Pronuclear Embryo Yield in Canindé and Saanen Goats for DNA Microinjection

The objective of this study was to examine the effect of donor breed on pronuclear‐stage embryo yield to be used for DNA microinjection in a transgenesis goat program. Twelve Canindé and twelve Saanen goats were heat synchronized using a progestagen‐cloprostenol treatment. Forty‐eight hours before the sponge removal, superovulation was induced with a total administration of 4.4 mg/kg bodyweight NIH‐FSH‐P1, given twice daily in decreasing doses over 3 days. In addition, goats received 100 μg of GnRH and they were hand‐mated at 36 and 48 h after progestagen removal. Embryo recovery was performed by oviduct flushing at 72 h after sponge removal. Embryos were microinjected with a DNA construct and noticeable swelling of the nuclei was the criterion for successful microinjection. The total diameter, cytoplasm diameter, zona pellucida thickness and pronuclei diameter were measured for each microinjected embryo. A higher (p < 0.05) percentage of fertilized ova was observed in Canindé (89.9%) than Saanen (36.2%) goats. In addition, Canindé donors produced a higher percentage of pronuclear embryos when compared with Saanen: 72.5% vs 20.6% (p < 0.05), respectively. Successful microinjection was verified in 96.7% and 73.3% of times in Canindé and Saanen embryos, respectively (p < 0.05). Significant differences were observed for all morphometric parameters except for cytoplasm diameter. In conclusion, under our study experimental conditions, Canindé were more efficient than Saanen goats concerning the pronuclear embryo yield and manipulation. The use of Canindé goats in transgenesis programs could be increase the interest in their breeding and could be contribute to saving them from extinction.     

Seed dormancy and germination in the summer annual Galeopsis speciosa

This study examined germination and dormancy in Galeopsis speciosa (Lamiaceae), a common summer annual weed in cold‐temperate areas. Seeds collected in southern Sweden were subjected to several experiments. The seeds were dormant at maturity. Seeds sown outdoors after collection produced a small number of seedlings that emerged early in the spring. After long cold stratification or stratification outdoors over two winters, the maximum germination was 40–50%; germination occurring over a wide range of temperatures. Warm stratification preceding cold stratification had no effect on germination, but repeated warm and cold periods seemed to promote germination. Gibberellic acid (GA) stimulated germination, but full germination was only achieved after more than 2 months of incubation at the most suitable temperature regime tested. Excised embryos grew and developed into normal seedlings. With these results, the species does not fit into the currently used system for seed dormancy classifications. The response to GA and the growth of excised embryos indicate non‐deep or intermediate physiological dormancy, but dormancy alleviation by stratification was not in line with the guiding principles for these classifications. Galeopsis speciosa has a strong dormancy that is sufficiently alleviated during the winter to allow germination of only part of a seed batch each year; hence a stepwise germination pattern occurs over a period of several years.     

Comparative evaluation of a commercial microdilution method and a conventional disk diffusion method for determination of antibiograms of gram-positive cocci

A commercial broth microdilution system for testing the antimicrobial susceptibility of gram-positive cocci was compared with the standardized disk agar-diffusion method by testing 254 clinical strains of staphylococci and streptococci using both methods. A total of 2,794 parallel determinations were made with 92.3% complete agreement between the 2 methods; of the discrepancies encountered, 3.0% were minor, 2.5% were major, and 2.1% were very major. The results indicate that the commercial microdilution system may provide a reliable quantitative method for antimicrobial susceptibility testing of clinical isolates from animals.     

Triallelic inheritance in Bardet-Biedl syndrome, a Mendelian recessive disorder

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous disorder characterized by multiple clinical features that include pigmentary retinal dystrophy, polydactyly, obesity, developmental delay, and renal defects. BBS is considered an autosomal recessive disorder, and recent positional cloning efforts have identified two BBS genes (BBS2 and BBS6). We screened our cohort of 163 BBS families for mutations in both BBS2 and BBS6 and report the presence of three mutant alleles in affected individuals in four pedigrees. In addition, we detected unaffected individuals in two pedigrees who carry two BBS2 mutations but not a BBS6 mutation. We therefore propose that BBS may not be a single-gene recessive disease but a complex trait requiring three mutant alleles to manifest the phenotype. This triallelic model of disease transmission may be important in the study of both Mendelian and multifactorial disorders.     

Ordered and dynamic assembly of single spliceosomes

The spliceosome is the complex macromolecular machine responsible for removing introns from precursors to messenger RNAs (pre-mRNAs). We combined yeast genetic engineering, chemical biology, and multiwavelength fluorescence microscopy to follow assembly of single spliceosomes in real time in whole-cell extracts. We find that individual spliceosomal subcomplexes associate with pre-mRNA sequentially via an ordered pathway to yield functional spliceosomes and that association of every subcomplex is reversible. Further, early subcomplex binding events do not fully commit a pre-mRNA to splicing; rather, commitment increases as assembly proceeds. These findings have important implications for the regulation of alternative splicing. This experimental strategy should prove widely useful for mechanistic analysis of other macromolecular machines in environments approaching the complexity of living cells.