Initial licking responses of mice to sweeteners: effects of tas1r3 polymorphisms

Recent studies have established that the T1R3 receptor plays a central role in the taste-mediated ingestive response to sweeteners by mice. First, transgenic mice lacking the gene for T1R3, Tas1r3, show dramatically reduced lick responsiveness to most sweeteners. Second, strains with the taster allele of Tas1r3 (T strains) are more sensitive to low sweetener concentrations than strains with the nontaster allele (NT strains) and consume greater quantities of low- to midrange concentrations of sweeteners during 24-h tests. We asked how Tas1r3 polymorphisms influence the initial licking responses of four T strains (FVB/NJ, SWR/J, SM/J, and C57BL/6J) and four NT strains (BALB/cJ, 129P3/J, DBA/2J, and C3H/HeJ) to two sweeteners (sucrose and SC-45647, an artificial sweetener). We used the initial licking response as a measure of the taste-mediated ingestive response because its brief duration minimizes the potential contribution of nontaste factors (e.g., negative and positive postingestive feedback). Further, we used two complimentary short-term intake tests (the brief-access taste test and a novel 1-min preference test) to reduce the possibility that our findings were an epiphenomenon of a specific testing procedure. In both tests, the T strains were more responsive than the NT strains to low concentrations of each sweetener. At higher concentrations, however, there was considerable overlap between the T and NT strains. In fact, the initial licking response of several NT strains was more vigorous than (or equivalent to) that of several T strains. There was also considerable variation among strains with the same Tas1r3 allele. We conclude that Tas1r3 polymorphisms contribute to strain differences in initial lick responsiveness to low but not high concentrations of sweeteners.     

The Effect of <Emphasis Type="Italic">Tas1r3</Emphasis> Gene Polymorphism on Preference and Consumption of Sucrose and Low-Calorie Sweeteners in Interstrain Hybrid Mice of the First Filial Generation

Inter- and intra-species differences in consumption of sweet tastants formed during the evolution of vertebrates are thought to be due to polymorphism of the Tas1r3 gene encoding T1R3, a sweet taste receptor subunit. The aim of the study was to assess the effect of Tas1r3 polymorphism on nutritional behavior of laboratory mice using the first filial generation (F1) hybrids produced by crossing inbred strains with different sensitivity to sweet: 129P3/J males (129, carriers of a recessive SacD sweet taste receptor allele) and C57BL/6 females (B6, dominant SacB allele) or females of the Tas1r3 gene knockout strain, C57BL/6-Tas1r3KO (B6-Tas1r3KO). SacD/B and SacD/0 hybrids, sharing identical background genotypes, differed only by sets of Sac alleles. In a briefaccess test (BAT) or a 48-h two-bottle free choice test, the presence of the dominant SacD allele in SacD/B hybrids determined increased preference for low sucrose concentrations (1–4%) and higher concentrations of nonmetabolized sweeteners (saccharin Na, sucralose, acesulfame K). A comparison between the 129 parental strain and SacD/0 hybrids or between the B6 parental strain and hybrids from crossing B6 × B6-Tas1r3KO revealed no influence of hemizygosity of SacD or SacB on preference for sweeteners in BAT. A small decrease in sucrose and saccharin preference associated with the lack of the SacB allele was observed during long-term exposure to solutions with low concentrations of these substances. The data obtained indicate the relevance of studying the Tas1r3 polymorphism effects on preference and consumption of sweet tastants using F1 interstrain hybrids and BAT.     

No relationship between sequence variation in protein coding regions of the Tas1r3 gene and saccharin preference in rats

Nearly all mammalian species like sweet-tasting foods and drinks, but there are differences in the degree of 'sweet tooth' both between species and among individuals of the same species. Some individual differences can be explained by genetic variability. Polymorphisms in a sweet taste receptor (Tas1r3) account for a large fraction of the differences in consumption of sweet solutions among inbred mouse strains. We wondered whether mice and rats share the same Tas1r3 alleles, and whether this gene might explain the large difference in saccharin preference among rats. We conducted three experiments to test this. We examined DNA sequence differences in the Tas1r3 gene among rats that differed in their consumption of saccharin in two-bottle choice tests. The animals tested were from an outbred strain (Sprague-Dawley; experiment 1), selectively bred to be high- or low-saccharin consumers (HiS and LoS; experiment 2), or from inbred strains with established differences in saccharin preference (FH/Wjd and ACI; experiment 3). Although there was considerable variation in saccharin preference among the rats there was no variation in the protein-coding regions of the Tas1r3 gene. DNA variants in intronic regions were detected in 1 (of 12) outbred rat with lower-than-average saccharin preference and in the ACI inbred strain, which also has a lower saccharin preference than the FH/Wjd inbred partner strain. Possible effects of these intronic nucleotide variants on Tas1r3 gene expression or the presence of T1R3 protein in taste papillae were evaluated in the ACI and FH/Wjd strains. Based upon the results of these studies, we conclude that polymorphisms in the protein-coding regions of the sweet receptor gene Tas1r3 are uncommon and do not account for individual differences in saccharin preference for these strains of rats. DNA variants in intron 4 and 5 are more common but appear to be innocuous.     

The involvement of the T1R3 receptor protein in the control of glucose metabolism in mice at different levels of glycemia

The heterodimeric protein T1R2/T1R3 is a chemoreceptor mediating taste perception of sugars, several amino acids, and non-caloric sweeteners in humans and many other vertebrate species. The T1R2 and T1R3 proteins are expressed not only in the oral cavity, but also in the intestine, pancreas, liver, adipose tissue, and in structures of the central nervous system, which suggests their involvement in functions other than gustatory perception. In this study, we analyzed the role of the T1R3 protein in regulation of glucose metabolism in experiments with the gene-knockout mouse strain C57BL/6J-Tas1r3 ^tm1Rfm (Tas1r3-/-), with a deletion of the Tas1r3 gene encoding T1R3, and the control strain C57BL/6ByJ with the intact gene. Glucose tolerance was measured in euglycemic or food-deprived mice after intraperitoneal or intragastric glucose administration. We have shown that in the Tas1r3-/- strain, in addition to the disappearance of taste preference for sucrose, glucose tolerance is also substantially reduced, and insulin resistance is observed. The effect of the Tas1r3 gene knockout on glucose utilization was more pronounced in the euglycemic state than after food deprivation. The baseline glucose level after food deprivation was lower in the Tas1r3-/- strain than in the control strain, which suggests that T1R3 is involved in regulation of endogenous glucose production. These data suggest that the T1R3-mediated glucoreception interacts with the K_ATP-dependent mechanisms of regulation of the glucose metabolism, and that the main role is likely played by T1R3 expressed in the pancreas and possibly in the central nervous system, but not in the intestinal mucosa, as it was suggested earlier.     

Allelic variation of the Tas1r3 taste receptor gene selectively affects taste responses to sweeteners: evidence from 129.B6-Tas1r3 congenic mice.

The Tas1r3 gene encodes the T1R3 receptor protein, which is involved in sweet taste transduction. To characterize ligand specificity of the T1R3 receptor and the genetic architecture of sweet taste responsiveness, we analyzed taste responses of 129.B6-Tas1r3 congenic mice to a variety of chemically diverse sweeteners and glucose polymers with three different measures: consumption in 48-h two-bottle preference tests, initial licking responses, and responses of the chorda tympani nerve. The results were generally consistent across the three measures. Allelic variation of the Tas1r3 gene influenced taste responsiveness to nonnutritive sweeteners (saccharin, acesulfame-K, sucralose, SC-45647), sugars (sucrose, maltose, glucose, fructose), sugar alcohols (erythritol, sorbitol), and some amino acids (D-tryptophan, D-phenylalanine, L-proline). Tas1r3 genotype did not affect taste responses to several sweet-tasting amino acids (L-glutamine, L-threonine, L-alanine, glycine), glucose polymers (Polycose, maltooligosaccharide), and nonsweet NaCl, HCl, quinine, monosodium glutamate, and inosine 5'-monophosphate. Thus Tas1r3 polymorphisms affect taste responses to many nutritive and nonnutritive sweeteners (all of which must interact with a taste receptor involving T1R3), but not to all carbohydrates and amino acids. In addition, we found that the genetic architecture of sweet taste responsiveness changes depending on the measure of taste response and the intensity of the sweet taste stimulus. Variation in the T1R3 receptor influenced peripheral taste responsiveness over a wide range of sweetener concentrations, but behavioral responses to higher concentrations of some sweeteners increasingly depended on mechanisms that could override input from the peripheral taste system.     

Identification of a novel member of the T1R family of putative taste receptors

In the gustatory system, the recognition of sugars, amino acids and bitter-tasting compounds is the function of specialized G protein-coupled receptors. Recently, two members of novel subfamily of G protein-coupled receptors were proposed to function as taste receptors based on their specific expression in taste receptor cells. Here, we report the identification of a third member, T1R3, of this family of receptors. T1R3 maps near the telomere of mouse chromosome 4 rendering it a candidate for the Sac locus, a primary determinant of sweet preference in mice. Consistent with its candidacy for the Sac locus, T1R3 displays taste receptor cell-specific expression. In addition, taster and non-taster strains of mouse harbor different alleles of T1R3.     

Stimulus processing of glycine is dissociable from that of sucrose and glucose based on behaviorally measured taste signal detection in Sac 'taster' and 'non-taster' mice

Mouse strains have been divided into 'tasters' and 'non-tasters' based on their relatively high and low preference, respectively, for low concentrations of sucrose and saccharin. These phenotypic differences appear to be due to a polymorphism in the gene at the Sac locus encoding for the T1R3 taste receptor selectively affecting the functionality of the T1R2+3 heterodimer. To psychophysically examine whether these phenotypes are due to sensory sensitivity as opposed to hedonic responsiveness, we measured taste signal detection of sucrose, glucose, and glycine by Sac taster (C57BL/6J and SWR/J) and non-taster (129P3/J and DBA/2J) strains in an operant conditioning paradigm using a gustometer. The taster mice had lower detection thresholds for sucrose and glucose compared with the non-taster mice. The detection thresholds corresponded well with reported responsiveness to low concentrations of these sugars in two-bottle intake tests suggesting that the Sac taster phenotype has a sensory basis and is not simply a matter of strain differences in the hedonic evaluation of weak intensities of the stimuli. Taster status did not entirely account for the strain differences in detection thresholds for glycine, a 'sweet' tasting amino acid. Collapsed across strains, detection thresholds for sucrose and glucose were highly correlated with each other (r = 0.81), but only modestly correlated with those for glycine (r < or = 0.43). This suggests that stimulus processing of glycine in the perithreshold intensity domain can be dissociated from that of sucrose and glucose. The mechanism underlying this difference may be related to the ability of glycine to bind with the T1R1+3 heterodimer.     

Mouse strain differences in Gurmarin-sensitivity of sweet taste responses are not associated with polymorphisms of the sweet receptor gene, Tas1r3

Gurmarin (Gur) is a peptide that selectively inhibits responses of the chorda tympani (CT) nerve to sweet compounds in rodents. In mice, the sweet-suppressing effect of Gur differs among strains. The inhibitory effect of Gur is clearly observed in C57BL/6 mice, but only slightly, if at all, in BALB/c mice. These two mouse strains possess different alleles of the sweet receptor gene, Sac (Tas1r3) (taster genotype for C57BL/6 and non-taster genotype for BALB/c mice), suggesting that polymorphisms in the gene may account for differential sensitivity to Gur. To investigate this possibility, we examined the effect of Gur in another Tas1r3 non-taster strain, 129 X 1/Sv mice. The results indicated that unlike non-taster BALB/c mice but similar to taster C57BL/6 mice, 129 X 1/Sv mice exhibited significant inhibition of CT responses to various sweet compounds by Gur. This suggests that the mouse strain difference in the Gur inhibition of sweet responses of the CT nerve may not be associated with polymorphisms of Tas1r3.     

High-resolution genetic mapping of the saccharin preference locus (Sac) and the putative sweet taste receptor (T1R1) gene (Gpr70) to mouse distal Chromosome 4

The Sac (saccharin preference) locus affecting mouse behavioral and neural responsiveness to sweeteners has been mapped to distal Chr 4. A putative sweet taste receptor, T1R1, has been recently cloned, and the gene encoding it, Gpr70, has also been mapped to mouse distal Chr 4. To assess Gpr70 as a candidate gene for Sac, we compared the Gpr70 sequences of C57BL/6ByJ and 129P3/J mouse strains with different alleles of Sac. Using Gpr70 sequence variation between the C57BL/6ByJ and 129P3/J strains, we conducted a high-resolution analysis of the chromosomal localization of the Gpr70 and Sac loci in the F₂ hybrids and 129.B6-Sac partially congenic mice originating from these two strains. The Gpr70 gene maps proximal to Sac, which demonstrates that they are different loci. Received: 24 April 2000 / Accepted: 14 September 2000     

Pseudogenization of a sweet-receptor gene accounts for cats' indifference toward sugar

Although domestic cats (Felis silvestris catus) possess an otherwise functional sense of taste, they, unlike most mammals, do not prefer and may be unable to detect the sweetness of sugars. One possible explanation for this behavior is that cats lack the sensory system to taste sugars and therefore are indifferent to them. Drawing on work in mice, demonstrating that alleles of sweet-receptor genes predict low sugar intake, we examined the possibility that genes involved in the initial transduction of sweet perception might account for the indifference to sweet-tasting foods by cats. We characterized the sweet-receptor genes of domestic cats as well as those of other members of the Felidae family of obligate carnivores, tiger and cheetah. Because the mammalian sweet-taste receptor is formed by the dimerization of two proteins (T1R2 and T1R3; gene symbols Tas1r2 and Tas1r3), we identified and sequenced both genes in the cat by screening a feline genomic BAC library and by performing PCR with degenerate primers on cat genomic DNA. Gene expression was assessed by RT-PCR of taste tissue, in situ hybridization, and immunohistochemistry. The cat Tas1r3 gene shows high sequence similarity with functional Tas1r3 genes of other species. Message from Tas1r3 was detected by RT-PCR of taste tissue. In situ hybridization and immunohistochemical studies demonstrate that Tas1r3 is expressed, as expected, in taste buds. However, the cat Tas1r2 gene shows a 247-base pair microdeletion in exon 3 and stop codons in exons 4 and 6. There was no evidence of detectable mRNA from cat Tas1r2 by RT-PCR or in situ hybridization, and no evidence of protein expression by immunohistochemistry. Tas1r2 in tiger and cheetah and in six healthy adult domestic cats all show the similar deletion and stop codons. We conclude that cat Tas1r3 is an apparently functional and expressed receptor but that cat Tas1r2 is an unexpressed pseudogene. A functional sweet-taste receptor heteromer cannot form, and thus the cat lacks the receptor likely necessary for detection of sweet stimuli. This molecular change was very likely an important event in the evolution of the cat's carnivorous behavior.     

Cloning of genes related to exo-beta-glucanase production in Saccharomyces cerevisiae: characterization of an exo-beta-glucanase structural gene

The EXG1 gene of Saccharomyces cerevisiae was cloned and identified by complementation of a mutant strain (exg1-2) with highly reduced extracellular exo-beta-1,3-glucanase (EXG) activity. Two recombinant plasmids containing an overlapping region of 5.2 kb were isolated from a genomic DNA library and characterized by restriction mapping. The coding region was located by subcloning the original DNA inserts in a 2.7-kb HindIII-XhoI fragment. Exg+ strains and Exg- mutants transformed with yeast multicopy plasmids containing this DNA fragment showed an EXG activity 5- to 20-fold higher than for the untransformed Exg+ wild-type (wt) strains. The overproduced EXG had the same enzymic activity on different substrates, and showed the same electrophoretic behaviour on polyacrylamide gels and identical properties upon filtration through Sephacryl S-200 as those of the main EXG from Exg+ wt strains. The EXG1 gene transformed Schizosaccharomyces pombe, yielding extracellular EXG activity which showed cross-reactivity with anti-S. cervisiae EXG antibodies. A fragment including only a part of the EXG1 region was subcloned into the integrating vector YIp5, and the resulting plasmid was used to transform an Exg+ strain. Genetic and Southern analysis of several stable Exg- transformants showed that the fragment integrated by homology with the EXG1 locus. The chromosomal DNA fragment into which the plasmid integrated has a restriction pattern identical to that of the fragment on which we had previously identified the putative EXG1 gene. Only one copy of the EXG1 gene per genome was found in several strains tested by Southern analysis. Furthermore, two additional recombinant plasmids sharing a yeast DNA fragment of about 4.1 kb, which partially complements the exg1-2 mutation but which shows no homology with the 2.7-kb fragment containing the EXG1 gene, were also identified in this study. This 4.1-kb DNA fragment does not appear to contain an extragenic suppressor and could be related in some way to EXG production in S. cerevisiae.     

Impaired Glucose Metabolism in Mice Lacking the Tas1r3 Taste Receptor Gene

The G-protein-coupled sweet taste receptor dimer T1R2/T1R3 is expressed in taste bud cells in the oral cavity. In recent years, its involvement in membrane glucose sensing was discovered in endocrine cells regulating glucose homeostasis. We investigated importance of extraorally expressed T1R3 taste receptor protein in age-dependent control of blood glucose homeostasis in vivo, using nonfasted mice with a targeted mutation of the Tas1r3 gene that encodes the T1R3 protein. Glucose and insulin tolerance tests, as well as behavioral tests measuring taste responses to sucrose solutions, were performed with C57BL/6ByJ (Tas1r3+/+) inbred mice bearing the wild-type allele and C57BL/6J-Tas1r3^tm1Rfm mice lacking the entire Tas1r3 coding region and devoid of the T1R3 protein (Tas1r3-/-). Compared with Tas1r3+/+ mice, Tas1r3-/- mice lacked attraction to sucrose in brief-access licking tests, had diminished taste preferences for sucrose solutions in the two-bottle tests, and had reduced insulin sensitivity and tolerance to glucose administered intraperitoneally or intragastrically, which suggests that these effects are due to absence of T1R3. Impairment of glucose clearance in Tas1r3-/- mice was exacerbated with age after intraperitoneal but not intragastric administration of glucose, pointing to a compensatory role of extraoral T1R3-dependent mechanisms in offsetting age-dependent decline in regulation of glucose homeostasis. Incretin effects were similar in Tas1r3+/+ and Tas1r3-/- mice, which suggests that control of blood glucose clearance is associated with effects of extraoral T1R3 in tissues other than the gastrointestinal tract. Collectively, the obtained data demonstrate that the T1R3 receptor protein plays an important role in control of glucose homeostasis not only by regulating sugar intake but also via its extraoral function, probably in the pancreas and brain.     

A Pseudomonas stutzeri gene cluster encoding the biosynthesis of the CCl4-dechlorination agent pyridine-2,6-bis(thiocarboxylic acid)

A spontaneous mutant of Pseudomonas stutzeri strain KC lacked the carbon tetrachloride (CCl₄) transformation ability of wild-type KC. Analysis of restriction digests separated by pulsed-field gel electrophoresis (PFGE) indicated that the mutant strain CTN1 differed from strain KC by deletion of approximately 170 kb of chromosomal DNA. CTN1 did not produce pyridine-2,6-bis(thiocarboxylic acid) (PDTC), the agent determined to be responsible for CCl₄ dechlorination in cultures of strain KC. Cosmids from a genomic library of strain KC containing DNA from within the deleted region were identified by hybridization with a 148 kb genomic Spe I fragment absent in strain CTN1. Several cosmids identified in this manner were further screened for complementation of the PDTC biosynthesis-negative (Pdt⁻) phenotype. One cosmid (pT31) complemented the Pdt⁻ phenotype of CTN1 and conferred CCl₄ transformation activity and PDTC production upon other pseudomonads. Southern analysis showed that none of three other P. stutzeri strains representing three genomovars contained DNA that would hybridize with the 25 746 bp insert of pT31. Transposon mutagenesis of pT31 identified open reading frames (ORFs) whose disruption affected the ability to make PDTC in the strain CTN1 background. These data describe the pdt locus of strain KC as residing in a non-essential region of the chromosome subject to spontaneous deletion. The pdt locus is necessary for PDTC biosynthesis in strain KC and is sufficient for PDTC biosynthesis by other pseudomonads but is not a common feature of P. stutzeri strains.     

用富集文库克隆人胰岛素基因组基因

通过构建可富集人胰岛素基因的λ噬菌体文库,克隆了人胰岛素基因组基因．首先从中国人血液白细胞中提取到人基因组ＤＮＡ,用ＥｃｏＲⅠ和ＢｇｌⅡ对基因组ＤＮＡ进行全酶切,经０．４％琼脂糖凝胶电泳,特异回收９．５ｋｂ左右的ＤＮＡ片段．将该片段与λＥＭＢＬ３／ＢａｍＨⅠ臂连接,构建成一个特殊的人基因组λ噬菌体文库（富集文库）,效价为２×１０４．同时采用ＰＣＲ方法及用引物Ⅰ：５′ＧＧＡＣＡＧＧＣＴＡＣＡＴＣＡＧＧＡＡＧＡＧＧ３′,引物Ⅱ：５′ＣＴＧＣＧＴＣＴＡＡＴＴＧＣＡＧＴＡＧＴＴＣ３′,从人基因组ＤＮＡ中扩增出一段含胰岛素基因的１．３６ｋｂＤＮＡ片段,做为放射性标记探针,对文库进行了噬菌斑原位杂交筛选,从１×１０４个噬菌斑中筛选到一个含人胰岛素基因组基因的阳性克隆,并进一步完成了亚克隆和该基因１７３２ｂｐＤＮＡ序列的测定．结果该基因的１７３２ｂｐＤＮＡ序列包括部分５′端和３′端与国外发表的人胰岛素α型等位基因的序列相同     

Genetic studies of cleavage-initiated mRNA decay and processing of ribosomal 9S RNA show that the Escherichia coli ams and rne loci are the same

We show in the present paper that the cleavages initiating decay of the ompA mRNA are suppressed both in the Escherichia coli ams(ts) strain (originally defined by a prolonged bulk mRNA half-life) and in the me(ts) strain (originally defined by aberrant 9S RNA processing). The temperature-sensitive defects of both these strains are complemented by a recombinant lambda phage containing a genomic segment that carries the putative ams locus. A 5.8 kb fragment from this genomic DNA segment was cloned into a low-copy plasmid and used to transform the ams(ts) and rne(ts) strains. This resulted in growth at the non-permissive temperature and a reoccurrence of the cleavages initiating decay of the ompA mRNA. Deletion analyses of this 5.8 kb fragment indicated that the putative ams open reading frame could complement both the Ams(ts) and the Rne(ts) phenotype with regard to the ompA cleavages. In addition we showed that the ams(ts) strain suppresses 9S RNA processing to 5S RNA to the same extent as the rne(ts) strain, and that the rne(ts0 strain has a prolonged bulk mRNA half-life, as was reported for the ams(ts) strain. Therefore we suggest that ams and rne reflect the same gene locus; one which is involved both in mRNA decay and RNA processing. We discuss how this gene locus may related to the previously characterized endoribonucleolytic activities of RNase E and RNase K.     

Detection and functional characterization of a large genomic deletion resulting in decreased pathogenicity in Ralstonia solanacearum race 3 biovar 2 strains

Bacterial wilt (brown rot) disease of potato caused by Ralstonia solanacearum is one of the most important bacterial diseases and a major constraint on potato production worldwide. Through a comparative genomic analysis between R.?solanacearum'race 3 biovar 2' (R3bv2) strains, we identified a 77 kb region in strain UW551 which is specifically absent in the hypoaggressive strain IPO1609. We proved that IPO1609 indeed carries a 77 kb genomic deletion and provide genetic evidence that occurrence of this deletion is responsible for almost complete loss of pathogenicity of this strain. We carried out a functional analysis of this 77 kb region in strain UW551 using a combination of gene deletion and functional complementation approaches which identified the methionine biosynthesis genes metER as having a major contribution to IPO1609 pathogenesis. Deletion of the metER genes significantly impacts pathogenicity of R3bv2 strains but does not lead to methionine auxotrophy nor reduced ability to multiply in planta. In addition, this study indicated that three type III secretion system effectors or a type VI secretion system present within the 77 kb region have no or very minor contribution to pathogenicity.