2-Methylthio-N7-methyl-cis-zeatin, a new antimalarial natural product isolated from a Streptomyces culture.

2-Methylthio-N7-methyl-cis-zeatin (1) was isolated from the culture broth of Streptomyces sp. 80H647 along with 2 known purine derivatives, 5'-methylthioinosine (2) and AT-265 (dealanylascamycin, 3). The structure elucidation of compound 1 was accomplished by high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) analyses. It inhibited the growth of Plasmodium falciparum 3D7 with a GI50 of 2.4 µm and had no effect on the growth of Arabidopsis at 2 µm. This is the first report of an N7-methylated zeatin-type natural product from Streptomyces and as an antimalarial compound.

Tenuazonic acid production is dispensable for virulence, but its biosynthetic gene expression pattern is associated with the infection of Pyricularia oryzae.

Tenuazonic acid (TeA) is a toxin produced by the rice blast fungus Pyricularia oryzae. Although knockout of the TeA biosynthetic gene TAS1 did not affect the virulence of P. oryzae, constitutive TAS1 expression suppressed its infection. TAS1 expression was induced alongside transition of P. oryzae infection behavior. The results suggested that controlling TeA biosynthesis is important for P. oryzae infection.

Anthocyanin synthesis potential in betalain-producing Caryophyllales plants.

Although anthocyanins are widely distributed in higher plants, betalains have replaced anthocyanins in most species of the order Caryophyllales. The accumulation of flavonols in Caryophyllales plants implies that the late step of anthocyanin biosynthesis from dihydroflavonols to anthocyanins may be blocked in Caryophyllales. The isolation and characterization of functional dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) from Caryophyllales plants has indicated a lack of anthocyanins due to suppression of DFR and ANS. In this study, we demonstrated that overexpression of DFR and ANS from Spinacia oleracea (SoDFR and SoANS, respectively) with PhAN9, which encodes glutathione S-transferase (required for anthocyanin sequestration) from Petunia induces ectopic anthocyanin accumulation in yellow tepals of the cactus Astrophytum myriostigma. A promoter assay of SoANS showed that the Arabidopsis MYB transcription factor PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1) activated the SoANS promoter in Arabidopsis leaves. The overexpression of Arabidopsis transcription factors with PhAN9 also induced ectopic anthocyanin accumulation in yellow cactus tepals. PAP homologs from betalain-producing Caryophyllales did not activate the promoter of ANS. In-depth characterization of Caryophyllales PAPs and site-directed mutagenesis in the R2R3-MYB domains identified the amino acid residues affecting transactivation of Caryophyllales PAPs. The substitution of amino acid residues recovered the transactivation ability of Caryophyllales PAPs. Therefore, loss of function in MYB transcription factors may suppress expression of genes involved in the late stage of anthocyanin synthesis, resulting in a lack of anthocyanin in betalain-producing Caryophyllales plants.

Two R2R3-MYB proteins are broad repressors of flavonoid and phenylpropanoid metabolism in poplar.

The phenylpropanoid pathway leads to the production of many important plant secondary metabolites including lignin, chlorogenic acids, flavonoids, and phenolic glycosides. Early studies have demonstrated that flavonoid biosynthesis is transcriptionally regulated, often by a MYB, bHLH, and WDR transcription factor complex. In poplar, several R2R3 MYB transcription factors are known to be involved in flavonoid biosynthesis. Previous work determined that poplar MYB134 and MYB115 are major activators of the proanthocyanidin pathway, and also induce the expression of repressor-like MYB transcription factors. Here we characterize two new repressor MYBs, poplar MYB165 and MYB194, paralogs which comprise a subgroup of R2R3-MYBs distinct from previously reported poplar repressors. Both MYB165 and MYB194 repressed the activation of flavonoid promoters by MYB134 in transient activation assays, and both interacted with a co-expressed bHLH transcription factor, bHLH131, in yeast two-hybrid assays. Overexpression of MYB165 and MYB194 in hybrid poplar resulted in greatly reduced accumulation of several phenylpropanoids including anthocyanins, proanthocyanidins, phenolic glycosides, and hydroxycinnamic acid esters. Transcriptome analysis of MYB165- and MYB194-overexpressing poplars confirmed repression of many phenylpropanoid enzyme genes. In addition, other MYB genes as well as several shikimate pathway enzyme genes were downregulated by MYB165-overexpression. By contrast, leaf aromatic amino acid concentrations were greater in MYB165-overexpressing poplars. Our findings indicate that MYB165 is a major repressor of the flavonoid and phenylpropanoid pathway in poplar, and may also affect the shikimate pathway. The coordinated action of repressor and activator MYBs could be important for the fine tuning of proanthocyanidin biosynthesis during development or following stress.

Synthesis of All Stereoisomers of RK460 and Evaluation of Their Activity and Selectivity as Abscisic Acid Receptor Antagonists.

The PYR/PYL/RCAR protein families have recently emerged as receptors of the phytohormone abscisic acid (ABA, 1), which regulates plant responses to environmental stress. These families have multiple members with different physiological actions, and so selective agonists or antagonists are needed both as tools to elucidate functional differences and as lead compounds for agrochemicals. We previously identified RK460 (rac-3 a) as a PYR1-selective antagonist, and showed that it possesses five stereocenters on a 6,5-cis-bicyclo skeleton. Here, we synthesized all the stereoisomers of RK460 and evaluated their activity towards a panel of receptors. Relative stereochemistry as well as absolute stereochemistry was important for selective action.

Poplar MYB115 and MYB134 Transcription Factors Regulate Proanthocyanidin Synthesis and Structure.

The accumulation of proanthocyanidins is regulated by a complex of transcription factors composed of R2R3 MYB, basic helix-loop-helix, and WD40 proteins that activate the promoters of biosynthetic genes. In poplar (genus Populus), MYB134 is known to regulate proanthocyanidin biosynthesis by activating key flavonoid genes. Here, we characterize a second MYB regulator of proanthocyanidins, MYB115. Transgenic poplar overexpressing MYB115 showed a high-proanthocyanidin phenotype and reduced salicinoid accumulation, similar to the effects of MYB134 overexpression. Transcriptomic analysis of MYB115- and MYB134-overexpressing poplar plants identified a set of common up-regulated genes encoding proanthocyanidin biosynthetic enzymes and several novel uncharacterized MYB transcriptional repressors. Transient expression experiments demonstrated the capacity of both MYB134 and MYB115 to activate flavonoid promoters, but only in the presence of a basic helix-loop-helix cofactor. Yeast two-hybrid experiments confirmed the direct interaction of these transcription factors. The unexpected identification of dihydromyricetin in leaf extracts of both MYB115- and MYB134-overexpressing poplar led to the discovery of enhanced flavonoid B-ring hydroxylation and an increased proportion of prodelphinidins in proanthocyanidin of the transgenics. The dramatic hydroxylation phenotype of MYB115 overexpressors is likely due to the up-regulation of both flavonoid 3',5'-hydroxylases and cytochrome b5 Overall, this work provides new insight into the complexity of the gene regulatory network for proanthocyanidin synthesis in poplar.

The MYB182 protein down-regulates proanthocyanidin and anthocyanin biosynthesis in poplar by repressing both structural and regulatory flavonoid genes.

Trees in the genus Populus (poplar) contain phenolic secondary metabolites including the proanthocyanidins (PAs), which help to adapt these widespread trees to diverse environments. The transcriptional activation of PA biosynthesis in response to herbivory and ultraviolet light stress has been documented in poplar leaves, and a regulator of this process, the R2R3-MYB transcription factor MYB134, has been identified. MYB134-overexpressing transgenic plants show a strong high-PA phenotype. Analysis of these transgenic plants suggested the involvement of additional MYB transcription factors, including repressor-like MYB factors. Here, MYB182, a subgroup 4 MYB factor, was found to act as a negative regulator of the flavonoid pathway. Overexpression of MYB182 in hairy root culture and whole poplar plants led to reduced PA and anthocyanin levels as well as a reduction in the expression of key flavonoid genes. Similarly, a reduced accumulation of transcripts of a MYB PA activator and a basic helix-loop-helix cofactor was observed in MYB182-overexpressing hairy roots. Transient promoter activation assays in poplar cell culture demonstrated that MYB182 can disrupt transcriptional activation by MYB134 and that the basic helix-loop-helix-binding motif of MYB182 was essential for repression. Microarray analysis of transgenic plants demonstrated that down-regulated targets of MYB182 also include shikimate pathway genes. This work shows that MYB182 plays an important role in the fine-tuning of MYB134-mediated flavonoid metabolism.

Novel Abscisic Acid Antagonists Identified with Chemical Array Screening.

Abscisic acid (ABA) signaling is involved in multiple processes in plants, such as water stress control and seed dormancy. Major regulators of ABA signaling are the PYR/PYL/RCAR family receptor proteins, group A protein phosphatases 2C (PP2Cs), and subclass III of SNF1-related protein kinase 2 (SnRK2). Novel ABA agonists and antagonists to modulate the functions of these proteins would not only contribute to clarification of the signaling mechanisms but might also be used to improve crop yields. To obtain small molecules that interact with Arabidopsis ABA receptor PYR1, we screened 24 275 compounds from a chemical library at the RIKEN Natural Products Depository by using a chemical array platform. Subsequent SnRK2 and PP2C assays narrowed down the candidates to two molecules. One antagonized ABA in a competitive manner and inhibited the formation of the PYR1-ABA-PP2C ternary complex. These compounds might have potential as bioprobes to analyze ABA signaling.

Characterization of an apple TT2-type R2R3 MYB transcription factor functionally similar to the poplar proanthocyanidin regulator PtMYB134.

MAIN CONCLUSION: The apple MdMYB9 gene encodes a positive regulator of proanthocyanidin synthesis that activates anthocyanidin reductase promoters from apple and poplar via interaction with basic helix-loop-helix proteins. The regulation of proanthocyanidins (PAs, condensed tannins) is of great importance in food plants due to the many benefits of PAs in the human diet. Two candidate flavonoid MYB regulators, MdMYB9 and MdMYB11, were cloned from apple (Malus × domestica) based on their similarity to known MYB PA regulators. Transcript accumulation of both MdMYB9 and MdMYB11 was induced by high light and wounding, similar to the poplar (Populus spp) PA regulator PtMYB134. In transient activation assays with various basic helix-loop-helix (bHLH) co-regulators, MdMYB9 activated apple and poplar anthocyanidin reductase (ANR) promoters, while MdMYB11 showed no activity. Potential transcription factor binding elements were found within several ANR promoters, and the importance of the bHLH binding site (E-box) on ANR promoter activation was demonstrated via mutational analysis. The ability of MdMYB9 and PtMYB134 to reciprocally activate ANR promoters from both apple and poplar and to partner with heterologous bHLH co-factors from these plants confirms the high degree of conservation of PA regulatory complexes across species. The similarity in apple and poplar PA regulation suggests that regulatory genes from poplar could be effectively employed for metabolic engineering of the PA pathway in apple.

New Abscisic Acid Derivatives Revealed Adequate Regulation of Stomatal, Transcriptional, and Developmental Responses to Conquer Drought.

The phytohormone abscisic acid (ABA) plays an important role in plant stress response, mainly against desiccation. Hence, ABA receptor agonists may function as agents to enhance drought tolerance in crops. ABA exhibits diverse functions that impact plant development and are regulated by various ABA receptor subfamilies. Indeed, we previously reported that 3'-alkyl ABAs exhibit diverse receptor specificities and that 3'-butyl ABA induced a drought stress response without eliciting growth inhibitory effects in Arabidopsis seedlings. Thus, to further investigate plant responses induced by 3'-butyl ABA, as well as the receptors that control the opposing stress and growth responses, we designed new 3'-alkyl ABA derivatives. In addition to the 3'-alkyl chain, a cyclopropyl group was attached to position 3 of ABA to occupy the C6 cleft in the ABA-binding pocket of the receptors, which served to increase the binding affinity and specificity to a certain receptor set. Additionally, the inhibitory activity of pyrabactin resistance 1 (PYR1) and PYR1-like (PYL1) proteins against type 2C protein phosphatase increased following incorporation of the 3-cyclopropyl group in all tested 3'-alkyl ABAs. Interestingly, 3'-butyl ABA induced the highest tolerance against drought stress, compared with 3-cyclopropyl derivatives. To investigate the molecular mechanism underlying the effects elicited by different chemical treatments, those of ABA derivatives on stomatal closure, growth, and gene expression were studied. Evaluation of the receptors activated by ABA derivatives and the plant responses revealed the induction of PYR1, PYL1, PYL2, and PYL5, mediated stomatal closure, and regulated transcription, consequently leading to drought tolerance in plants.

Comparative analysis of the triplicate proathocyanidin regulators in Lotus japonicus.

Proanthocyanidins (PAs), which are flavonoid compounds widely distributed in the plant kingdom, protect against environmental stress. The accumulation of PAs is regulated by a ternary transcriptional complex comprising the R2R3-MYB transcription factor, a basic helix-loop-helix (bHLH) transcription factor and a WD40 repeat (WDR) protein. Recently, multigene families of the R2R3-MYB-type PA regulators from Lotus japonicus, LjTT2a, b and c, were isolated and characterized. Although their roles as transcription factors that up-regulate PA biosynthetic genes have been elucidated, the significance of their redundancies and functions in planta is unknown. In this study, we characterized LjTT2a, b and c to elucidate their functions in planta and determine differences in transcriptional activation properties. Transgenic studies demonstrated that LjTT2a could induce ectopic PA accumulation in Arabidopsis. Further analysis of the LjTT2 multigene family using a transient expression system revealed differences in transcriptional activities in cooperation with WDR and bHLH proteins isolated from L. japonicus. In-depth characterization of chimeric constructs of three LjTT2s, as well as site-directed mutagenesis in R2-MYB domains, identified the amino acid residues that affect the level of transcriptional activation of LjTT2.

Transcriptional control of the dihydroflavonol 4-reductase multigene family in Lotus japonicus.

In the genome of the model legume Lotus japonicus, dihydroflavonol 4-reductase (DFR), which is the first committed enzyme of the anthocyanin and proanthocyanidin (PA) pathways, is encoded as a tandemly arrayed five-gene family. Expression analysis revealed that both organ specificity and stress responsiveness differ among the DFRs. To elucidate the regulatory mechanisms underlying the expression of DFRs, we investigated the transcriptional control of each member of the DFR multigene family. Ectopic expression of a combination of the transcription factors MYB, bHLH, and WDR showed that only the DFR2 promoter was activated, indicating that each member of the DFR gene family is regulated independently.

Upregulation of c-myc gene accompanied by PU.1 deficiency in radiation-induced acute myeloid leukemia in mice.

OBJECTIVE: High-dose radiation exposure induces acute myeloid leukemia (AML) in C3H mice, most of which have a frequent hemizygous deletion around the D2Mit15 marker on chromosome 2. This region includes PU.1, a critical candidate gene for initiation of leukemogenesis. To identify novel cooperative genes with PU.1, relevant to radiation-induced leukemogenesis, we analyzed the copy number alterations of tumor-related gene loci by array CGH, and their expressions in primary and transplanted AMLs. MATERIALS AND METHODS: For the induction of AMLs, C3H/He Nrs mice were exposed to 3 Gy of x-rays or gamma-rays. The genomic alterations of 35 primary AMLs and 34 transplanted AMLs obtained from the recipient mice transplanted the primary AMLs were analyzed by array CGH. According to the genomic alterations and mutations of the 235th arginine of PU.1 allele, we classified the radiogenic AMLs into three types such as Chr2(del) PU.1(del/R235-) AML, Chr2(del) PU.1(del/R235+) AML and Chr2(intact) PU.1(R235+/R235+) AML, to compare the expression levels of 8 tumor-related genes quantitatively by real-time polymerase chain reaction and cell-surface antigen expression. Results. In addition to well-known loss of PU.1 with hemizygous deletion of chromosome 2, novel genomic alterations such as partial gain of chromosome 6 were recurrently detected in AMLs. In this study, we found similarity between cell-surface antigen expressions of bone marrows and those of spleens in AML mice and significantly higher expressions of c-myc and PU.1 expression, especially in the PU.1-deficient (Chr2(del) PU.1(del/R235-)) AML and Chr2(del) PU.1(del/R235+) compared to Chr2(intact) PU.1(R235+/R235+) AMLs. CONCLUSION: The new finding on upregulation of c-myc and PU.1 in both and hemizygous PU.1-deficient AMLs and different genomic alterations detected by array CGH suggests that the molecular mechanism for development of radiation-induced AML should be different among three types of AML.

Abscisic Acid Derivatives with Different Alkyl Chain Lengths Activate Distinct Abscisic Acid Receptor Subfamilies.

The plant hormone abscisic acid (ABA) regulates the development of various plant organs including seeds, roots, and fruits, and significantly contributes to abiotic stress responses, especially to drought. Since recent climate changes are adversely affecting crop cultivation, enhancement of plant stress tolerance by regulation of ABA signaling would be an important strategy. In the plant genome, ABA receptors are encoded by multiple genes constituting three subfamilies; however, functional differences among them remain unclear. To enhance desired effects of ABA, the biological functions of the receptor family warrant clarification. This study aimed to determine the functional differences among ABA receptors in plants. We screened small-molecule ligands binding to specific receptors, using a chemical array. In vitro evaluation of hit compounds using 11 Arabidopsis ABA receptors revealed that (+)-3'-alkyl ABAs served as agonists for different receptors depending on the length of their 3'-alkyl chains. Combinatorial in vitro and physiological effects of these compounds on the stomata, seeds, and seedlings indicated that, along with subfamily III, receptors of subfamily II are important to induce strong drought responses. Among (+)-3'-alkyl ABAs assessed herein, (+)-3'-butyl ABA induced a transcriptional response and stomatal closure but only slightly inhibited seed germination and growth, suggesting that it enhances drought tolerance. In silico docking simulation and site-directed mutagenesis revealed the amino acid residues contributing to the selective agonist activity of the (+)-3'-alkyl ABAs. These results provide novel insights into the structure and biological effects of 3'-derivatives of ABA and a basis for agrochemical development.

Functional differentiation of Lotus japonicus TT2s, R2R3-MYB transcription factors comprising a multigene family.

Leguminous plants have many paralogous genes encoding enzymes involved in the flavonoid biosynthetic pathway. Duplicate genes are predicted to contribute to the production of various flavonoid compounds and to have resulted in a diversity of legume species. We identified gene duplication in the transcription factors regulating flavonoid biosynthesis in the model legume Lotus japonicus. Three copies of a homolog of Arabidopsis thaliana TRANSPARENT TESTA2 (TT2), which is a MYB transcription factor that regulates proanthocyanidin biosynthesis, were present in the L. japonicus genome. The organ specificity and stress responsiveness differed among the three LjTT2s, and correlations between proanthocyanidin accumulation and the expression levels of LjTT2s were observed during seedling development. Moreover, three LjTT2s functionally complemented TT2 in transient expression experiments in A. thaliana leaf cells. The different reporter activity caused by LjTT2a was consistent with the affinity of physical interactions with TT8 and TTG1 in yeast two-hybrid experiments as well as the branching pattern of the phylogenetic tree. These results suggest that LjTT2 factors have diverse functions in the tissues in which they are expressed; in particular, LjTT2a is predicted to have evolved flexibility in interaction with other transcription regulators to resist environmental stresses.

[Outcomes of Pharmacists' Involvement with Residents of Special Nursing Homes for the Elderly].

　The current study aimed to examine the outcomes of pharmacists' involvement with elderly people in special nursing homes. We analyzed 58 cases involving regular visits by community pharmacists to 41 residents. The residents' mean age was 87.8±6.9 years, and 68.3% were prescribed 6 or more types of medication. Antipsychotic and insomnia medication was taken by 24.4% and 31.8% of residents, respectively. Pharmaceutical consultation following medication use accounted for 60.3% of pharmacists' involvement with residents. The outcomes of these consultations included improvements in prescription content; the identification and prevention of adverse drug events; improvement in activities of daily living; and improvement in test results, sleep, and urination/bowel control. The results also suggested that pharmacists' intervention reduced drug costs. Information that facilitated involvement was most frequently acquired via conversations (67.2%) and conferences (24.1%) in the facilities. The most common information sources were care workers (72.4%), followed by nurses (37.9%), physicians (6.9%), and functional training instructors (6.9%). Information was also acquired from patients (3.4%) and their family members (5.2%). The findings indicated that regular visits by pharmacists to facilities for elderly people and conversations between residents, their family members, and physicians, nurses and various other professionals improved various pharmacotherapy outcomes.

p53 (TRP53) deficiency-mediated antiapoptosis escape after 5 Gy X irradiation still induces stem cell leukemia in C3H/He mice: comparison between whole-body assay and bone marrow transplantation (BMT) assay.

Mice exposed to a lethal dose of radiation were repopulated with heterozygous p53(+/-) (TRP53(+/-)) bone marrow cells and then exposed to doses of 1, 3 and 5 Gy 1 month later. This resulted in the transplanted bone marrow-specific diseases other than competitively induced nonhematopoietic neoplasms. Interestingly, the present study showed a high frequency of stem cell leukemia, i.e., leukemias characterized by a lack of differentiation due also to p53 deficiency, even after 5 Gy irradiation. The frequencies of stem cell leukemias (and those of total hematopoietic malignancies) were 16% (24%) at 1 Gy and 45% (75%) at 3 Gy. Furthermore, markedly high incidences of stem cell leukemias were observed at 5 Gy in p53(+/-) mice, i.e., 87% (100%) in the transplantation assay and 60% (83.3%) in the whole-body assay, whereas a conventional whole-body assay induced only 14% in wild-type mice. The high incidence of stem cell leukemias observed in this study using heterozygous p53-deficient mice agrees with results of a previous study of homozygous p53-deficient mice and is consistent with the high frequency of loss of heterozygosity in the p53 wild-type allele observed in leukemias. This suggests that the target cells for radiation-induced stem cell leukemias may be p53-deficient hematopoietic stem cells.

Direct detection of p53 -/- thymocyte appearing at an early stage of radiation-induced thymic lymphomagenesis in p53 +/- heterozygous B10 mice.

PURPOSE: The appearance of tumor suppressor protein 53 (p53) -/- thymocytes at an early stage of radiation-induced lymphomagenesis was investigated in the p53 heterozygous (+/-) B10 mice following a single dose of irradiation, since most thymic lymphomas manifested the loss of the wild-type p53 allele and the loss of heterozygosity was thought to be an early event critical for radiation-induced thymic lymphomagenesis in p53 +/- mice. MATERIALS AND METHODS: The mice were exposed to a single dose (6 Gy) of irradiation to induce thymic lymphomas and, at various times after irradiation, treated with an extremely high dose (30 Gy) of whole-body irradiation to enrich p53 -/- thymocytes and, 24 h later, the remaining thymocytes were assayed for cell surface markers and p53 genotype. RESULTS: In a significant fraction of the p53 +/- mice 5 weeks after 6 Gy irradiation, there was a relative increase in the number of cluster of differentiation (CD) 4+CD8+ thymocyte subpopulation among thymocytes remaining after 30 Gy irradiation. The CD4+CD8+ double-positive (DP) thymocytes were shown to contain p53-/- cells, and the number of p53 -/- thymocytes was more than 10(5) in those individuals. CONCLUSIONS: The results clearly indicated that an extremely high dose (30 Gy) of whole-body irradiation enabled us to directly detect p53 -/- thymocytes in an abundant p53 +/- thymocyte population and that proliferative p53 -/- thymocytes develop in a CD4+CD8+ DP thymocyte subpopulation within a few weeks after a single dose (6 Gy) of irradiation.

Intestinal tumorigenicity of mice carrying hemizygous Pax6, Pax6(Sey-4H).

The genotype-phenotype relationship in mice was examined experimentally using one of the small eye mutants, Pax6(Sey-4H), which deletes the chromosome 2 middle region, hemizygously. The genotyping indicated that the deleted region starts at a site 102.60 Mb from the centromere and has a length of 6.51 Mb, in which 12 known and 27 novel genes are located. Expecting the development of myeloid leukemia, gamma-irradiation was performed to female mutants at the age of 10 weeks. The mutants did not develop myeloid leukemia during the observation period of 18 months. Instead, they developed tumors in the alimentary tract spontaneously (56.0%). The tumor latency was shortened by the radiation exposure, but the tumor incidence of the gamma-irradiated group (62.5%) was as high as that of spontaneously developing tumors. Intraductal proliferation of the epithelium of the Wirsung duct was observed in the gamma-irradiated mutants (18.8%). Considering the results of the Pax6(Sey-4H) mutant together with those of another small eye mutant, Pax6(Sey-3H), the anomaly and the tumorigenicity of the intestinal tract were closely related to the hemizygosity of the 3.2 Mb segment of chromosome 2, where both mutants show a common deletion.