Dynamic molecular combing: stretching the whole human genome for high-resolution studies.

DNA in amounts representative of hundreds of eukaryotic genomes was extended on silanized surfaces by dynamic molecular combing. The precise measurement of hybridized DNA probes was achieved directly without requiring normalization. This approach was validated with the high-resolution mapping of cosmid contigs on a yeast artificial chromosome (YAC) within yeast genomic DNA. It was extended to human genomic DNA for precise measurements ranging from 7 to 150 kilobases, of gaps within a contig, and of microdeletions in the tuberous sclerosis 2 gene on patients' DNA. The simplicity, reproducibility, and precision of this approach makes it a powerful tool for a variety of genomic studies.

Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by the widespread development of distinctive tumors termed hamartomas. TSC-determining loci have been mapped to chromosomes 9q34 (TSC1) and 16p13 (TSC2). The TSC1 gene was identified from a 900-kilobase region containing at least 30 genes. The 8.6-kilobase TSC1 transcript is widely expressed and encodes a protein of 130 kilodaltons (hamartin) that has homology to a putative yeast protein of unknown function. Thirty-two distinct mutations were identified in TSC1, 30 of which were truncating, and a single mutation (2105delAAAG) was seen in six apparently unrelated patients. In one of these six, a somatic mutation in the wild-type allele was found in a TSC-associated renal carcinoma, which suggests that hamartin acts as a tumor suppressor.

Advances in fluorescent in situ hybridisation.

Recent advances in fluorescent in situ hybridisation included the generation of allele-specific probes, bar-coded chromosomes, and the visualisation of chromosome territories and genes within the nucleus. One major advance has been our ability to visualise and make precise and reproducible measurements from stretched DNA molecules prepared directly from human cells.

The human glucosamine-6-phosphate deaminase gene: cDNA cloning and expression, genomic organization and chromosomal localization.

When mammalian eggs are fertilized by sperm, a distinct series of calcium oscillations are generated which serve as the essential trigger for egg activation and early embryo development. The identification of a soluble hamster sperm 33-kDa protein that co-migrated with calcium oscillation-inducing activity was recently described by Parrington et al. (Parrington, J., Swann, K., Shevchenko, V.I., Sesay, A.K. and Lai, F.A., 1996. Calcium oscillations in mammalian eggs triggered by a soluble sperm protein. Nature 379, 364-368). The hamster sperm 33 kDa protein was termed oscillin because it correlated with calcium oscillation-inducing activity in mammalian eggs. Sequence analysis of the hamster sperm 33 kDa protein indicated no similarity to any known cell signalling molecule, however, it displayed extensive homology with a bacterial glucosamine-6-phosphate deaminase. We have isolated the corresponding human testis homologue of the hamster sperm 33 kDa cDNA. Nucleotide sequence analysis reveals a high level of sequence identity between the hamster and human genes. The deduced protein sequence of the human gene also shares extensive amino acid identity with the bacterial glucosamine-6-phosphate deaminase enzyme. Heterologous expression of the human testis 33 kDa protein produced a glucosamine-6-phosphate deaminase activity. The genomic structure of the human glucosamine-6-phosphate deaminase has been mapped and the gene was localized by fluorescence in situ hybridization (FISH) to chromosome 5q31.

Comparison of the in vitro activities of quassinoids with activity against Plasmodium falciparum, anisomycin and some other inhibitors of eukaryotic protein synthesis.

Using the inhibition of incorporation of [3H]hypoxanthine as an index of viability of malaria parasites, it was shown that a chloroquine-sensitive strain of Plasmodium falciparum (T9-96) and a chloroquine-resistant strain (K1) did not differ in their sensitivities to the quassinoids ailanthinone, bruceantin and chaparrin. Similarly, there were no differences between the strains in their sensitivities to the protein synthesis inhibitors anisomycin, deacetylanisomycin, cephalotaxine, homoharringtonine, cycloheximide, puromycin and puromycin aminonucleoside. The IC50 values derived for ailanthinone and bruceantin, cycloheximide, homoharringtonine and puromycin were in the nanomolar range, whereas those for the anisomycins, cephalotaxine and the aminonucleoside of puromycin were micromolar or greater. Those drugs tested which contain an ester moiety (ailanthinone, bruceantin, anisomycin, homoharringtonine) were more active than the related drugs (chaparrin, deacetylanisomycin, cephalotaxine) that do not. Cross-resistance to inhibitors of protein synthesis appeared not to accompany resistance to chloroquine.

The chemotherapy of rodent malaria. XLV. Reversal of chloroquine resistance in rodent and human Plasmodium by antihistaminic agents.

The inherent blood schizontocidal activities of five antihistaminic compounds, cyproheptadine hydrochloride (CYP), ketotifen hydrogen fumarate (KET), pizotyline hydrogen maleate (PIZ), azatadine maleate (AZAT) and loratadine (LOR) were examined against the following organisms: chloroquine-sensitive (CS) Plasmodium berghei and chloroquine-resistant (CR) P. yoelii ssp. NS in mice; and CS Tak 9 clone 96 and CR K1 strain of P. falciparum in vitro. Chloroquine, verapamil and desipramine were used as comparison standards. CYP, KET, PIZ were active against the CS strain in vivo with ED90 levels between 20 and 30 mg kg-1 (given sc daily for four days). They were slightly more active against the CR strain. AZA was active, but much less so than the other compounds. LOR, verapamil and desipramine were inactive in vivo at the doses tested. Against CS P. falciparum in vitro, all five antihistaminics and desipramine were active at EC50 concentrations ranging from about 50-80 mumol l-1, while verapamil was only active at 175 mumol l-1. Against the CR strain of this parasite, CYP, PIZ and LOR were slightly more active than against the CS strain, but KET, AZAT, desipramine and verapamil were significantly less active. The action of all these compounds in combination with chloroquine was then examined both in vivo and in vitro. The ability of verapamil and desipramine to reverse chloroquine resistance in vitro was confirmed, but only a low level of reversal was seen with these compounds in vivo. However, CYP, KET, PIZ and AZAT produced a marked reversal of chloroquine resistance both in vivo and in vitro. The implications of these observations in relation to further laboratory and clinical research are discussed.

Transcripts of the multidrug resistance genes in chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum.

Homologues of the mammalian multidrug resistance gene have been identified in isolates and clones of Plasmodium falciparum and designated pfmdr1 and pfmdr2. Mutations in pfmdr1 have been associated with chloroquine resistance but confirmation could not be obtained in a genetic cross. We have examined the copy number and expression of pfmdr1 and pfmdr2 in chloroquine-sensitive and -resistant P. falciparum and have found no relationship between the copy number of either gene and chloroquine resistance. However, a marked correlation was seen between levels of mRNA transcribed for each gene and chloroquine resistance. Two transcripts of pfmdr1 were detected, and in the asexual blood cycle an 8 kb transcript appeared first, followed by the appearance of a 7 kb species.

A mutation screen of the TSC1 gene reveals 26 protein truncating mutations and 1 splice site mutation in a panel of 79 tuberous sclerosis patients.

The entire coding region of the TSC1 gene has been screened for mutations in 79 unrelated patients with tuberous sclerosis. Causative mutations have been found in 27 of these patients and five other variations in the gene have been identified. 26 of the mutations are predicted to cause premature truncation of the protein product of the gene and one mutation is in a splice site. The mutation screen has revealed that TSC1 mutations are rarer in sporadic tuberous sclerosis patients than in familial cases. We have also found that the only previously described case of non-penetrance can no longer be described as such, and that a single ungual fibroma is not necessarily diagnostic of tuberous sclerosis, important findings for the genetic counselling of tuberous sclerosis patients.

Construction of a radiation hybrid map of chromosome 9p.

A radiation hybrid panel has been constructed for chromosome 9 using the somatic cell hybrid GM10611 as the donor cell line fused to the hamster cell line A23. The hybrid GM10611 was characterized by fluorescence in situ hybridization and reverse painting onto spreads of normal human metaphase chromosomes; it contains human chromosome 9 as the only cytogenetically detectable human material. GM10611 was irradiated with 6000 rads of X rays prior to fusion, a total of 93 independent clones were selected, and frozen stocks and DNA were prepared from each clone. These clones were screened by PCR amplification with oligonucleotide primers for sequence-tagged sites specific for 50 single-copy loci mapping to the short arm of chromosome 9. The average retention frequency of these hybrids was approximately 23%. The markers were ordered into a framework map by analyzing coretention patterns, minimizing the number of obligatory chromosome breaks, and finally confirming the order by maximum likelihood methods. A framework map ordering 27 markers with odds greater than 1000:1 was constructed. A further 16 markers that could not be uniquely placed on the map with the required support were positioned within a range of adjacent intervals.

Plasmodium falciparum: effects of phaeanthine, a naturally-occurring bisbenzylisoquinoline alkaloid, on chloroquine-resistant and -sensitive parasites in vitro, and its influence on chloroquine activity.

Phaeanthine, a bisbenzylisoquinoline alkaloid which occurs naturally in Triclisia species, was extracted from Triclisia patens (Menispermaceae) obtained from Sierra Leone (West Africa). In vitro, phaeanthine was found to be twice as potent against a chloroquine-resistant Plasmodium falciparum strain (K1), as against a chloroquine-sensitive clone (T9-96), with 50% inhibitory concentrations of 365.85 (+/- 11.41) nM and 704.87 (+/- 81.48) nM respectively. At a sub-inhibitory concentration of 80.35 nM, chloroquine resistance was not reversed by phaeanthine. Isobolograms constructed from experiments with chloroquine/phaeanthine combinations showed antagonism in T9-96 and an additive effect in K1. In a 48-hour microtest, phaeanthine at antimalarial concentrations showed no cytotoxicity to mammalian (KB) cells in vitro.

Construction and characterization of a highly stable human: rodent monochromosomal hybrid panel for genetic complementation and genome mapping studies.

Human:rodent somatic cell hybrids carrying a single, intact, selectable human chromosome are valuable both for functional somatic cell genetic analysis and genome mapping procedures. Here, we describe the construction and detailed molecular cytogenetic characterization of a panel of 23 stable hybrids, representing all 22 human autosomes plus the X-chromosome. Individual normal human chromosomes have been tagged with a selectable fusion gene (Hytk) introduced into the chromosome in a small (4.2 kbp) retroviral vector. Use of the Hytk marker permits both positive and negative ("in-out") selection to be applied to the human chromosome in any mammalian cell background. The panel includes 18 new hybrids isolated by direct microcell transfer from normal human diploid fibroblasts into mouse A9 cells.

A 1.7-megabase sequence-ready cosmid contig covering the TSC1 candidate region in 9q34.

The disease gene TSC1 has been genetically mapped to human chromosome region 9q34, in a 4-cM interval between the markers D9S149 and D9S114. Within this interval there is conflicting genetic evidence as to the finer localization of the gene. We have used finger-printing methods and hybridization to produce a 1.7-Mb overlapping clone map covering the TSC1 candidate region, with a single gap of 20 kb. We have localized 12 previously cloned genes and 17 genetic markers on this map and have confirmed the order of the genetic map. This deep set of overlapping clones is now ready to be used for candidate gene isolation, for transcription studies, or for sequencing.