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AFLP-Based mRNA Fingerprinting

AFLP, a robust and rapid technique for displaying large numbers of DNA polymorphisms, is being used extensively for genetic mapping and fingerprinting in plants (1). Use of this technique avoids problems which may be encountered with reproducibility and optimisation of reaction conditions when using arbitrarily primed PCR. Since the primer sets are readily available (Life Technologies and Perkin Elmer), we have explored whether AFLP could be applied to generating mRNA fingerprints in polyploid crop plants. In polyploid species, mutant stocks can be created by the deletion of short chromosome segments on one of the constituent homoeologous genomes. Messenger RNA fingerprints would be useful for identifying genes located within these deleted segments. However, the expression of monomorphic mRNAs from homoeologous genes in polyploids would make fingerprints indistinguishable when comparing normal and deletion stocks. If the homoeologous gene sequences were polymorphic, e.g. for a restriction enzyme site, then their AFLP fingerprints would be easily distinguishable and the expressed sequences could be genetically mapped. To test the sensitivity of this approach we used hexaploid wheat (2n = 6x arranged in seven groups of three homoeologous chromosome pairs) and one of its deletion mutants. Messenger RNA was extracted from immature spikes taken from wheat cv Chinese Spring and a mutant of this variety with deletions on chromosomes 3A and 5B (2,3). We used an mRNA Quickprep kit (Pharmacia) following the manufacturer’s instructions, but including treatment with DNase I before final precipitation. The mRNA was dissolved in 100 μl dH2O and purified using an RNeasy kit (Qiagen). Synthesis of double-stranded cDNA was performed according to instructions supplied with Superscript reverse transcriptase (Life Technologies). An equimolar mixture of three oligonucleotides with the sequence 5′-AGTCTGCAGT12V-3′ (where V denotes A, C or G) was used to prime first strand cDNA synthesis. As this primer contains a recognition sequence for PstI, the doublestranded cDNA can be cut with this restriction enzyme. This dual-purpose primer was designed to enable us to make use of available AFLP adapter and primer stocks without having to modify the PCR conditions used in the technique. The variable 3′ nucleotide adjacent to the T12 tract ensures that synthesis begins at the junction between the poly A tail and the sequence at the 3′ end of the mRNA (4,5). First strand cDNA synthesis was performed at 42 C using 0.5 μg of the oligonucleotide mix, 1 μg mRNA and 200 U reverse transcriptase, omitting [α-32P]dCTP but including RNAguard RNase inhibitor (Pharmacia). The cDNA was purified using a QiaQuick column (Qiagen). After second strand synthesis, cDNA was digested with 5.0 U each of PstI (NBL) and MseI (New England Biolabs). Digested cDNA was ligated with an MseI-adapter and a biotinylated PstI-adapter [patent application, Zabeau and Vos (1993), EP 0534858] and affinity-purified using streptavidin-linked paramagnetic beads (Dynal). Preamplification was carried out using non-selective primers to conserve purified cDNA stocks. All subsequent steps were performed as previously reported for genomic DNA AFLP (1). Labelled selective amplification products were run on standard 6% acrylamide sequencing gels and visualised by exposure to Kodak BioMax-MR film for ∼50 h. RNA fingerprints were generated from Chinese Spring and the deletion mutant cDNA templates using 49 MseI-primers with two or three selective bases. Amplification products ranged in size from 600 bp. Examples of the fingerprints obtained are shown in Figure 1a. Sixteen amplification products, which differed between Chinese Spring and the deletion mutant, were excised and reamplified. Purified reamplification products were labelled and hybridised with various digests of genomic DNA from Chinese Spring and the deletion mutant. Five of the products gave similar hybridisation patterns and these five probes were subsequently found to cross-hybridise with the 18S–5.8S–26S ribosomal RNA genes of wheat. The remaining 11 probes hybridised to single or low copy sequences. Two of these (M12B and M48A) detected DNA fragments that are present in Chinese Spring but absent in the deletion mutant (Fig. 1b). Further analysis revealed that the fragments are located on chromosome 3A (data not shown). The reproducibility of the technique was demonstrated by synthesising fresh cDNA from Chinese Spring and deletion mutant mRNA and processing this (including non-selective preamplification) as described. In the selective amplification, 10 different MseI primers were used for direct comparison of the banding patterns obtained using the duplicate cDNA templates. There was little or no variation between the two batches of cDNA and differences between Chinese Spring and the mutant were consistent (Fig. 1a). We have shown that the AFLP technique can be modified to allow display of mRNAs and used to isolate sequences mapping to deleted chromosome segments in hexaploid wheat. Since our protocol used existing MseI-adapters and primers, cDNA sequences forming an MseI recognition site (5′-TTAA-3′) at the junction of

A simple PCR-based method for scoring the ph1b deletion in wheat

The amplified fragment length polymorphism (AFLP) technique was used to isolate DNA sequences present in the euploid wheat Chinese Spring but not in the Chinese Spring ph1b mutant (which has a deletion of the Ph1 gene, a suppressor of homoeologous chromosome pairing). The polymorphic DNA fragments identified by AFLP were then cloned, sequenced, and used to design two primer pairs. These primers were used in a PCR-based assay to specifically amplify products from the Chinese Spring euploid but not from the ph1b mutant. This PCR assay can be carried out from extracted genomic DNA or directly from alkaline-treated wheat leaves, and the reaction products can be scored on a plus-minus basis, making the screening amenable to automation. The reliability of the assay was tested using a F1-derived doubled-haploid population of 55 lines which segregate for the ph1b deletion. This PCR-screening technique is less time and labour consuming, and more accurate and reliable, than cytologically based conventional methods.

Protein-protein interactions in the complex between the enhancer binding protein NIFA and the sensor NIFL from Azotobacter vinelandii.

The enhancer binding protein NIFA and the sensor protein NIFL from Azotobacter vinelandii comprise an atypical two-component regulatory system in which signal transduction occurs via complex formation between the two proteins rather than by the phosphotransfer mechanism, which is characteristic of orthodox systems. The inhibitory activity of NIFL towards NIFA is stimulated by ADP binding to the C-terminal domain of NIFL, which bears significant homology to the histidine protein kinase transmitter domains. Adenosine nucleotides, particularly MgADP, also stimulate complex formation between NIFL and NIFA in vitro, allowing isolation of the complex by cochromatography. Using limited proteolysis of the purified proteins, we show here that changes in protease sensitivity of the Q linker regions of both NIFA and NIFL occurred when the complex was formed in the presence of MgADP. The N-terminal domain of NIFA adjacent to the Q linker was also protected by NIFL. Experiments with truncated versions of NIFA demonstrate that the central domain of NIFA is sufficient to cause protection of the Q linker of NIFL, although in this case, stable protein complexes are not detectable by cochromatography.

HpaII library indicates ‘methylation-free islands’ in wheat and barley

SummaryA library of wheat genomic DNA HpaII tiny fragments (HTF), sized below 500 bp, has been constructed. Of the clones in the library 80% belong to the single/low-copy category, while 12% of the clones are nuclear repetitive sequences and 8% originate from the chloroplast and mitochondrial DNA. This result shows a substantial enrichment in the single/low-copy sequences of the wheat genome, which contains at least 80% repetitive sequences. Twenty-nine random single/lowcopy clones were analysed further for wheat chromosome location, cross-hybridisation to barley DNA and their association with rare-cutting, C-methylation-sensitive restriction sites. The results show that the HTF clones are associated more frequently than expected with NotI, MluI, NruI and PstI sites in wheat and barley genomic DNA. The 12% repetitive fraction of the clones contain both moderately and highly repetitive sequences, but no tandemly repeated sequences. The level of enrichment for single/low-copy sequences indicates that libraries of this type are a valuable source of probes for RFLP mapping. In addition, the close association of the HTF clones with rare-cutting restriction enzyme sites ensures that HTF clones will have a useful role in the construction of long-range physical maps in wheat.

A FAMILY OF RELATED SEQUENCES ASSOCIATED WITH (TTTAGGG)N REPEATS ARE LOCATED IN THE INTERSTITIAL REGIONS OF WHEAT CHROMOSOMES

A family of related sequences associated with (TTTAGGG)n repeats has been cloned from the wheat cultivar Chinese Spring. These sequences reveal a high level of polymorphism between wheat varieties when used as restriction fragment length polymorphism (RFLP) probes. Although this family of sequences contains motifs homologous to the repeats in the telomeres of wheat, they are located at interstitial sites on wheat chromosomes.

Changing the spatial pattern of TFL1 expression reveals its key role in the shoot meristem in controlling Arabidopsis flowering architecture

Highlight Plants carefully control where and when flowers are made through activators and repressors. We show that spatially the shoot meristem is key in responding to the repressors of flowering TFL1.