Biologically relevant effects of mRNA amplification on gene expression profiles

Background  

Gene expression microarray technology permits the analysis of global gene expression profiles. The amount of sample needed limits the use of small excision biopsies and/or needle biopsies from human or animal tissues. Linear amplification techniques have been developed to increase the amount of sample derived cDNA. These amplified samples can be hybridised on microarrays. However, little information is available whether microarrays based on amplified and unamplified material yield comparable results.     

Gβ Regulates Coupling between Actin Oscillators for Cell Polarity and Directional Migration

For directional movement, eukaryotic cells depend on the proper organization of their actin cytoskeleton. This engine of motility is made up of highly dynamic nonequilibrium actin structures such as flashes, oscillations, and traveling waves. In Dictyostelium, oscillatory actin foci interact with signals such as Ras and phosphatidylinositol 3,4,5-trisphosphate (PIP₃) to form protrusions. However, how signaling cues tame actin dynamics to produce a pseudopod and guide cellular motility is a critical open question in eukaryotic chemotaxis. Here, we demonstrate that the strength of coupling between individual actin oscillators controls cell polarization and directional movement. We implement an inducible sequestration system to inactivate the heterotrimeric G protein subunit Gβ and find that this acute perturbation triggers persistent, high-amplitude cortical oscillations of F-actin. Actin oscillators that are normally weakly coupled to one another in wild-type cells become strongly synchronized following acute inactivation of Gβ. This global coupling impairs sensing of internal cues during spontaneous polarization and sensing of external cues during directional motility. A simple mathematical model of coupled actin oscillators reveals the importance of appropriate coupling strength for chemotaxis: moderate coupling can increase sensitivity to noisy inputs. Taken together, our data suggest that Gβ regulates the strength of coupling between actin oscillators for efficient polarity and directional migration. As these observations are only possible following acute inhibition of Gβ and are masked by slow compensation in genetic knockouts, our work also shows that acute loss-of-function approaches can complement and extend the reach of classical genetics in Dictyostelium and likely other systems as well.     

Relaxin family peptide receptors Rxfp1 and Rxfp2: mapping of the mRNA and protein distribution in the reproductive tract of the male rat

Background  

Relaxin is the endogenous ligand of the G-protein coupled receptor RXFP1, previously known as LGR7. In humans relaxin can also activate, but with lower affinity, the closely related receptor for the insulin-like peptide from Leydig cells, RXFP2, previously known as LGR8. The lack of relaxin impairs male fertility but the precise distribution and the function of relaxin receptors in the male reproductive tract is not known. We investigated the distribution of Rxfp1 and Rxfp2 in the reproductive tract of the male rat and the function of relaxin in the vas deferens, a tissue with high expression of both receptors.     

Isozyme and plastid DNA assessment of pedigrees of nineteenth century potato cultivars

Summary Isozyme and ctDNA RFLP patterns were determined for ten historically important potato cultivars (Solanum tuberosum ssp. tuberosuni) in order to relate and confirm their pedigrees. Isozyme polymorphism was detected at 11 of 13 loci examined, whereas only T-type cytoplasm, the predominant ctDNA of S. tuberosum ssp. tuberosum, was observed. Isozyme analysis indicated that potato cultivars previously presumed to be derived from open-pollinated berries of Garnet Chili and Early Rose were in fact the result of hybridizations. In addition, putative pedigrees of Irish Cobbler, White Rose, and Bliss Triumph were not supported. Garnet Chili, the first derivative of Rough Purple Chili, carries allozmyes at Mdh-1 and Pgm-2, which supports the Chilean origin of Rough Purple Chili. The identical ctDNA pattern among the cultivars may indicate a common maternal lineage that traces through Garnet Chili to Rough Purple Chili. The allozyme frequencies estimated from these cultivars provide a base from which subsequent introductions of Solanum species into the ssp. tuberosum gene pool can be assessed.Journal Article No. 000123     

Efficiency of potato breeding using FDR 2n gametes for multitrait selection and progeny testing

Summary The objective of this research was to compare the efficiency of the 4x×2x breeding scheme with the traditional 4x×4x method with respect to potato improvement. The basis for such a comparison was the parental value of four 2x and four 4x male parents from the International Potato Center (CIP) as measured by multitrait selection and progeny testing. The 2x parents produced 2n pollen by parallel spindles at anaphase II, which is genetically equivalent to a first division restitution (FDR) mechanism. Both 2x and 4x parents were crossed with four common 4x female parents. Thus, 32 families were evaluated over 2 years at four Peruvian locations. A selection index which considered tuber yield, tuber number, average tuber weight and specific gravity was used for multitrait selection. Three FDR 2x parents had better selection index scores than the 4x parents over the four locations. Estimates of broad-sense heritability for total yield using different number of replications and locations were calculated by using the variance components. The 4x × 2x breeding scheme was found to be better than the traditional 4x × 4x method since fewer replications and locations are required to evaluate tuber yield in 4x × 2x progenies than in 4x × 4x progenies. The FDR 2x parents were also better material than the 4x parents for testing combining ability for tuber yield of the 4x progenitors. This could be the result of the mode of FDR 2n pollen formation. The pollen of FDR 2x parents is more heterozygous, but more homogenous than n pollen from 4x parents.Paper from the Laboratory of Genetics. Research supported by the College of Agricultural and Life Sciences; International Potato Center; USDA-CRGO-88-37234 3619, and Frito-Lay, Inc., USA     

Towards an integrated linkage map of common bean. 4. Development of a core linkage map and alignment of RFLP maps

 Three RFLP maps, as well as several RAPD maps have been developed in common bean (Phaseolus vulgaris L.). In order to align these maps, a core linkage map was established in the recombinant inbred population BAT93×Jalo EEP558 (BJ). This map has a total length of 1226 cM and comprises 563 markers, including some 120 RFLP and 430 RAPD markers, in addition to a few isozyme and phenotypic marker loci. Among the RFLPs mapped were markers from the University of California, Davis (established in the F₂ of the BJ cross), University of Paris-Orsay, and University of Florida maps. These shared markers allowed us to establish a correspondence between the linkage groups of these three RFLP linkage maps. In total, the general map location (i.e., the linkage group membership and approximate location within linkage groups) has been determined for some 1070 markers. Approaches to align this core map with other current or future maps are discussed. Received: 10 March 1998 / Accepted: 22 April 1998     

Toxoplasma gondii: protection against colonization of the brain and muscles in a rat model

The objective was to test immune protection against the formation of Toxoplasma gondii tissue cysts in rats. It has been previously shown that 50 T. gondii tissue cysts of strain Me49 are not pathogenic for CF-1 mice, whereas 1 T. gondii tissue cyst of strain M-7741, can be lethal for mice 11-13 days after subcutaneous or oral administration. In the present study, ten rats were fed T. gondii oocysts of strain Me49 and after a further 30 days they were each orally challenged with T. gondii oocysts of strain M-7741. Thirty days after this, they were euthanased and brain and muscle samples inoculated subcutaneously or orally dosed, respectively, to mice for bioassay. None of the mice died, whereas all the mice that were inoculated with brain homogenates or were fed muscle samples from four non-immunized rats that had been inoculated with T. gondii oocysts of strain M-7741, died. These results encourage further research towards achieving vaccinal protection against the formation of T. gondii tissue cysts in meat animals and people.     

Very high expression of an anti-carcinoembryonic antigen single chain Fv antibody fragment in the yeast Pichia pastoris

In this paper we report the development of a recombinant strain of the yeast Pichia pastoris, which secretes an anti-carcinoembryonic antigen single chain Fv (scFv) antibody fragment to the culture supernatant as a biologically active protein, at levels of 1.2 g l(-1). The yeast scFv was purified by IMAC, with a final yield of approximately 0.440 g of 93% pure scFv per liter of culture supernatant. The specific activity in ELISA of the yeast scFv was almost three times higher than that of a bacterial periplasmic counterpart. These results reaffirm that the yeast P. pastoris is a suitable host for high level production of scFv antibody fragments with potential in vivo diagnostic and therapeutic applications.     

Patterns of reproductive isolation in nolana (chilean bellflower)

We examined reproductive isolating barriers at four postmating stages among 11 species from the morphologically diverse genus Nolana (Solanaceae). At least one stage was positively correlated with both genetic and geographic distance between species. Postzygotic isolation was generally stronger and faster evolving than postmating prezygotic isolation. In addition, there was no evidence for mechanical isolation, or for reproductive character displacement in floral traits that can influence pollinator isolation. In general, among the potential isolating stages examined here, postzygotic barriers appear to be more effective contributors to reducing gene flow, including between sympatric species.