Contrasting effects of ENU induced embryonic lethal mutations of the quaking gene.

Multiple alleles of the quaking (qk) gene have a variety of phenotypes ranging in severity from early embryonic death to viable dysmyelination. A previous study identified a candidate gene, QKI, that contains an RNA-binding domain and encodes at least three protein isoforms (QKI-5, -6 and -7). We have determined the genomic structure of QKI, identifying an additional alternative end in cDNAs. Further we have examined the exons and splice sites for mutations in the lethal alleles qkl-1, qkkt1, qkk2, and qkkt3. The mutation in qkl-1 creates a splice site in the terminal exon of the QKI-6 isoform. Missense mutations in the KH domain and the QUA1 domains in qkk2 and qkkt3, respectively, indicate that these domains are of critical functional importance. Although homozygotes for each ENU induced allele die as embryos, their phenotypes as viable compound heterozygotes with qkv differ. Compound heterozygous qkv animals carrying qkkt1, qkk2, and qkkt3 all exhibit a permanent quaking phenotype similar to that of qkv/qkv animals, whereas qkv/qkl-1 animals exhibit only a transient quaking phenotype. The qkl-1 mutation eliminates the QKI-5 isoform, showing that this isoform plays a crucial role in embryonic survival. The transient quaking phenotype observed in qkv/qkl-1 mice indicates that the QKI-6 and QKI-7 isoforms function primarily during myelination, but that QKI-5 may have a concentration-dependent role in early myelination. This mutational analysis demonstrates the power of series of alleles to examine the function of complex loci and suggests that additional mutant alleles of quaking could reveal additional functions of this complex gene.     

A method of one-step enzyme labelling of short oligonucleotide probes for filter hybridisation.

Here we describe a method of labelling short oligonucleotide probes with enzyme without purification or chemical modifications. Biotinylated oligonucleotides as short as 10 nt are coupled with streptavidin-conjugated enzyme, hybridised and detected with enzyme-triggered chemiluminescence. The detection of hybridisation signal is linear for two orders of magnitude of target dilution. It is shown to be comparable in sensitivity with standard procedures and with radioactive detection. The method is quick, simple and has potential for automation of large-scale oligo-nucleotide hybridisation and multiplex sequencing.     

Effect of 5-bromo- and 5-methyldeoxycytosine on duplex stability and discrimination of the NotI octadeoxynucleotide. Quantitative measurements using thin-layer chromatography

Octadeoxynucleotides based on the recognition sequence of the restriction endonuclease NotI were synthesized containing unmodified nucleotides and nucleotides with methyl and bromide additions at the C5 position of the pyrimidine ring of deoxycytosine. On annealing to single-stranded DNA bearing one NotI site, thin-layer chromatography (TLC) of the different oligonucleotides was used quantitatively to determine differences in dissociation temperature (Td) and binding equilibrium. Buffers used in filter hybridization experiments could be used in this TLC system. In addition, actual hybridizations were carried out to filter-bound DNA with and without a NotI site. The incorporation of 5-methyldeoxycytosine and 5-bromodeoxycytosine led to a significant increase in stability of homoduplex formation during hybridization, due to a shift in the binding equilibrium and an increase of the Td, thereby improving discrimination considerably. Some implications of the results for several techniques involving oligomer hybridization are discussed.