The novel p.E89K mutation in the SRY gene inhibits DNA binding and causes the 46,XY disorder of sex development

Male sex determination in humans is controlled by the SRY gene, which encodes a transcriptional regulator containing a conserved high mobility group box domain (HMG-box) required for DNA binding. Mutations in the SRY HMG-box affect protein function, causing sex reversal phenotypes. In the present study, we describe a 19-year-old female presenting 46,XY karyotype with hypogonadism and primary amenorrhea that led to the diagnosis of 46,XY complete gonadal dysgenesis. The novel p.E89K missense mutation in the SRY HMG-box was identified as a de novo mutation. Electrophoretic mobility shift assays showed that p.E89K almost completely abolished SRY DNA-binding activity, suggesting that it is the cause of SRY function impairment. In addition, we report the occurrence of the p.G95R mutation in a 46,XY female with complete gonadal dysgenesis. According to the three-dimensional structure of the human SRY HMG-box, the substitution of the conserved glutamic acid residue by the basic lysine at position 89 introduces an extra positive charge adjacent to and between the positively charged residues R86 and K92, important for stabilizing the HMG-box helix 2 with DNA. Thus, we propose that an electrostatic repulsion caused by the proximity of these positive charges could destabilize the tip of helix 2, abrogating DNA interaction.

The novel p.E89K mutation in the SRY gene inhibits DNA binding and causes the 46,XY disorder of sex development.

Male sex determination in humans is controlled by the SRY gene, which encodes a transcriptional regulator containing a conserved high mobility group box domain (HMG-box) required for DNA binding. Mutations in the SRY HMG-box affect protein function, causing sex reversal phenotypes. In the present study, we describe a 19-year-old female presenting 46,XY karyotype with hypogonadism and primary amenorrhea that led to the diagnosis of 46,XY complete gonadal dysgenesis. The novel p.E89K missense mutation in the SRY HMG-box was identified as a de novo mutation. Electrophoretic mobility shift assays showed that p.E89K almost completely abolished SRY DNA-binding activity, suggesting that it is the cause of SRY function impairment. In addition, we report the occurrence of the p.G95R mutation in a 46,XY female with complete gonadal dysgenesis. According to the three-dimensional structure of the human SRY HMG-box, the substitution of the conserved glutamic acid residue by the basic lysine at position 89 introduces an extra positive charge adjacent to and between the positively charged residues R86 and K92, important for stabilizing the HMG-box helix 2 with DNA. Thus, we propose that an electrostatic repulsion caused by the proximity of these positive charges could destabilize the tip of helix 2, abrogating DNA interaction.

A naturally occurring deletion in the SRY promoter region affecting the Sp1 binding site is associated with sex reversal.

Male to female sex reversal results from failure of testis development. Mutations in the SRY gene or in other genes involved in the sexual differentiation pathway are considered to cause XY gonadal dysgenesis. The majority of the mutations in the SRY described so far are located within the SRY coding region, mainly in the HMG-box conserved domain. Comparison of 5' flanking SRY gene sequences among different species indicated the presence of several putative conserved consensus sequences for different transcription regulators. In this study, we investigated a 360 bp sequence encompassing the SRY putative core promoter, in 17 patients with variable degrees of 46,XY sex reversal, which have been previously shown not to bear mutations in the SRYcoding region. Sequencing analysis of the SRYpromoter in one patient with complete XY gonadal dysgenesis revealed a three base pair deletion in one of the Sp1 binding sites. The deletion abolished Sp1 binding in vitro. This is the first report on a naturally occurring mutation affecting the Sp1 regulatory element in the SRY promoter region, which is associated with sex reversal. Additionally, upon familial investigation the father, who had 18 genital surgeries due to severe hypospadia without cryptorchidism, was found to bear the same deletion and several relatives were referred to have sexual ambiguity.

Novel mutations affecting SRY DNA-binding activity: the HMG box N65H associated with 46,XY pure gonadal dysgenesis and the familial non-HMG box R30I associated with variable phenotypes.

The SRY gene (sex-determining region of the Y chromosome) initiates the process of male sex differentiation in mammalians. In humans mutations in the SRY gene have been reported to account for 10-15% of the XY sex reversal cases. We describe here two novel missense mutations in the SRY gene after the screening of 17 patients, including 3 siblings, with 46,XY gonadal dysgenesis and 4 true hermaphrodites. One of the mutations, an A to C transversion within the HMG box, causes the N65H substitution and it was found in a patient presenting 46,XY pure gonadal dysgenesis. The Escherichia coli expressed SRY(N65H) protein did not present DNA-binding activity in vitro. The other mutation, a G to T transversion, causes the R30I substitution. This mutation was found in affected and nonaffected members of a family, including the father, two siblings with partial gonadal dysgenesis, a phenotypic female with pure gonadal dysgenesis, and three nonaffected male siblings. The G to T base change was not found in the SRY sequence of 100 normal males screened by ASO-PCR. The R30I mutation is located upstream to the HMG box, within the (29)RRSSS(33) phosphorylation site. The E. coli expressed SRY(R30I) protein was poorly phosphorylated and consequently showed reduced DNA-binding capacity in vitro.

On the biosynthesis of Rhodnius prolixus heme-binding protein.

The biosynthesis of Rhodnius prolixus heme-binding protein (RHBP), which is present in the hemolymph and oocytes of Rhodnius prolixus, was investigated. Fat bodies of female insects incubated in vitro with 14C-leucine were able to synthesize and secrete 14C-RHBP to the culture medium. Titrtion of synthesized RHBP with hemin showed that the protein secreted by the fat bodies is bound to heme, despite the presence of apo-RHBP in the hemolymph. The sequence of the RHBP cDNA encodes a pre-protein of 128 amino acids with no significant homology to any known protein. Northern-blot assays revealed that RHBP expression was limited to fat bodies. The levels of both RHBP mRNA and secreted protein increased in response to blood meal. In addition, the time-course of RHBP secretion in vitro paralleled mRNA accumulation observed in vivo. The inhibition of the de novo heme biosynthesis by treatment of fat bodies with succinyl acetone (SA), an irreversible inhibitor of delta-aminolevulinic acid-dehydratase, led to a significant decrease of heme-RHBP secretion. Nevertheless, the levels of RHBP mRNA were not modified by SA treatment, suggesting that the heme availability is involved in a post-transcriptional control of the RHBP synthesis.

COI1 affects myrosinase activity and controls the expression of two flower-specific myrosinase-binding protein homologues in Arabidopsis.

Two cDNA clones homologous to myrosinase-binding proteins (MBPs) were identified by differential display in Arabidopsis thaliana (L.) Heynh. The cDNAs (MBP1 and MBP2) correspond to two open-reading frames found in a gene cluster of seven putative MBP genes located on chromosome 1. The predicted proteins MBP1 and MBP2 are similar to lectins and plant aggregating factors. In addition. MBP2 contains a region of high content of proline and alanine residues, commonly found in arabinogalactan proteins and hydroxyproline-rich glycoproteins. Transcripts corresponding to MBP1 and MBP2 genes are exclusively and abundantly expressed in flowers but are not detected in male-sterile flowers of coi1 plants, insensitive to jasmonic acid. Northern analysis and in situ hybridization revealed that MBP mRNAs are present in higher levels in immature flowers and are localized in several floral organs, including the ovary, ovules, style, anthers and filament. Transcripts of the Arabidopsis myrosinase gene TGG1 show a pattern of expression similar to that observed for the MBP genes during flower development; however, they are also abundant in green tissues and are only partially affected by COI1. Crude preparations of soluble proteins from leaf and flower extracts of wild-type Arabidopsis showed myrosinase activity when sinigrin was used as substrate. In contrast, coi1 plants showed significantly reduced myrosinase activities in both leaves and flowers. The results show that COI1 controls MBP expression in flowers and significantly affects the expression and activity of myrosinase in Arabidopsis.

Lack of changes in serum prolactin, FSH, TSH, and estradiol after melatonin treatment in doses that improve sleep and reduce benzodiazepine consumption in sleep-disturbed, middle-aged, and elderly patients.

An open pilot study on the safety and efficacy of melatonin in the treatment of insomniac patients was conducted in 22 subjects (16 females), mean +/- S.D. age 60.1 +/- 9.5 years. All patients received 3 mg of gelatin melatonin capsules per os daily for 6 months, 30 min before expected sleep time. Twenty of 22 patients were on benzodiazepine treatment and they continued this treatment for part of or for the entire melatonin administration period. Serum concentrations of prolactin, follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), or estradiol were measured by radioimmunoassay (RIA) in morning samples at the beginning and after 6 months of melatonin administration, and standard clinical laboratory tests for blood components were performed. Urinary 6-sulphatoxymelatonin (aMT6s) excretion was measured by RIA before treatment. Serum concentrations of prolactin, FSH, TSH, or estradiol did not exhibit changes after 6 months of melatonin administration, nor were any indications of hematologic or blood biochemistry alteration found. Melatonin augmented significantly the quality and duration of sleep, and decreased sleep latency and the number of awakening episodes, as assessed from sleep logs filled by the patients (first 21 days) and from structured interviews performed by incumbent physicians (up to 6 months). Estimates of next-day function (i.e., alertness in the morning and during the day) also improved significantly during melatonin treatment. The observed effect lasted for the entire period examined (up to 6 months), with 22 out of 22 patients showing improved sleep at the end of treatment. The urinary excretion of aMT6s before starting administration of melatonin correlated negatively and significantly with age, but not with the intensity of sleep the disorder or the outcome of treatment. In 13 of 20 patients taking benzodiazepines together with melatonin, benzodiazepine use could be stopped, and in another four patients, benzodiazepine dose could be decreased to 25-66% of the initial dose. The results of this open, subacute administration trial indicate that melatonin is a safe and useful treatment for sleep disturbances in middle-aged or elderly patients, either by itself or together with benzodiazepines.

An Arabidopsis gene induced by wounding functionally homologous to flavoprotein oxidoreductases.

The regulation of genes in response to wounding is mediated in part by the octadecanoids 12-oxo-phytodienoic acid (OPDA), jasmonic acid (JA) and its methyl ester methyl jasmonate (MeJA). We identified, by differential display, an Arabidopsis gene (OPR3) induced after wounding. OPR3 is homologous to members of the flavin mononucleotide (FMN) binding proteins, including the old yellow enzyme (OYE) from yeast and 12-oxophytodienoate-10,11-reductase (OPR) from Arabidopsis. Transcripts of OPR3 rapidly accumulated in leaves after wounding and MeJA treatment, but they were detected in various tissues of unwounded plants at relatively low levels. Expression of the OPR3 gene was significantly reduced in wounded leaves of the coil mutant, indicating partial dependence on jasmonate perception for full induction of the gene. The recombinant protein of OPR3 cross-reacted with an antiserum raised against the OYE protein, and showed oxidation of beta-NADPH when OPDA or 15-deoxy-delta(12,14) prostaglandin J2 (PGJ2), an analogue of OPDA, was used as substrate. Beta-NADPH oxidation was not observed when MeJA, which lacks the double bond in the ketone ring, was used as substrate. The recombinant OPR3 protein also showed beta-NADPH oxidation activity in the presence of cyclohexenone, but not cyclohexanone, suggesting that the enzyme has specificity to cleavage of olefinic bonds in cyclic enones. The results show that the OPR3 gene product represents a new OPR of Arabidopsis induced after wounding.

AtPUMP: an Arabidopsis gene encoding a plant uncoupling mitochondrial protein.

A cDNA clone (AtPUMP) encoding a plant uncoupling mitochondrial protein was isolated from Arabidopsis thaliana. The cDNA contains an open reading frame of 921 nucleotides encoding 306 amino acids (predicted molecular weight 32,708). The predicted polypeptide is 81% identical and 89% similar to the potato UCP-like protein, and includes an energy transfer protein motif common to mitochondrial transporters. The AtPUMP gene exists as a single copy in the Arabidopsis genome. The corresponding transcript was expressed in all tissues and was strongly induced by cold treatment. We suggest that the putative AtPUMP protein may play a role in heat-requiring physiological events in Arabidopsis.

Differential expression of a novel gene in response to coronatine, methyl jasmonate, and wounding in the Coi1 mutant of Arabidopsis.

Coronatine is a phytotoxin produced by some plant-pathogenic bacteria. It has been shown that coronatine mimics the action of methyl jasmonate (MeJA) in plants. MeJA is a plant-signaling molecule involved in stress responses such as wounding and pathogen attack. In Arabidopsis thaliana, MeJA is essential for pollen grain development. The coi1 (for coronatine-insensitive) mutant of Arabidopsis, which is insensitive to coronatine and MeJA, produces sterile male flowers and shows an altered response to wounding. When the differential display technique was used, a message that was rapidly induced by coronatine in wild-type plants but not in coi1 was identified and the corresponding cDNA was cloned. The coronatine-induced gene ATHCOR1 (for A. thaliana coronatine-induced) is expressed in seedlings, mature leaves, flowers, and siliques but was not detected in roots. The expression of this gene was dramatically reduced in coi1 plants, indicating that COI1 affects its expression. ATHCOR1 was rapidly induced by MeJA and wounding in wild-type plants. The sequence of ATHCOR1 shows no strong homology to known proteins. However, the predicted polypeptide contains a conserved amino acid sequence present in several bacterial, animal, and plant hydrolases and includes a potential ATP/GTP-binding-site motif (P-loop).