Fluorescence in situ hybridisation of multiple probes on a single microscope slide.

We report a method to analyse multiple samples by fluorescence in situ hybridisation on a single glass microscope slide. Wells were formed in which independent hybridisation reactions could proceed by sealing a silicon rubber gasket to the slide. In the largest format tested, different probes were hybridised simultaneously by applying them directly from a 96-well microtitre dish which was inverted on a glass plate. This technique will increase the rate of analysis of multiple probes against a standard set of chromosomes and could also be used to analyse different karyotypes using a panel of probes such as single chromosome paints during a single operation. It should be useful for both chromosomal mapping projects and screening for chromosome abnormalities in clinical diagnostic laboratories.     

Isolation of the human sex determining region from a Y-enriched yeast artificial chromosome library

We describe the isolation and characterization of yeast artificial chromosome (YAC) clones spanning the male sex determining region on the short arm of the human Y chromosome. The clones were isolated by hybridizing probes in the interval between the genes MIC2 and ZFY to a Y chromosome-enriched YAC library. The YAC clones were consistent with the order of probes established for this interval and may be useful for functional studies of the region in male sex determination. However, many of the YAC clones from this library carried only one arm of the vector ("half-YACs"), deleted sequences from one end, and contained much smaller inserts (148 kb average) than the size of ligated fragments selected by pulsed-field gel electrophoresis (greater than 440 kb). These problems were overcome by protecting DNA with polyamines during YAC library construction and a second Y-enriched YAC library was constructed with an average insert size of 627 kb.     

Genetic diversity of <Emphasis Type="Italic">Escherichia coli</Emphasis> in gut microbiota of patients with Crohn’s disease discovered using metagenomic and genomic analyses

Background

Crohn’s disease is associated with gut dysbiosis. Independent studies have shown an increase in the abundance of certain bacterial species, particularly Escherichia coli with the adherent-invasive pathotype, in the gut. The role of these species in this disease needs to be elucidated.

Methods

We performed a metagenomic study investigating the gut microbiota of patients with Crohn’s disease. A metagenomic reconstruction of the consensus genome content of the species was used to assess the genetic variability.

Results

The abnormal shifts in the microbial community structures in Crohn’s disease were heterogeneous among the patients. The metagenomic data suggested the existence of multiple E. coli strains within individual patients. We discovered that the genetic diversity of the species was high and that only a few samples manifested similarity to the adherent-invasive varieties. The other species demonstrated genetic diversity comparable to that observed in the healthy subjects. Our results were supported by a comparison of the sequenced genomes of isolates from the same microbiota samples and a meta-analysis of published gut metagenomes.

Conclusions

The genomic diversity of Crohn’s disease-associated E. coli within and among the patients paves the way towards an understanding of the microbial mechanisms underlying the onset and progression of the Crohn’s disease and the development of new strategies for the prevention and treatment of this disease.

     

Generation of large insert yeast artificial chromosome libraries

The development of YAC cloning technology has directly enhanced the relationship among genetic, physical, and functional mapping of genomes. Because of their large size, YACs have enabled the rapid construction of physical maps by ordered clone mapping and contig building, and they complement other molecular approaches for mapping complex genomes. Large insert libraries are constructted by size fractionating large DNA embedded in agarose and protecting DNA from degradation with polyamines.     

MEDIATION BY β-ADRENERGIC RECEPTORS OF EFFECT OF NOREPINEPHRINE ON PINEAL SYNTHESIS OF [14C]SEROTONIN AND [14C]MELATONIN

Rat pineal organs maintained in organ culture converted [¹⁴C]tryptophan to [¹⁴C]serotonin and [¹⁴C]melatonin. The synthesis of both indoles was stimulated by the presence of norepinephrine or dibutyryl adenosine 3′,5′-monophosphate. This effect of norepinephrine could be blocked by the α-adrenergic blocking drug, propranolol, but was not modified by the a-adrenergic blocking agent, phenoxybenzamine. Neither blocking agent modified the pineal response to dibutyryl adenosine 3′,5′-monophosphate. Unlike dibutyryl adenosine 3′,5′-monophosphate, the naturally occurring adenosine phosphates did not stimulate synthesis of [¹⁴C]melatonin in vitro.     

Pattern of Resource Allocation of Six Plantago Species with Different Breeding Systems

Plantago exhibit great deal of differences in the breeding system. The reproductive effort calculated on the basis of, (i) dry biomass of foliar and floral parts and (ii) seed output-weight (mg) per unit leaf area (cm²), exhibits relation with breeding system. The predominantly inbreeding taxa invest higher reproductive effort compared to their outbreeding allies. In terms of sex allocation strategies, the outbreeding species like P. lanceolata, P. lagopus invest more to the development of floral features and to male functions. On the contrary, inbreeding species such as P. patagonica, P. drummondii, and P. ovata invest greater resources to the female function. Received 13 April 1998/ Accepted in revised form 6 November 1998