An expression vector inhibits gene expression in Xenopus embryos by antisense RNA

Summary An expression vector was constructed containing the entire bovine papilloma virus (BPV-1) genome and part of the a-actin gene of Xenopus laevis cloned in the antisense orientation into the neomycin resistance gene under the control of the herpes simplex virus (HSV) thymidine kinase (TK) promoter. When this vector is microinjected into X. laevis embryos it replicates extrachromosomally, at least up to the tadpole stage, and a fusion RNA is synthesized after the mid blastula transition (MBT). The expression of the antisense gene results in a morphological abnormality of somites demonstrating that antisense RNA generated by an episomal replicating expression vector can inhibit the expression of a selected gene during early embryogenesis of X. laevis.     

Inactivation of a testis-specific Lis1 transcript in mice prevents spermatid differentiation and causes male infertility

Lis1 protein is the non-catalytic component of platelet-activating factor acetylhydrolase 1b (PAF-AH 1B) and associated with microtubular structures. Hemizygous mutations of the LIS1 gene cause type I lissencephaly, a brain abnormality with developmental defects of neuronal migration. Lis1 is also expressed in testis, but its function there has not been determined. We have generated a mouse mutant (LIS1GT/GT) by gene trap integration leading to selective disruption of a Lis1 splicing variant in testis. Homozygous mutant males are infertile with no other apparent phenotype. We demonstrate that Lis1 is predominantly expressed in spermatids, and spermiogenesis is blocked when Lis1 is absent. Mutant spermatids fail to form correct acrosomes and nuclei appear distorted in size and shape. The tissue architecture in mutant testis appears severely disturbed displaying collapsed seminiferous tubules, mislocated germ cells, and increased apoptosis. These results provide evidence for an essential and hitherto uncharacterized role of the Lis1 protein in spermatogenesis, particularly in the differentiation of spermatids into spermatozoa.     

Identification of a critical control element directing expression of the muscle-specific transcription factor MRF4 in the mouse embryo

Skeletal muscle development in the vertebrate embryo critically depends on the myogenic regulatory factors (MRFs) including MRF4 and Myf5. Both genes exhibit distinct expression patterns during mouse embryogenesis, although they are genetically closely linked with multiple regulatory elements dispersed throughout the common gene locus. MRF4 has a biphasic expression profile, first in somites and later in foetal skeletal muscles. Here, we demonstrate by transgenic analysis that elements within a 7.5-kb promoter fragment of the MRF4 gene are sufficient to drive the embryonic wave of expression very similar to the endogenous gene in somites of mouse embryos. In contrast, a 3-kb fragment of the proximal promoter fails to support expression in the myotome, suggesting that essential cis-acting elements are located between -7.5 and -3 kb upstream of MRF4. Further analysis of this sequence delimits an essential region between -6.6 and -5.6 kb that together with the 3-kb promoter fragment directs transgene expression in the epaxial myotome of all somites during the appropriate developmental period. These data provide evidence that the partly overlapping expression patterns of Mrf4 and Myf5 in somites are controlled by distinct regulatory elements. We also show that 11.4 kb sequence upstream of MRF4, including the promoter and the somitic control region identified in this study, is not sufficient to elicit target specificity towards the strong Myf5 (-58/-48 kb) enhancer, suggesting that additional yet unidentified elements are necessary to convey promoter selectivity and protect the MRF4 gene from this enhancer.     

Jekyll encodes a novel protein involved in the sexual reproduction of barley

Cereal seed development depends on the intimate interaction of filial and maternal tissues, ensuring nourishment of the new generation. The gene jekyll, which was identified in barley (Hordeum vulgare), is preferentially expressed in the nurse tissues. JEKYLL shares partial similarity with the scorpion Cn4 toxin and is toxic when ectopically expressed in Escherichia coli and tobacco (Nicotiana tabacum). In barley, jekyll is upregulated in cells destined for autolysis. The gene generates a gradient of expression in the nucellar projection, which mediates the maternal-filial interaction during seed filling. Downregulation of jekyll by the RNA interference technique in barley decelerates autolysis and cell differentiation within the nurse tissues. Flower development and seed filling are thereby extended, and the nucellar projection no longer functions as the main transport route for assimilates. A slowing down in the proliferation of endosperm nuclei and a severely impaired ability to accumulate starch in the endosperm leads to the formation of irregular and small-sized seeds at maturity. Overall, JEKYLL plays a decisive role in the differentiation of the nucellar projection and drives the programmed cell death necessary for its proper function. We further suggest that cell autolysis during the differentiation of the nucellar projection allows the optimal provision of basic nutrients for biosynthesis in endosperm and embryo.     

N-Terminal pyroglutamate formation of Aβ38 and Aβ40 enforces oligomer formation and potency to disrupt hippocampal long-term potentiation

Pyroglutamate (pGlu)-modified amyloid peptides have been identified in sporadic and familial forms of Alzheimer's disease (AD) and the inherited disorders familial British and Danish Dementia (FBD and FDD). In this study, we characterized the aggregation of amyloid-β protein Aβ37, Aβ38, Aβ40, Aβ42 and ADan species in vitro, which were modified by N-terminal pGlu (pGlu-Aβ3-x, pGlu-ADan) or possess the intact N-terminus (Aβ1-x, ADan). The pGlu-modification confers rapid formation of oligomers and short fibrillar aggregates. In accordance with these observations, the pGlu-modified Aβ38, Αβ40 and Αβ42 species inhibit hippocampal long term potentiation of synaptic response, but pGlu-Aβ3-42 showing the highest effect. Among the unmodified Aβ peptides, only Aβ1-42 exhibites such propensity, which was similar to pGlu-Aβ3-38 and pGlu-Aβ3-40. Likewise, the amyloidogenic peptide pGlu-ADan impaired synaptic potentiation more pronounced than N-terminal unmodified ADan. The results were validated using conditioned media from cultivated HEK293 cells, which express APP variants favoring the formation of Aβ1-x, Aβ3-x or N-truncated pGlu-Aβ3-x species. Hence, we show that the ability of different amyloid peptides to impair synaptic function apparently correlates to their potential to form oligomers as a common mechanism. The pGlu-modification is apparently mediating a higher surface hydrophobicity, as shown by 1-anilinonaphtalene-8-sulfonate fluorescence, which enforces potential to interfere with neuronal physiology.     

Separation of enantiomers of drugs by capillary electrophoresis,part 7: Gamma-cyclodextrin as chiral solvating agent

Following an extended chiral drug screening program by capillary zone electrophoresis (CZE), the enantioseparation of 86 racemic drugs was tested with γ-cyclodextrin as a chiral solvating agent. Unified conditions were applied to all experiments. In total, 18 drug racemates were separated, 13 entries thereof that had not been separated at the lower CSA concentration applied in an earlier stage of the project. A comparison of the data with the results obtained for α- and β-cyclodextrin points to the significance of partial penetration (“side-on binding”) of aryl groups into the cyclodextrin cavity. Chirality 10:548–554, 1998. © 1998 Wiley-Liss, Inc.