The effect of the nucleotides immediately upstream of the AUG start codon on the efficiency of translation initiation in sperm cells.

It is widely known that an optimal nucleotide sequence context immediately upstream of the AUG start codon greatly improves the efficiency of translation initiation of mRNA in mammalian and plant somatic cells, which in turn increases protein levels. However, it is still unclear whether a similar regulatory mechanism is also present in highly differentiated cells. Here, we surveyed this issue in Arabidopsis thaliana sperm cells and found that the sequence context-mediated regulation of translation initiation in sperm cells is generally similar to that in somatic cells. A simple motif of four adenine nucleotides at positions - 1 to - 4 greatly improved the efficiency of translation initiation, and when the motif was present there, translation was even initiated at some non-AUG codons in sperm cells. However, unlike that in mammalian cells, a mainly effective nucleotide site to regulate the efficiency of translation initiation was not present at positions - 1 to - 4 in sperm cells. Meanwhile, different from somatic cells, sperm cells did not use eukaryotic translation initiation factor 1 to regulate the efficiency in a poor context consisting of the lowest frequency nucleotides. All these results contribute to our understanding of the cytoplasmic event of translation initiation in highly differentiated sperm cells.

A competitive PCR-based method to detect a single copy of T-DNA insertion in transformants.

Gene function studies benefit from the availability of mutants. In plants, Agrobacterium-mediated genetic transformation is widely used to create mutants. These mutants, also called transformants, contain one or several transfer-DNA (T-DNA) copies in the host genome. Quantifying the copy number of T-DNA in transformants is beneficial to assess the number of mutated genes. Here, we developed a competitive polymerase chain reaction (PCR)-based method to detect a single copy of a T-DNA insertion in transformants. The competitor line BHK- -1 that contains a single copy of competitor BHK- (BHK, Basta, Hygromycin, Kanamycin-resistant genes) was crossed with test transformants and the genomic DNA of F1 plants was subjected to competitive PCR. By analyzing the gray ratio between two PCR products, we were able to determine whether or not the test transformants contained a single copy of T-DNA insertion. We also generated the control lines BHK±1:1 and BHK±2:1 , which contain the target (BHK+ ) and competitor (BHK- ) in a ratio of 1:1 and 2:1, respectively. The ratios of their PCR products are useful references for quantitative analysis. Overall, this method is reliable and simple in experimental manipulations and can be used as a substitute for Southern-blot analysis to identify a single copy of T-DNA insertion in transformants.