upcroft ask: is this the beginning of another chapter of drug resistance? or is the situation likely to remain as a ‘few refractory cases'? Should we wait to find out or can we act positivelyto avert the possibility of yet another valuable drug in our limited pharmacopoeia becoming obsolete?

Evidence for drug resistance in giardiasis is reviewed and biochemical studies undertaken to determine the basis for this resistance are discussed. Metronidazole and furazolidone, which produce toxic radicals within the cell, have different biochemical mechanisms of action. Resistance to metronidazole is negatively correlated with the intracellular concentration of pyruvateferredoxin oxidoreductase leading to a concomitant decrease in the uptake of free metronidazole into the cell, while resistance to furazolidone appears to be due to an increase in thiol cycling enzymes. At the molecular level resistance to metronidazole is associated with DNA changes. DNA probes which hybridize with specific chromosomes and repetitive sequences indicate that rearrangements both at the chromosome and repetitive DNA level occurred concurrently with the development of metronidazole resistance. The problems of cross-resistance and treatment failures that occur in the absence of resistance are additional difficulties which have important implications for the management of individual patients. New drugs such as azithromycin, while showing great variation in activity against different stocks may be useful in treating some refractory cases of giardiasis. In the community, it is important to recognize the occurrence and spread of drug resistant Giardia, and markers, such as DNA probes, provide methods to monitor potential epidemics and the spread of drug resistant Giardia.

A DNA fingerprint procedure has been established for the intestinal parasite, Giardia duodenalis. This permits the identification of individual strains from both human and animal sources. Analysis of strain movement, resurgence and variation is now possible. The fingerprint probe is based on the tandem repetitive sequence found in bacteriophage M13 which hybridizes to a set of hypervariable polymorphic minisatellite sequences found in higher eukaryotes. This probe recognizes a weakly homologous set of hypervariable regions in the Giardia genome to provide a DNA fingerprint comparable to those seen in higher eukaryotes.

The advantages and limitations of determining mechanisms of drug resistance in Giardia duodenalis laboratory isolates, which have been generated in a number of ways, is weighed against the difficulty of analysing mechanisms in clinical isolates with a large diversity of genetic and expression capabilities. Using isogenic strains to follow changes in enzyme regulation involved in drug resistance, we have been able to assess the full capability of the parasite in developing drug resistance mechanisms. The complementarity of the two approaches, clinical versus laboratory induced drug resistance, and continuing comparison with other organisms, particularly the anaerobic bacteria with which Giardia has strong affiliations, is emphasized. These considerations lead to the study of the population genetics of drug resistance, and strategies critical for rational drug usage, design and therapy.

cDNA and genomic DNA of Giardia intestinalis have been cloned in pUC vectors and used to express Giardia antigens in Escherichia coli. Several expression libraries have been produced and positive clones identified by immuno-colony assays with antisera raised against whole parasites and partially purified antigen(s). Those clones which express G. intestinalis antigens have been used to raise antisera in mice and the antisera used in immunofluorescence assays. The proteins expressed by the clones have been shown to represent a 32 kDa protein of the flagellae and axonemes, a protein associated with the spiral part of the ventral disc, proteins covering the surface of the trophozoite or associated with the coat, and other proteins associated with axonemes of posterolateral flagellae, kinetosomes and funis, and the anterolateral axonemes. mRNA was purified from G. intestinalis and translated in a cell free lysate. A rabbit antiserum raised against trophozoites immunoprecipitated several translation products while an antiserum raised against a purified 32 kDa protein only immunoprecipitated this protein. G. intestinalis rRNA subunits also were examined in the course of mRNA purification. Two rRNA species were evident, the small rRNA and the post-transcriptionally processed large rRNA.

Chromosomes of 41 stocks of Giardia duodenalis derived from humans and 14 stocks from other animal species were analysed by field inversion gel electrophoresis (FIGE). These stocks have two predominant karyotypes as judged by FIGE which appear to fit a geographic distribution. Under FIGE conditions used to optimize the detection of size variation in Giardia chromosomes, five or six major chromosomes could be identified. Most of the stocks derived from North America have three major chromosomes smaller than 800 kb while most of the Australian stocks have four. A few exceptions, and minor variations, of these karyotypes were observed. It was estimated that not all of the DNA entered the gel, the remainder being trapped conformations or very large chromosomes. Karyotypes of Giardia stocks from different animal hosts and human sources within a geographical region are similar.

Three stocks of Blastocystis hominis were adapted to monophasic culture in minimal essential medium (MEM) and the chromosomes of these stocks separated by field inversion gel electrophoresis (FIGE). Ten-twelve chromosomes were distinguished in the electrophoretic karyotype of these three stocks over the range 200 kilobase pairs to > 1 megabase pairs. The karyotype of each stock was different. Three DNA probes, B10, B30 and B31, derived from the Netsky stock isolated in America were used as chromosome markers. Probe B10 hybridized to chromosomes of the same size in two of the stocks, one of which was isolated in the U.S.A. and the other in Queensland. B30 and B31 hybridized to a similar number of chromosomes of different sizes in these two stocks. The third stock, from Australia, did not hybridize at all with probes B10 and B30 and only weakly with probe B31.

Transfection of African green monkey kidney cells directly with recombinant DNA excised from, but still present in, Seaplaque agarose after electrophoresis, is described. Efficiencies of transfection increased by 30% when the gel was present compared with transfection in the absence of the agarose. Extraction of the DNA from the gel was not necessary, thereby obviating a purification step and the concomitant losses. To generate recombinant molecules bacterial plasmid sequences are not necessary, thereby reducing considerably the size of the recombinant molecule and removing extraneous and deleterious sequences, e.g., “poison sequences.” Linear or circular DNA molecules could be transfected in the melted and diluted agarose with the same ease as in its absence. Hence linear partial ligation products can be excised from the gel after electrophoresis to generate recombinant DNA molecules directly in mammalian cells.

We describe a system to generate cDNA or genomic libraries from DNA segments that have blunt termini. Background and rearrangement levels are low, but efficiencies are high and the procedural times very short. T4 ligase in the presence of polyethylene glycol produces high Mr oligomers of vector and insert. These concatemers are reduced to vector-insert monomers at a high frequency by subsequent cleavage with a restriction endonuclease, which recognises the insert rarely, if at all, and the vector once. The monomers are recircularised under standard ligation conditions prior to transformation. Thus insertion conditions are optimised independently of those for recircularisation. All reading frames for expression libraries are generated by short BAL 31 cleavage followed by the blunt-end cloning procedure. Similarly, genomic expression libraries can be made by BAL 31 or mung-bean nuclease treatment after cleavage with DNase I is the presence of Mn2+. The technique is suitable for any DNA segment that is blunt-ended or can be made so. When the vector is treated with alkaline phosphatase, recombinants are generated at a frequency greater than 90% and have single inserts. Yields are 3-5 X 10(6) colony-forming units per micrograms of insert.

A simple method is described which permits both the separation and concentration of circular recombinant plasmids from smaller plasmid vectors that are an undesirable by-product of a ligation reaction. SeaPlaque agarose plugs are used to entrap open-circular forms of recombinant plasmids during electrophoresis. In the example described over 98% of supercoiled, open-circular and linear forms of the 2.9-kb Bluescript plasmid vector, as well as the equivalent dimer forms, pass through the 1.4% SeaPlaque plug. Circular recombinant plasmids greater in length than the vector dimer are entrapped within the plug. By increasing the concentration of SeaPlaque, recombinants smaller than the vector dimer are retained in the trap, but with a concomitant increase in contamination by open-circular vector dimers. For most library constructions the high ratio of insert to vector used during the ligation reaction reduces the formation of vector dimers and makes this level of contamination inconsequential. The recombinant plasmids can be extracted readily from the SeaPlaque plug by excising it, melting the agarose and extracting with phenol. Alternatively, the excised plug can be melted and the recombinant plasmids used to transform bacteria, or mammalian cells, directly in the agarose. The procedure should be valuable for cloning large inserts for ‘jumping’ and ‘linking’ libraries, for large inserts in general where recircularisation is a low-frequency event e.g., minichromosomes, for pulsed-field gel electrophoresis applications, and for hosts and vectors where genetic selection of the recombinant is not possible.

It is estimated that approximately 100 gene loci are involved in coding for functions concerned with the processing of DNA, such as replication, repair and recombination. Diseases associated with mutations in this gene class include the autosomal recessive disorders xeroderma pigmentosum, ataxia telangiectasia, Cockayne’s syndrome, Bloom’s syndrome, Rothmund-Thompson syndrome and Fanconi’s anaemia. Other disorders in this group, inherited as dominant traits, include some forms of retinoblastoma (D-deletion), Gardiner’s syndrome, familial melanoma and some ‘cancer-prone’ families. Recently evidence has accrued which suggests there is a high correlation between ionizing radiosensitivity and Huntington’s disease, while this may also be true of some other types of dementia including a proportion of cases of Alzheimer’s disease. Some selective developmental anomalies of the immune system may also be related to mutants of this general class. Emphasis to date has been on the sensitivity of these mutants to extrinsically generated DNA damage. There is reason, however, to believe that some of the pleotropic effects observed clinically in those with an early onset of symptoms are due to developmental anomalies related to abnormal DNA replication or recombination events in embryo. Where neuronal loss occurs at a much later age, both programmed degeneration and accumulation of damage must be considered.