A genetic mapping system in Caenorhabditis elegans based on polymorphic sequence-tagged sites.

We devised an efficient genetic mapping system in the nematode Caenorhabditis elegans which is based upon the differences in number and location of the transposable element Tc1 between the Bristol and Bergerac strains. Using the nearly completed physical map of the C. elegans genome, we selected 40 widely distributed sites which contain a Tc1 element in the Bergerac strain, but not in the Bristol strain. For each site a polymerase chain reaction assay was designed that can distinguish between the Bergerac Tc1-containing site and the Bristol "empty" site. By combining appropriate assays in a single reaction, one can score multiple sites within single worms. This permits a mutation to be rapidly mapped, first to a linkage group and then to a chromosomal subregion, through analysis of only a small number of progeny from a single interstrain cross.

A survey of expressed genes in Caenorhabditis elegans.

As an adjunct to the genomic sequencing of Caenorhabditis elegans, we have investigated a representative cDNA library of 1,517 clones. A single sequence read has been obtained from the 5' end of each clone, allowing its characterization with respect to the public databases, and the clones are being localized on the genome map. The result is the identification of about 1,200 of the estimated 15,000 genes of C. elegans. More than 30% of the inferred protein sequences have significant similarity to existing sequences in the databases, providing a route towards in vivo analysis of known genes in the nematode. These clones also provide material for assessing the accuracy of predicted exons and splicing patterns and will lead to a more accurate estimate of the total number of genes in the organism than has hitherto been available.

YACs and the C. elegans genome.

During the past decade, it has become apparent that it is within our grasp to understand fully the development and functioning of complex organisms. It is widely accepted that this undertaking must include the elucidation of the genetic blueprint - the genome sequence - of a number of model organisms. As a prelude to the determination of these sequences, clone-based physical maps of the genomes of a number of multicellular animals and plants are being constructed. Yeast artificial chromosome (YAC) vectors, by virtue of their relatively unbiased cloning capabilities and capacity to carry large inserts, have come to play a central role in the construction of these maps. The application of YACs to the physical map of the Caenorhabditis elegans genome has enabled cosmid clone 'islands' to be linked together in an efficient manner. The long-range continuity has improved the linkage between the genetic and physical maps, greatly increasing its utility. Since the genome can be represented by a relatively small number of YACs, it has been possible to make replica filters of genomically ordered YACs available to the community at large.

Studies of human pituitary LH containing internally cleaved beta subunit.

We have investigated the origin of the internal peptide bond cleavage found in the beta subunit of a proportion of pituitary human LH (hLH) molecules, as well as the effects of this cleavage (or nick) on the interaction between alpha and beta subunits and on binding of hLH to its receptor. The content of cleaved beta subunit, assessed by the intensity of staining of an approximately 10 kDa component on sodium dodecyl sulphate-polyacrylamide gel electrophoresis of reduced hLH, was variable in batches of hLH prepared from pooled acetone-preserved human pituitary glands. There was evidence of a similar cleavage in purified hTSH, but not in the purified hFSH or human chorionic gonadotrophin examined. Although intact hLH was relatively resistant to cleavage in solution, urea dissociation of hormone followed by dialysis resulted in an increased content of nicked beta subunit, which was largely prevented by incorporation of the proteolytic enzyme inhibitor phenylmethylsulphonyl fluoride (PMSF) and the metal-chelating agent EDTA. Hormone that was virtually free of nicked beta subunit was obtained by urea dissociation of hLH subunits in the presence of PMSF and EDTA followed by dialysis to remove urea, reassociation of subunits at 37 degrees C (pH 7) and purification of reassociated hLH dimer by gel filtration on Sephadex G-100 in the presence of 1-anilinonaphthalene-8-sulphonic acid (ANS).(ABSTRACT TRUNCATED AT 250 WORDS)

Studies of the heterogeneity of human pituitary LH by fast protein liquid chromatography.

The Pharmacia fast protein liquid chromatography system was employed to fractionate a purified preparation of human LH (hLH) on the anion exchanger Mono Q at pH 7 X 8 into 14 sub-fractions. Each of the sub-fractions was characterized by its behaviour on polyacrylamide gel electrophoresis, sodium dodecyl sulphate (SDS) gel electrophoresis, LH receptor binding activity and sialic acid content. All sub-fractions contained sialic acid, were active in binding to LH receptors, and exhibited components typical of hLH subunits on SDS gel electrophoresis. None of the subfractions was homogeneous with respect to charge. There is evidence that part of the heterogeneity results from the presence in some molecules of an internal proteolytic cleavage within the beta-subunit, and fractions enriched in species containing such cleavages were prepared by this method.

Studies of the human chorionic gonadotrophin-like substance of human pituitary glands and its significance.

Radioimmunoassays utilizing antisera specific for the carboxyl-terminal portion of human chorionic gonadotrophin (hCG) beta-subunit were used to measure the concentration in human pituitary extracts of an immunoactive hCG factor (hCG') which was different from human LH (hLH). The content of hCG' from different human pituitary pools collected between 1966 and 1979 was relatively constant at 0.5-1.1 microgram per gland. The hCG' concentrations observed in acetone-dried powder of individual human pituitary glands (0.4-26 ng/mg) were close to those reported for full-term placental powder. After separation and partial purification of human pituitary glycoprotein hormones, pituitary hCG' was found mainly in the crude human FSH (hFSH) fraction. It was separated from hFSH by diethylaminoethyl-cellulose chromatography at pH 7 and by gel filtration on Sephadex G-100. On gel filtration its molecular size was larger than that of hLH or hFSH and it was strongly bound to Concanavalin A-Sepharose. The most active preparations of pituitary hCG' obtained by these procedures were approximately 5 per cent as potent by specific radioimmunoassay as hCG purified from pregnancy urine. Although the hCG' content in individual pituitary glands was more variable than the hLH content, on average pituitary hCG' was estimated to be around 25- to 50-fold less than the content of hLH, hFSH or human TSH in the human pituitary gland.

Studies of a human growth hormone preparation used for clinical treatment in Great Britain.

This report describes some of the properties of a clinical-grade preparation of human growth hormone (hGH) extracted from acetone-preserved autopsy human pituitary glands and used in Great Britain from 1967 to 1980. Gel filtration of this hGH on Sephadex G-100 yielded (on a weight basis) an average of 48% of a high molecular weight fraction, 10% of an intermediate fraction expected to contain dimeric forms of the hormone and 33% of a fraction considered to be the hGH monomer. The immunoassay potency of the monomer fraction was twice that of the clinical-grade preparation and the amino-acid composition of the monomer fraction agreed well with that obtained from published hGH sequence data. The results of polyacrylamide-gel electrophoresis (under reducing and dissociating conditions) and amino-acid analysis of the high molecular weight fraction suggest that it contains around 30% of aggregated hGH as well as other material not separated from hGH by the purification procedure.

Physiological actions of human follicle-stimulating hormone and its beta-subunit in reptiles.

The actions of human follicle-stimulating hormone (hFSH) and its beta-subunit were examined in several assays in reptiles, including effects on lizard testicular activity (growth and androgen production) in vivo, and stimulation of androgen production by snake testes and competition for binding of 125I-labeled hFSH in lizards and snakes in vitro. Binding was also examined with mammalian tissues. The hFSH was highly steroidogenic in the snake and lizard; otherwise results were similar to those observed in mammals. In all cases, the potency of the beta-subunit was only a few per cent of the intact hormone. The potency of hFSH in vivo compared with NIH-FSH ovine standards was several 100 times greater than in vitro. Results for stimulation of androgen production in vivo closely paralleled those for binding assays in both reptiles and mammals. In contrast to previous results for ovine FSH beta-subunit, human FSH beta-subunit has little if any FSH biological activity in reptiles.

Purification and properties of the subunits of human pituitary follicle-stimulating hormone.

Highly purified human pituitary FSH was partially dissociated by treatment with 8M-urea, and alpha- and beta-subunits were isolated by ion-exchange chromatography and gel filtration. Tests of biological activity by in-vivo assays and in-vitro radioreceptor assays were in good agreement and showed that preparations of isolated alpha-subunit had less than 1%, and beta-subunit from 2 to 10% of the FSH activity of the intact hormone. In contrast to results reported elsewhere, most of the subunit preparations reassociated with counterpart subunit to regain biological activity equal to that of intact FSH (around 160 mg NIH-FSH-S1/mg). The intact FSH recovered as a by-product after isolation of subunits was of high biological activity, and its LH contamination was reduced by more than 90% when compared with thepurified FSH starting material. The subunits are relatively inactive in a radioimmunoassay specific for intact FSH. Sialic acid and tryptophan determinations indicated that both subunits contain sialic acid and that tryptophan is present only in the beta-subunit.