Long-range walking techniques in positional cloning strategies

Conclusion The past several years have seen an explosive growth in our understanding of the organization and structure of mammalian genomes, and refinements of existing techniques for genetic analysis, physical mapping, and large-fragment cloning techniques may well be enough to continue the momentum of that explosion for some time to come. Although refinement of existing techniques will certainly be necessary, the development of new and better cloning techniques may, perhaps, no longer be our most urgent need. The most important challenge that we face at present may in fact be that of finding efficient ways to share existing resources and information rapidly and equitably throughout the scientific community so that progress can continue unimpeded, and to catalog, correlate, and interpret the wealth of new data that is so rapidly accumulating.New strategies aimed at whole-genome mapping (Coulson et al. 1986, 1988; Michiels et al. 1987; Brenner and Livak 1989; Carrano et al. 1989; Lehrach et al. 1991) and sequencing (Church and Keifer-Higgins 1988; Bains and Smith 1988; Drmanac et al. 1989; Strzoska et al. 1991) may someday make the current method of long-range walking and physical mapping nearly passe. For example, since most of the relatively small nematode genome is now stored as ordered sets of cosmid and YAC clones (Coulson et al. 1986, 1988), a walk between a mapped marker and an uncloned gene can be accomplished rapidly, through a request for the appropriate series of clones from the ordered library. Vigorous drives by many laboratories to produce ordered clone libraries for murine and human chromosomes (Lehrach et al. 1991) may transform the process of cloning mammalian genes into a relatively trivial matter within the foreseeable future. The remarkable number of positional-cloning successes that have been reported in recent years may indicate that most of the best-defined, simply inherited mouse mutations and human hereditary disorders will have already been cloned by that time. When that is accomplished, the true challenging task will just begin: we must learn to decipher the complex biological programs encoded by our large and ever-growing storehouse of cloned, mapped and sequenced genes, before we can begin to understand what might be held in the vast silent mass of mammalian genomes. Offprint requests to: L. Stubbs     

Transgenic animals as a tool for studying the effect of the c-myc proto-oncogene on cardiac development

Transgenic animals provide a model system to elucidate the role of specific proteins in development. This model is now being used increasingly in the cardiovascular system to study cardiac growth and differentiation. During cardiac myocyte development a transition occurs from hyperplastic to hypertrophic growth. In the heart the switch from myocyte proliferation to terminal differentiation is synchronous with a decrease in c-myc mRNA abundance. To determine whether c-myc functions to regulate myocyte proliferation and/or differentiation, we examined the in vivo effect of increasing c-myc expression during fetal development and of preventing the decrease in c-myc mRNA expression that normally occurs during myocyte development. The model system used was a strain of transgenic mice exhibiting constitutive expression of c-myc mRNA in cardiac myocytes throughout development. Increased c-myc mRNA expression is associated with both atrial and ventricular enlargement in the transgenic mice. This increase in cardiac mass is secondary to myocyte hyperplasia, with the transgenic hearts containing greater than twice as many myocytes as nontransgenic hearts. The results of this study indicate that constitutive expression of c-myc mRNA in the heart during development results in enhanced hyperplastic growth, and suggest a regulatory role for the c-myc protooncogene in cardiac myogenesis.     

A molecular genetic approach to the identification of isochromosomes of chromosome 21

Summary The largest class of de novo chromosomal rearrangements in Down syndrome are rea(21q21q). Classically, these rearrangements have been termed Robertsonian translocations, implying an attachment of two different chromosome 21 homologues. Additionally, a Robertsonian translocation between two chromosomes 21 cannot be distinguished from an isochromosome composed of genetically identical arms by cytogenetic analyses. Therefore, we have used molecular techniques to differentiate between true Robertsonian translocations and isochromosomes. Samples were obtained from 12 probands, ascertained for de novo rearrangements between homologous chromosomes 21 [11 rea(21q21q) and 1 rea (21;21)(q22;q22)], their parents (n = 24) and available siblings (n = 7). The parental origins of the de novo rearrangements were assigned using molecular and cytogenetic analyses. Although not statistically significant, there was a two-fold increase in the number of paternally derived de novo rearrangements (n = 8) as compared with maternally derived rearrangements (n = 4). To distinguish between rob(21q21q) and i(21q), we used restriction fragment length polymorphisms (RFLPs) spanning the length of chromosome 21. Using all informative and partially informative RFLPs, we used the method of maximum likelihood to assign the most likely rearrangement definition (i or rob) and parental origin in each family. The maximum likelihood estimates indicated that all rearrangements tested (n = 8) were isochromosomes. C-banding revealed two centromeres in three cases indicating that a U-type exchange occurred between sister chromatids in these rearrangements. Our results suggest that the majority of de novo rea(21q21q) are isochromosomes derived from a single parental chromosome 21.     

Effects of various experimental manipulations on neostriatal acetylcholine and dopamine release

The release of endogenous acetylcholine and dopamine and the appearance of their metabolites, choline and dihydroxyphenylacetic acid (DOPAC), from neostriatal slices prepared from Fischer 344 rats was examined under various experimental conditions. There was a dose-dependent increase in the amount of neurotransmitter or metabolite as the medium potassium concentration was increased from 5 to 50 mM. Over an eight minute period in Krebs Ringer bicarbonate buffer containing 25 mM potassium, the rate of release of acetylcholine was 6 to 13 times greater than that of dopamine. The dopamine endogenous to the slice preparation appeared to have little effect on the release of endogenous acetylcholine since manipulations that significantly altered dopamine release (depletion with 6-hydroxydopamine or uptake inhibition with nomifensine) had minimal effects on the cholinergic neurons. In contrast, increasing the endogenous acetylcholine in the preparation by inhibiting acetylcholinesterase resulted in a 1.2 to 12 fold increase in dopamine release depending upon the incubation time and the potassium concentration. These studies indicate that within the neostriatal slices there is minimal influence of the endogenous dopamine on the cholinergic neurons, whereas the extracellular acetylcholine can influence dopamine release when its concentration is increased by inhibition of acetylcholinesterase.     

Chemotaxis in the Marine Fungus Rhizophydium littoreum

Zoospores of the marine chytrid Rhizophydium littoreum are attracted to a variety of substances common to their environment. In general, carbohydrates and polysaccharides elicited strong concentration-dependent positive responses. There was no direct correlation between all substances used as foods and those stimulating positive responses. The chemotactic activities of this organism should, however, tend to bring it toward concentrated food sources.     

Packing of the 30 nm chromatin fiber in the human metaphase chromosome

The human genetic material is packed hierarchically within the metaphase chromosome: the DNA moleculet together with histone proteins form 11 nm diameter nucleosomes, which are then ordered into the 30 nm thick chromatin fiber. Little is known about the packing of this fiber within the chromosome. We have developed a tracking algorithm with which we followed its path within a three-dimensional reconstruction of a human chromosome computed from a series of electron micrographie projections. Fiber segments were seen to form loops of 100–350 nm diameter. Our observations indicate that these loops — which themselves show no preferred orientation — are organised into regions of roughly 200 nm axial extent.     

Stathmokinetic Analysis of Human Epidermal Cells in vitro

Proliferation kinetics of cultured human epidermal cells is characterized in quantitative terms. Three distinct subpopulations of keratinocytes, two of which are cycling have been discriminated by two parameter DNA/RNA flow cytometry. Based on mathematical modelling, the cell cycle parameters of the cycling subpopulations have been assessed from stathmokinetic data collected at different time points after initiation of cultures (7–15 days). the first subpopulation is composed of low-RNA cells which resemble basal keratinocytes of epidermis and which show some characteristics of stem cells; these cells have a mean generation time of approximately 100 hr. the second subpopulation consists of high-RNA cells, resembling stratum spinosum cells of epidermis, which have an average generation time of approximately 40 hr. the third subpopulation consists of non-cycling cells with G_o/G₁ DNA content, with cytochemical features similar to those of cells in granular layer of epidermis. The results based on modelling can reproduce with acceptable accuracy the actual growth curve of the cultured cell population. Analysis of kinetics and differentiation of human keratinocytes is of interest in view of the recent application of cultured epidermal cell sheets for transplantation onto burn wounds. the results of this study also reveal the existence of regulatory mechanisms associated with proliferation and differentiation in the cultured epidermal cell population.     

CAM-idling in Hoya carnosa (Asclepiadaceae)

In the leaf succulent Asclepiad Hoya carnosa (L.) R. Br., CAM photosynthesis occurred under well-watered conditions, as characterized by diurnal gas exchange and changes in titratable acidity. Following 10–12 days of severe water stress, the plants shifted from CAM to a modified CAM-idling mode of metabolism. CAM-idling was characterized by complete or almost complete stomatal closure accompanied by CAM-like diurnal changes in titratable acidity. H. carnosa plants maintained this CAM-idling mode of photosynthesis for at least 8 weeks. Upon reirrigation, the plants returned to the original CAM mode within 1 week. These results suggested that CAM-idling is a reversible, intermediate form of sustained metabolism which enables plant survival under conditions of extended drought.This work was supported in part by NSF Grant PCM 8200366 and in part by the Science and Education Administration of the United States Department of Agriculture under Competitive Grant 5901-0420-8-0018-0.     

Nucleotide sequence of the Rickettsia prowazekii ATP/ADP translocase-encoding gene

The Rickettsia prowazekii ATP/ADP translocase (Tlc) gene (tlc), previously cloned in Escherichia coli was localized to a 1.6-kb chromosomal fragment. Nucleotide sequence analysis of this fragment revealed an open reading frame of 1494 bp that could encode a hydrophobic protein of 497 amino acids (aa) with an M_r of 56 668. Analysis of the deduced aa sequence revealed that it contained twelve potential membrane-spanning regions. Comparisons between the deduced aa sequence of the R. prowazekii ATP/ADP Tlc and the sequences of mitochondrial (mt) Tic revealed no detectable homologies between the rickettsial and mt sequences. The major protein synthesized in E. coli minicells containing the rickettsial gene exhibited an M_r of approx. 34000.