Mammalian DAI (DNA-dependent activator of IFN-regulatory factors), an activator of the innate immune response, senses cytosolic DNA by using 2 N-terminal Z-DNA binding domains (ZBDs) and a third putative DNA binding domain located next to the second ZBD. Compared with other previously known ZBDs, the second ZBD of human DAI (hZ␤DAI) shows significant variation in the sequence of the residues that are essential for DNA binding. In this article, the crystal structure of the hZ␤DAI/Z-DNA complex reveals that hZ␤DAI has a similar fold to that of other ZBDs, but adopts an unusual binding mode for recognition of Z-DNA. A residue in the first ␤-strand rather than residues in the ␤-loop contributes to DNA binding, and part of the (␣3) recognition helix adopts a 310 helix conformation. The role of each residue that makes contact with DNA was confirmed by mutational analysis. The 2 ZBDs of DAI can together bind to DNA and both are necessary for full B-to-Z conversion. It is possible that binding 2 DAIs to 1 dsDNA brings about dimerization of DAI that might facilitate DNA-mediated innate immune activation. circular dichroism ͉ hydrogen bonding ͉ interferon induction ͉ X-ray crystallography ͉ innate immunity T he innate immune response is essential for protection from foreign invasion, acting as an immediate cellular defense mechanism. Nucleic acids are known as one of the triggers for activation of the innate immune response (1-3). Recent reports have indicated the presence of a new cytosolic DNA sensor that can initiate an innate immune response independent of the endosomic Toll-like receptor 9 (4, 5). Z-DNA binding protein 1 (ZBP1), also known as DLM-1, was identified as the first innate immune activator that senses cytosolic DNAs (4). In response to foreign DNA, this protein activates type I IFN and other immune responses and was therefore named DAI (DNA-dependent activator of IFN-regulatory factors; ref 4). DAI contains 2 tandem Z-DNA binding domains (ZBDs or Z␣ and Z␤) at its N terminus and a third DNA binding region (D3) located next to the second ZBD. D3 is a novel domain and is reported to bind right-handed B-DNA (4). Upon activation, the C terminus of DAI binds to Tank binding kinase 1 (TBK1), a serine/threonine kinase, and to IFN regulatory factor 3 (IRF3), a transcription factor (4). The N-terminal region, including D3, is thought to be essential for sensing DNA, as shown by its ability to bind to Z-DNA and synthetic B-DNA (4). For the full activation of an in vivo DNA-dependent immune response, all 3 DNA-binding regions are required (5). At the molecular level, it has been suggested that dimerization of DAI results in activation of the innate immune response (5). At the cellular level, it is known that the localization of DAI and its association with stress granules is regulated by ZBDs (6,7).ZBDs that are found in DAI are also found in the editing enzyme dsRNA adenosine deaminase (ADAR1) in vertebrates and in fish PKZ protein kinase containing Z-DNA-binding domains and in the E3L of pox viruses (Fig. 1). They ...

A high-resolution physical map targeting a cluster of yield-related QTLs on the long arm of rice chromosome 9 has been constructed across a 37.4 kb region containing seven predicted genes. Using a series of BC3F4 nearly isogenic lines (NILs) derived from a cross between the Korean japonica cultivar Hwaseongbyeo and Oryza rufipogon (IRGC 105491), a total of seven QTLs for 1,000-grain weight, spikelets per panicle, grains per panicle, panicle length, spikelet density, heading date and plant height were identified in the cluster (P

A quantitative trait locus (QTL) for grain weight (GW) was detected near SSR marker RM210 on chromosome 8 in backcross populations derived from a cross between the Korean japonica cultivar Hwaseongbyeo and Oryza rufipogon (IRGC 105491). The O. rufipogon allele increased GW in the Hwaseongbyeo background despite the fact that O. rufipogon was the small-seeded parent. Using sister BC(3)F(3) near-isogenic lines (NILs), gw8.1 was validated and mapped to a 6.1 cM region in the interval between RM42 and RM210 (P < or = 0.0001). Substitution mapping with eight BC(3)F(4) sub-NILs further narrowed the interval containing gw8.1 to about 306.4 kb between markers RM23201.CNR151 and RM30000.CNR99. A yield trial using homozygous BC(3)F(4) sister sub-NILs and the Hwaseongbyeo recurrent parent indicated that the NIL carrying an O. rufipogon chromosome segment across the entire gw8.1 target region out-yielded its sister NIL (containing Hwaseongbyeo chromosome in the RM42-RM210 interval) by 9% (P=0.029). The higher-yielding NIL produced 19.3% more grain than the Hwaseongbyeo recurrent parent (P=0.018). Analysis of a BC(3)F(4) NIL indicated that the variation for GW is associated with variation in grain shape, specifically grain length. The locus, gw8.1 is of particular interest because of its independence from undesirable height and grain quality traits. SSR markers tightly linked to the GW QTL will facilitate cloning of the gene underlying this QTL as well as marker-assisted selection for variation in GW in an applied breeding program.

Mutations in the MTR gene, which encodes methionine synthase on human chromosome 1p43, result in the methylcobalamin deficiency G (cblG) disorder, which is characterized by homocystinuria, hyperhomocysteinemia, and hypomethioninemia. To investigate the molecular basis of the disorder, we have characterized the structure of the MTR gene, thereby identifying exon-intron boundaries. This enabled amplification of each of the 33 exons of the gene, from genomic DNA from a panel of 21 patients with cblG. Thirteen novel mutations were identified. These included five deletions (c.12-13delGC, c.381delA, c.2101delT, c.2669-2670delTG, and c.2796-2800delAAGTC) and two nonsense mutations (R585X and E1204X) that would result in synthesis of truncated proteins that lack portions critical for enzyme function. One mutation was identified that resulted in conversion of A to C of the invariant A of the 3' splice site of intron 9. Five missense mutations (A410P, S437Y, S450H, H595P, and I804T) were identified. The latter mutations, as well as the splice-site mutation, were not detected in a panel of 50 anonymous DNA samples, suggesting that these sequence changes are not polymorphisms present in the general population. In addition, a previously described missense mutation, P1173L, was detected in 16 patients in an expanded panel of 24 patients with cblG. Analysis of haplotypes constructed using sequence polymorphisms identified within the MTR gene demonstrated that this mutation, a C-->T transition in a CpG island, has occurred on at least two separate genetic backgrounds.