Point mutations result from errors made during DNA replication or repair, so they are usually expected to be homogeneous across all regions of a genome. However, we have found a region of chloroplast DNA in plants related to sweetpea (Lathyrus) whose local point mutation rate is at least 20 times higher than elsewhere in the same molecule. There are very few precedents for such heterogeneity in any genome, and we suspect that the hypermutable region may be subject to an unusual process such as repeated DNA breakage and repair. The region is 1.5 kb long and coincides with a gene, ycf4, whose rate of evolution has increased dramatically. The product of ycf4, a photosystem I assembly protein, is more divergent within the single genus Lathyrus than between cyanobacteria and other angiosperms. Moreover, ycf4 has been lost from the chloroplast genome in Lathyrus odoratus and separately in three other groups of legumes. Each of the four consecutive genes ycf4-psaI-accD-rps16 has been lost in at least one member of the legume ''inverted repeat loss'' clade, despite the rarity of chloroplast gene losses in angiosperms. We established that accD has relocated to the nucleus in Trifolium species, but were unable to find nuclear copies of ycf4 or psaI in Lathyrus. Our results suggest that, as well as accelerating sequence evolution, localized hypermutation has contributed to the phenomenon of gene loss or relocation to the nucleus.

Polymorphisms in folate-related genes have emerged as important risk factors in a range of diseases including neural tube defects (NTDs), cancer and coronary artery disease (CAD). Having previously identified a polymorphism within the cytoplasmic folate enzyme, MTHFD1, as a maternal risk factor for NTDs; we considered the more recently identified mitochondrial paralogue, MTHFD1L as a candidate gene for NTD association. We identified a common deletion/ insertion polymorphism, rs3832406, c.781-6823ATT(7-9), that influences splicing efficiency and is strongly associated with NTD risk. Three alleles of rs3832406 were detected in the Irish population with varying number of ATT repeats; Allele 1 consists of ATT 7 , while Alleles 2 and 3 consist of ATT 8 and ATT 9 respectively. Allele 2 of this triallelic polymorphism showed a decreased case risk as demonstrated by case-control logistic regression (P= 0.002) and by transmission disequilibrium test (TDT) (P= 0.001); while Allele 1 showed an increased case risk. Allele 3 showed no influence on NTD risk and represents the lowest frequency allele (0.15). Additional SNP genotyping in the same genomic region provides additional supportive evidence of an association. We demonstrate that two of the three alleles of rs3832406 are functionally different and influence the splicing efficiency of the alternate MTHFD1L mRNA transcripts.

The possibility of altering the unsaturation level of fatty acids in plant lipids by genetic transformation has implications for the stress tolerance of higher plants as well as for their nutritional value and industrial utilisation. While the integration and expression of transgenes in the plastome has several potential advantages over nuclear transformation, very few attempts have been made to manipulate fatty acid biosynthesis using plastid transformation. We produced transplastomic tobacco plants that express a D 9 desaturase gene from either the wild potato species Solanum commersonii or the cyanobacterium Anacystis nidulans, using PEG-mediated DNA uptake by protoplasts. Incorporation of chloroplast antibioticinsensitive point mutations in the transforming DNA was used to select transformants. The presence of the transcript and the D 9 desaturase protein in transplastomic plants was confirmed by northern and western blot analyses. In comparison with control plants, transplastomic plants showed altered fatty acid profiles and an increase in their unsaturation level both in leaves and seeds. The two transgenes produced comparable results. The results obtained demonstrate the feasibility of using plastid transformation to engineer lipid metabolic pathways in both vegetative and reproductive tissues and suggest an increase of cold tolerance in transplastomic plants showing altered leaf fatty acid profiles. This is the first example of transplastomic plants expressing an agronomically relevant gene produced with the ''binding-type'' vectors, which do not contain a heterologous marker gene. In fact, the transplastomic plants expressing the S. commersonii gene contain only plant-derived sequences, a clear attraction from a public acceptability perspective.

A T7 promoter-controlled transgene, AbL, encoding a camel single-domain antibody fragment that binds to the model antigen chicken egg-white lysozyme was introduced into the plastid genome of tobacco. AbL expression was activated in the transplastomic line by introducing a nuclear transgene, ST7, encoding a light-regulated plastid-targeted T7RNAP by cross-pollination. The resulting AbL x ST7 progeny seedlings developed a pale-green phenotype and ceased growth soon after germination. High levels of AbL transcripts accumulated in AbL x ST7 seedlings and expression of functional AbL antibody was detected by ELISA. Transplastomic AbL plants were also crossed with nuclear-transformed tobacco plants containing a salicylic acid-inducible transgene encoding a plastid-targeted T7RNAP (PR-T7 transgene). The resulting AbL x PR-T7 progeny were wild-type in appearance but were slow growing and prone to wilting even when provided with adequate water. Although AbL transcription was inducible by treating AbL x PR-T7 leaves with salicylic acid, high levels of T7RNAP-dependent AbL transcripts also accumulated in the absence of induction. However, AbL antibody did not accumulate at levels detectable by immunoblotting or ELISA in AbL x PR-T7 plants despite the fact that total leaf RNA containing AbL transcripts was capable of directing AbL antibody synthesis in an E. coli-derived in vitro translation system.

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Classical or transferase-deficient galactosaemia is an inherited metabolic disorder caused by mutation in the human Galactose-1-phosphate uridyl transferase (GALT) gene. Of some 170 causative mutations reported, fewer than 10% are observed in more than one geographic region or ethnic group. To better understand the population history of the common GALT mutations, we have established a haplotyping system for the GALT locus incorporating eight single nucleotide polymorphisms and three short tandem repeat markers. We analysed haplotypes associated with the three most frequent GALT gene mutations, Q188R, K285N and Duarte-2 (D2), and estimated their age. Haplotype diversity, in conjunction with measures of genetic diversity and of linkage disequilibrium, indicated that Q188R and K285N are European mutations. The Q188R mutation arose in central Europe within the last 20 000 years, with its observed east-west cline of increasing relative allele frequency possibly being due to population expansion during the re-colonization of Europe by Homo sapiens in the Mesolithic age. K285N was found to be a younger mutation that originated in Eastern Europe and is probably more geographically restricted as it arose after all major European population expansions. The D2 variant was found to be an ancient mutation that originated before the expansion of Homo sapiens out of Africa.

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Transcription of plastid transgenes by plastid-targeted T7 RNA polymerase (ptT7RNAP) during early seedling development in tobacco was associated with a pale-green leaf phenotype, depletion of plastid rRNAs and arrest of shoot development. Extensive analysis of mutant seedlings at the transcript level using DNA microarrays and RNA gel blotting revealed severe disruption of plastid rRNA accumulation at 4-days post-germination and reduced transcript accumulation for the essential gene clpP. Several nuclear genes encoding plastid proteins were differentially regulated in mutant seedlings over time. Ef-Tu was upregulated at 4-days post-germination and then subsequently downregulated, while RbcS was already downregulated at this early time point. The downregulation of nuclear genes encoding plastid proteins suggests disruption of plastid-to-nucleus signalling. In contrast, transcripts of three plastid genes showed increased accumulation in mutant seedlings. Transcripts of ndhC and ndhK accumulated at high levels possibly due to T7RNAP-mediated enhancement of transcription, while ptT7RNAP-mediated transcription through the phage T7 Tphi terminator into the adjacent plastome increased the level of accD transcripts. The leakiness of the Tphi terminator has implications for the use of T7RNAP-based expression systems in plastid biotechnology.