Targeted mutagenesis of BnTT8 homologs controls yellow seed coat development for effective oil production in Brassica napus L.

Yellow seed is a desirable trait with great potential for improving seed quality in Brassica crops. Unfortunately, no natural or induced yellow seed germplasms have been found in Brassica napus, an important oil crop, which likely reflects its genome complexity and the difficulty of the simultaneous random mutagenesis of multiple gene copies with functional redundancy. Here, we demonstrate the first application of CRISPR/Cas9 for creating yellow-seeded mutants in rapeseed. The targeted mutations of the BnTT8 gene were stably transmitted to successive generations, and a range of homozygous mutants with loss-of-function alleles of the target genes were obtained for phenotyping. The yellow-seeded phenotype could be recovered only in targeted mutants of both BnTT8 functional copies, indicating that the redundant roles of BnA09.TT8 and BnC09.TT8b are vital for seed colour. The BnTT8 double mutants produced seeds with elevated seed oil and protein content and altered fatty acid (FA) composition without any serious defects in the yield-related traits, making it a valuable resource for rapeseed breeding programmes. Chemical staining and histological analysis showed that the targeted mutations of BnTT8 completely blocked the proanthocyanidin (PA)-specific deposition in the seed coat. Further, transcriptomic profiling revealed that the targeted mutations of BnTT8 resulted in the broad suppression of phenylpropanoid/flavonoid biosynthesis genes, which indicated a much more complex molecular mechanism underlying seed colour formation in rapeseed than in Arabidopsis and other Brassica species. In addition, gene expression analysis revealed the possible mechanism through which BnTT8 altered the oil content and fatty acid composition in seeds.

CRISPR/Cas9-mediated genome editing reveals differences in the contribution of INDEHISCENT homologues to pod shatter resistance in Brassica napus L.

The INDEHISCENT (IND) and ALCATRAZ (ALC) gene homologues have been reported to be essential for dehiscence of fruits in Brassica species. But their functions for pod shatter resistance in Brassica napus, an important oil crops, are not well understood. Here, we assessed the functions of these two genes in rapeseed using CRISPR/Cas9 technology. The induced mutations were stably transmitted to successive generations, and a variety of homozygous mutants with loss-of-function alleles of the target genes were obtained for phenotyping. The results showed that the function of BnIND gene is essential for pod shatter and highly conserved in Brassica species, whereas the BnALC gene appears to have limited potential for rapeseed shatter resistance. The homoeologous copies of the BnIND gene have partially redundant roles in rapeseed pod shatter, with BnA03.IND exhibiting higher contributions than BnC03.IND. Analysis of data obtained from the gene expression and sequence variations of gene copies revealed that cis-regulatory divergences alter gene expression and underlie the functional differentiation of BnIND homologues. Collectively, our results generate valuable resources for rapeseed breeding programs, and more importantly provide a strategy to improve polyploid crops.

Targeted mutagenesis of BnTTG1 homologues generated yellow-seeded rapeseed with increased oil content and seed germination under abiotic stress.

Yellow seed is one desirable trait with great potential to improve seed oil quality and yield. The present study surveys the redundant role of BnTTG1 genes in the proanthocyanidins (PA) biosynthesis, oil content and abiotic stress resistance. Stable yellow seed mutants were generated after mutating BnTTG1 by CRISPR/Cas9 genome editing system. Yellow seed phenotype could be obtained only when both functional homologues of BnTTG1 were simultaneously knocked out. Homozygous mutants of BnTTG1 homologues showed decreased thickness and PA accumulation in seed coat. Transcriptome and qRT-PCR analysis indicated that BnTTG1 mutation inhibited the expression of genes involved in phenylpropanoid and flavonoid biosynthetic pathways. Increased seed oil content and alteration of fatty acid (FA) composition were observed in homozygous mutants of BnTTG1 with enriched expression of genes involved in FA biosynthesis pathway. In addition, target mutation of BnTTG1 accelerated seed germination rate under salt and cold stresses. Enhanced seed germination capacity in BnTTG1 mutants was correlated with the change of expression level of ABA responsive genes. Overall, this study elucidated the redundant role of BnTTG1 in regulating seed coat color and established an efficient approach for generating yellow-seeded oilseed rape genetic resources with increase oil content, modified FA composition and resistance to multiple abiotic stresses.

ATM signals to TSC2 in the cytoplasm to regulate mTORC1 in response to ROS.

Ataxia-telangiectasia mutated (ATM) is a cellular damage sensor that coordinates the cell cycle with damage-response checkpoints and DNA repair to preserve genomic integrity. However, ATM also has been implicated in metabolic regulation, and ATM deficiency is associated with elevated reactive oxygen species (ROS). ROS has a central role in many physiological and pathophysiological processes including inflammation and chronic diseases such as atherosclerosis and cancer, underscoring the importance of cellular pathways involved in redox homeostasis. We have identified a cytoplasmic function for ATM that participates in the cellular damage response to ROS. We show that in response to elevated ROS, ATM activates the TSC2 tumor suppressor via the LKB1/AMPK metabolic pathway in the cytoplasm to repress mTORC1 and induce autophagy. Importantly, elevated ROS and dysregulation of mTORC1 in ATM-deficient cells is inhibited by rapamycin, which also rescues lymphomagenesis in Atm-deficient mice. Our results identify a cytoplasmic pathway for ROS-induced ATM activation of TSC2 to regulate mTORC1 signaling and autophagy, identifying an integration node for the cellular damage response with key pathways involved in metabolism, protein synthesis, and cell survival.

Intracellular Cu/Zn superoxide dismutase (Cu/Zn-SOD) from hard clam Meretrix meretrix: its cDNA cloning, mRNA expression and enzyme activity.

Hard clam (Meretrix meretrix) is an economically important bivalve in China. In the present study, a gene coding for an intracellular Cu/Zn-SOD was cloned and characterized from hard clam. The full-length cDNA of this Cu/Zn-SOD (designated as Mm-icCuZn-SOD) consisted of 1,383 bp, with a 462-bp of open reading frame (ORF) encoding 153 amino acids. Several highly conserved motifs, including the Cu/Zn binding sites [H(46), H(48), H(63), and H(119) for Cu binding; H(63), H(71), H(80), and D(83) for Zn binding], an intracellular disulfide bond and two Cu/Zn-SOD signatures were identified in Mm-icCu/Zn-SOD. The deduced amino acid sequence of Mm-icCu/Zn-SOD has a high degree of homology with the Cu/Zn-dependent SODs from other species, indicating that Mm-icCu/Zn-SOD should be a member of the intracellular Cu/Zn-dependent SOD family. Real-time PCR analysis showed that the highest level of Mm-icCu/Zn-SOD expression was in the hepatopancreas, while the lowest level occurred in the hemocytes. Hard clam challenged with Vibrio anguillarum showed a time-dependent increase in Mm-icCu/Zn-SOD expression that reached a maximum level after 6 h. Mm-icCu/Zn-SOD purified as a recombinant protein expressed in E. coli retained a high level of biological activity, 83 % after 10 min incubation at 10-50 °C, and more than 87 % after incubation in buffers with pH values between 2.2 and 10.2. These results indicated that Mm-icCu/Zn-SOD may play an important role in the innate immune system of hard clam.

Larval settlement and metamorphosis of the mussel Mytilus coruscus in response to natural biofilms.

Settlement and metamorphosis of pediveliger larvae of Mytilus coruscus in response to natural biofilms was investigated in the laboratory. Pediveliger larvae settled and metamorphosed in response to biofilms and post-larval settlement and metamorphosis increased with biofilm age. The activity of the biofilm was positively correlated with biofilm age, dry weight, bacterial density and diatom density, but had no apparent relationship with chlorophyll a concentration. The change in bacterial community composition corresponding to biofilm age may explain differences in the age-dependent inducing activities of biofilms, which in turn may play an important role in larval settlement in this species.

Activity of TSC2 is inhibited by AKT-mediated phosphorylation and membrane partitioning.

Loss of tuberin, the product of TSC2 gene, increases mammalian target of rapamycin (mTOR) signaling, promoting cell growth and tumor development. However, in cells expressing tuberin, it is not known how repression of mTOR signaling is relieved to activate this pathway in response to growth factors and how hamartin participates in this process. We show that hamartin colocalizes with hypophosphorylated tuberin at the membrane, where tuberin exerts its GTPase-activating protein (GAP) activity to repress Rheb signaling. In response to growth signals, tuberin is phosphorylated by AKT and translocates to the cytosol, relieving Rheb repression. Phosphorylation of tuberin at serines 939 and 981 does not alter its intrinsic GAP activity toward Rheb but partitions tuberin to the cytosol, where it is bound by 14-3-3 proteins. Thus, tuberin bound by 14-3-3 in response to AKT phosphorylation is sequestered away from its membrane-bound activation partner (hamartin) and its target GTPase (Rheb) to relieve the growth inhibitory effects of this tumor suppressor.

AMPK-mediated phosphorylation of murine p27 at T197 promotes binding of 14-3-3 proteins and increases p27 stability.

The tuberous sclerosis complex 2 (Tsc2) gene product, tuberin, acts as a negative regulator of mTOR signaling, and loss of tuberin function leads to tumors of the brain, skin, kidney, heart, and lungs. Previous studies have shown that loss of tuberin function affects the stability and subcellular localization of the cyclin-dependent kinase inhibitor (CKI) p27, although the mechanism(s) by which tuberin modulates p27 stability has/have not been elucidated. Previous studies have also shown that AMP-activated protein kinase (AMPK), which functions in an energy-sensing pathway in the cell, becomes activated in the absence of tuberin. Here we show that in Tsc2-null tumors and cell lines, AMPK activation correlates with an increase in p27 levels, and inhibition of AMPK signaling decreases p27 levels in these cells. In addition, activation of AMPK led to phosphorylation of p27 at the conserved terminal threonine residue of murine p27 (T197) in both in vitro kinase assays and in cells. Phosphorylation of p27 at T197 led to increased interaction between p27 and 14-3-3 proteins and increased the protein stability of p27. Furthermore, activation of AMPK signaling promoted the interaction between p27 and 14-3-3 proteins and increased the stability of the p27 protein in a manner that was dependent on T197. These data identify a conserved mechanism for the regulation of p27 stability via phosphorylation at the terminal threonine (mT197/hT198) and binding of 14-3-3 proteins, which when AMPK is activated results in stabilization of the p27 protein.

Cytoplasmic sequestration of p27 via AKT phosphorylation in renal cell carcinoma.

PURPOSE: p27 localization and expression has prognostic and predictive value in cancer. Little is known regarding expression patterns of p27 in renal cell carcinoma (RCC) or how p27 participates in disease progression or response to therapy. EXPERIMENTAL DESIGN: RCC-derived cell lines, primary tumors, and normal renal epithelial cells were analyzed for p27 expression, phosphorylation (T157 of the NLS), and subcellular localization. RCC-derived cell lines were treated with phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitors and effects on p27 localization were assessed. The potential contribution of cytoplasmic p27 to resistance to apoptosis was also evaluated. RESULTS: p27 was elevated in tumors compared with matched controls, and cytoplasmic mislocalization of p27 was associated with increasing tumor grade. Cytoplasmic localization of p27 correlated with phosphorylation at T157, an AKT phosphorylation site in the p27 NLS. In RCC cell lines, activated PI3K/AKT signaling was accompanied by mislocalization of p27. AKT activation and phosphorylation of p27 was associated with resistance to apoptosis, and small interfering RNA knockdown of p27 or relocalization to the nucleus increased apoptosis in RCC cells. Treatment with the PI3K inhibitors LY294002 or wortmannin resulted in nuclear relocalization of p27, whereas mTOR inhibition by rapamycin did not. CONCLUSIONS: In RCC, p27 is phosphorylated at T157 of the NLS, with increasing tumor grade associated with cytoplasmic p27. PI3K inhibition (which reduces AKT activity) reduces T157 phosphorylation and induces nuclear relocalization of p27, whereas mTOR inhibition does not. Clinical testing of these findings may provide a rational approach for use of mTOR and PI3K/AKT pathway inhibitors in patients with RCC.

Precision Genome Engineering Through Cytidine Base Editing in Rapeseed (Brassica napus. L).

Rapeseed is one of the world's most important sources of oilseed crops. Single nucleotide substitution is the basis of most genetic variation underpinning important agronomic traits. Therefore, genome-wide and target-specific base editing will greatly facilitate precision plant molecular breeding. In this study, four CBE systems (BnPBE, BnA3A-PBE, BnA3A1-PBE, and BnPBGE14) were modified to achieve cytidine base editing at five target genes in rapeseed. The results indicated that genome editing is achievable in three CBEs systems, among which BnA3A1-PBE had the highest base-editing efficiency (average 29.8% and up to 50.5%) compared to all previous CBEs reported in rapeseed. The editing efficiency of BnA3A1-PBE is ~8.0% and fourfold higher, than those of BnA3A-PBE (averaging 27.6%) and BnPBE (averaging 6.5%), respectively. Moreover, BnA3A1-PBE and BnA3A-PBE could significantly increase the proportion of both the homozygous and biallelic genotypes, and also broaden the editing window compared to BnPBE. The cytidine substitution which occurred at the target sites of both BnaA06.RGA and BnaALS were stably inherited and conferred expected gain-of-function phenotype in the T1 generation (i.e., dwarf phenotype or herbicide resistance for weed control, respectively). Moreover, new alleles or epialleles with expected phenotype were also produced, which served as an important resource for crop improvement. Thus, the improved CBE system in the present study, BnA3A1-PBE, represents a powerful base editor for both gene function studies and molecular breeding in rapeseed.

Discrepancy variation of dinucleotide microsatellite repeats in eukaryotic genomes.

To address whether there are differences of variation among repeat motif types and among taxonomic groups, we present here an analysis of variation and correlation of dinucleotide microsatellite repeats in eukaryotic genomes. Ten taxonomic groups were compared, those being primates, mammalia (excluding primates and rodentia), rodentia, birds, fish, amphibians and reptiles, insects, molluscs, plants and fungi, respectively. The data used in the analysis is from the literature published in the Journal of Molecular Ecology Notes. Analysis of variation reveals that there are no significant differences between AC and AG repeat motif types. Moreover, the number of alleles correlates positively with the copy number in both AG and AC repeats. Similar conclusions can be obtained from each taxonomic group. These results strongly suggest that the increase of SSR variation is almost linear with the increase of the copy number of each repeat motif. As well, the results suggest that the variability of SSR in the genomes of low-ranking species seem to be more than that of high-ranking species, excluding primates and fungi.

14-3-3 interacts with the tumor suppressor tuberin at Akt phosphorylation site(s).

Tuberin, the product of the tuberous sclerosis complex 2 tumor suppressor gene, is a phosphoprotein that negatively regulates phosphatidylinositol 3'-kinase signaling downstream of Akt. Several high stringency 14-3-3 binding sites that overlapped with Akt phosphorylation sites were identified in tuberin in silico. Recognition of tuberin by an alpha-14-3-3 binding site-specific antibody correlated with mitogen-induced phosphorylation of tuberin and recognition of tuberin by an alpha-Akt phosphorylation substrate antibody. Recognition of tuberin by both antibodies was blocked by inhibiting phosphatidylinositol 3'-kinase activity. Using a protein domain microarray, a tuberin peptide containing Ser(939) demonstrated phospho-specific binding to 14-3-3. Glutathione S-transferase pull-down assays with 14-3-3 fusion proteins revealed that all seven 14-3-3 isoforms (beta, gamma, zeta, epsilon, tau, eta, and sigma) could bind tuberin, and this interaction was abrogated by competition with phosphorylated but not unphosphorylated Ser(939) tuberin peptide. Tuberin also coimmunoprecipitated with 14-3-3, confirming the interaction between endogenous 14-3-3 and tuberin. These data establish the presence of functional and overlapping 14-3-3 and Akt recognition site(s) in tuberin.

Aberrant expression of HMGA2 in uterine leiomyoma associated with loss of TSC2 tumor suppressor gene function.

Unregulated proliferation of mesenchymal cells in leiomyomas, lipomas, hamartomas,and other diseases has been linked to the high mobility group (HMGA) family of DNA architectural proteins. HMGA genes are primarily expressed during embryonal development and silenced in adult tissues but can become reactivated in neoplasia as a result of chromosomal rearrangements. Although the genetic data suggesting a role for HMGA proteins in tumorigenesis are compelling, the biological role of these proteins in mesenchymal proliferation and differentiation is incompletely defined. Uterine myometria and spontaneous leiomyomas from the Eker rat, which carries a germ-line mutation in the tuberous sclerosis complex-2 (Tsc2) tumor suppressor gene, were analyzed for genetic defects in and expression of the Tsc2 and HMGA proteins. Eker leiomyomas exhibited a 50% incidence of loss of the wild-type Tsc2 allele and an almost uniform loss of protein expression, implicating loss of function of the Tsc2 gene in these tumors. Concomitantly, HMGA2 protein, which was completely absent in normal myometria, was expressed in 16 of 19 Eker leiomyomas. HMGA1 was expressed in both leiomyoma and normal myometria. No structural alterations were observed at the HMGA2 locus in either primary rat leiomyomas or leiomyoma-derived cell lines that expressed HMGA2. These data support a role for HMGA2 in the development of smooth muscle neoplasms and suggest HMGA2 expression is a point of convergence between the human disease and the Eker rat model. Furthermore, these data indicate that aberrant HMGA2 expression can result from dysfunction of the Tsc2 tumor suppressor gene, in the absence of structural alterations involving the HMGA2 locus.

TSC2, a key player in tumor suppression and cystic kidney disease.

Research into inherited cancer syndromes that involve defective tumor suppressor genes has increased our understanding of the genetic basis of this disease. Dramatic advances over the past decade have established the tuberous sclerosis complex 2 tumor suppressor gene (TSC2) as a key player in signal transduction pathways involved in the development of cancer. Importantly, the discovery of the functional link between TSC2 and the polycystic kidney disease 1 gene (PKD1) is beginning to build a foundation for understanding the heritable diseases associated with defects in each of these genes, namely, tuberous sclerosis complex and polycystic kidney disease. In this review, we summarize the latest findings about the functions of the TSC2 gene product, tuberin, and their implications for the development of cystic kidney disease.

mRNA of MAGE genes as specific markers in detection of tumor cells in the peripheral blood of patients with hepatocellular carcinoma.

OBJECTIVE: To detect tumor cells in the peripheral blood of patients with hepatocellular carcinoma (HCC) by using the mRNA of the MAGE-1 and MAGE-3 genes as specific tumor markers. METHODS: Peripheral blood was obtained from 25 HCC patients and 20 healthy volunteers. The mRNA of the MAGE-1 and MAGE-3 genes in the peripheral blood mononuclear cells (PBMCs) was detected by nested RT-PCR. The MAGE-1 and MAGE-3 transcripts in the tumor tissues of these HCC patients were also detected by RT-PCR. RESULTS: Of the 25 HCC patients, MAGE-1 and MAGE-3 mRNA were positive in 44% (11/25) and 36% (9/25) of PBMCs respectively, and in 68% (17/25) and 56% (14/25) of HCC tissues respectively. In the PBMCs of the 25 HCC patients, 16 (64%) samples were detected to express at least one type of MAGE mRNA. MAGE mRNA were not detected in the PBMCs from the patients whose tumors did not express the MAGE genes, nor in the PBMCs from the 20 healthy donors. The positive rate of MAGE mRNA in the PBMCs was closely correlated with the TNM stages and the diameter of tumors, but there was no correlation between the positive rate of MAGE mRNA in PBMCs and tumor differentiation degree or serum alpha-FP level. Of 9 HCC patients whose serum alpha-FP was normal or slightly elevated (< 50 ng/ml), 6 were MAGE-1 and/or MAGE-3 mRNA positive in their PBMCs. CONCLUSION: MAGE-1 and MAGE-3 mRNA could be specifically detected with high percentage in the PBMCs of HCC patients by our method. They can be used as specific tumor markers for the detection of the circulating HCC cells, and the detection results may be helpful to evaluate the prognosis of HCC patients.

Cloning of Hsp21 gene and its expression in Chinese shrimp Fenneropenaeus chinensis in response to WSSV challenge.

In the present study, a DNA sequence encoding a small heat shock protein gene (FcHsp21) in the Chinese shrimp, Fenneropenaeus chinensis, was cloned, and its expression was analyzed after white spot syndrome virus (WSSV) infection. The FcHsp21 gene contained an open reading frame (ORF) of 555 bp in length, encoding a 184 amino acid protein with a theoretical size of about 21 kDa and a predicted isoelectric point of 5.38. The mRNA of the Hsp21 had a long Poly(A) tail (748 bp) with six polyadenylation signals (AATAA) downstream from the terminator. In addition, the gene contained a relatively long intron (507 bp), which has not been described in shrimps. The intron contained a long compound type microsatellite repeat sequence. The analysis of the phylogenetics revealed that the Hsp21 was highly conserved among the genomes of animals. Our results show that the expression modes of FcHsp21 can be changed by different WSSV infection methods. The expression of FcHsp21 was inhibited by muscle-injecting WSSV, but induced by feeding WSSV.

A tuberous sclerosis complex signalling node at the peroxisome regulates mTORC1 and autophagy in response to ROS.

Subcellular localization is emerging as an important mechanism for mTORC1 regulation. We report that the tuberous sclerosis complex (TSC) signalling node, TSC1, TSC2 and Rheb, localizes to peroxisomes, where it regulates mTORC1 in response to reactive oxygen species (ROS). TSC1 and TSC2 were bound by peroxisomal biogenesis factors 19 and 5 (PEX19 and PEX5), respectively, and peroxisome-localized TSC functioned as a Rheb GTPase-activating protein (GAP) to suppress mTORC1 and induce autophagy. Naturally occurring pathogenic mutations in TSC2 decreased PEX5 binding, and abrogated peroxisome localization, Rheb GAP activity and suppression of mTORC1 by ROS. Cells lacking peroxisomes were deficient in mTORC1 repression by ROS, and peroxisome-localization-deficient TSC2 mutants caused polarity defects and formation of multiple axons in neurons. These data identify a role for the TSC in responding to ROS at the peroxisome, and identify the peroxisome as a signalling organelle involved in regulation of mTORC1.

PERMANENT GENETIC RESOURCES: Twelve new microsatellite markers for the Chinese shrimp Fenneropenaeus chinensis.

Twelve new microsatellite markers were isolated by sequencing random clones from a genomic library of Fenneropenaeus chinensis. The number of alleles per locus ranged from five to 15. The polymorphism information content ranged from 0.568 to 0.898, and observed and expected heterozygosities from 0.344 to 0.882 and from 0.691 to 0.915, respectively. All loci except one conformed to Hardy-Weinberg equilibrium. These markers, described here for Chinese shrimp, will be further used to analyse the species' population genetics.

AMP-activated protein kinase signaling results in cytoplasmic sequestration of p27.

Tuberin, the Tsc2 gene product, integrates the phosphatidylinositol 3-kinase/mitogen-activated protein kinase (mitogenic) and LKB1/AMP-activated protein kinase (AMPK; energy) signaling pathways, and previous independent studies have shown that loss of tuberin is associated with elevated AMPK signaling and altered p27 function. In Tsc2-null tumors and tumor-derived cells from Eker rats, we observed elevated AMPK signaling and concordant cytoplasmic mislocalization of p27. Cytoplasmic localization of p27 in Tsc2-null cells was reversible pharmacologically using inhibitors of the LKB1/AMPK pathway, and localization of p27 to the cytoplasm could be induced directly by activating AMPK physiologically (glucose deprivation) or genetically (constitutively active AMPK) in Tsc2-proficient cells. Furthermore, AMPK phosphorylated p27 in vitro on at least three sites including T170 near the nuclear localization signal, and T170 was shown to determine p27 localization in response to AMPK signaling. p27 functions in the nucleus to suppress cyclin-dependent kinase-2 (Cdk2) activity and has been reported to mediate an antiapoptotic function when localized to the cytoplasm. We found that cells with elevated AMPK signaling and cytoplasmic p27 localization exhibited elevated Cdk2 activity, which could be suppressed by inhibiting AMPK signaling. In addition, cells with elevated AMPK signaling and cytoplasmic p27 localization were resistant to apoptosis, which could be overcome by inhibition of AMPK signaling and relocalization of p27 to the nucleus. These data show that AMPK signaling determines the subcellular localization of p27, and identifies loss of integration of pathways controlling energy balance, the cell cycle, and apoptosis due to aberrant AMPK and p27 function as a feature of cells that have lost the Tsc2 tumor suppressor gene.

Spontaneous renal tubular hyperplastic and neoplastic lesions in three Sprague-Dawley rats from a 90-day toxicity study.

Multiple renal tubular cell adenomas and atypical tubular hyperplasia were diagnosed in 2 high-dose and 1 mid-dose female Sprague-Dawley (Crl:CD (SD)IGS BR) rats from a 90-day toxicity study of an amino acid found in green tea. The tumors were bilateral multicentric adenomas accompanied by atypical foci of renal tubular hyperplasia in both kidneys of the 3 animals. Toxic tubular changes that typically accompany renal carcinogenesis were not seen in any of the other animals of the study, suggesting rather, an underlying germline mutation of a tumor suppressor gene in these three rats. The histological appearance of these tumors and short latency was reminiscent of the spontaneous lesions reported to arise in Sprague-Dawley rats in the Nihon rat model. Nihon rats develop kidney tumors as a result of a spontaneous mutation in the rat homologue of the Birt-Hogg-Dubé gene (Bhd). Frozen samples of liver from two tumor-bearing rats were assayed for germline alterations in the Bhd gene. The entire coding region (exons 3-13) of the Bhd gene was sequenced, and a guanine (nt106G) to adenine (nt106A) polymorphism was detected resulting in a glycine to arginine (G36R) substitution in both tumor-bearing animals. In the study animals, the frequency of the A-allele (adenine) was determined to be 27% (19/70). Interestingly, rats obtained from two other sources (n = 17) only carried the nt106G-allele, consistent with the published rat sequence for this gene. Genetic fingerprinting of microsatellite loci indicated that the rats had a shared genetic background. Laser capture microdissection (LCM) of the tumor cells demonstrated a loss of heterozygosity in the Bhd gene in neoplastic cells of one of the two animals. Taken together, these data suggest that the tumors observed in these animals arose spontaneously as a result of a shared genetic susceptibility leading to the development of renal tubular neoplasms.