ABSTRACT
<p><b>OBJECTIVE</b>To analyze the expression of genes from chromosomal region 22q11.2 and assess the association between mutation(s) of particular gene(s) from this region and malformations of the urinary system.</p><p><b>METHODS</b>Expression of rat homologs of 33 genes from above region was determined in kidney tissues derived from rats of different fetal development ages (E13, E15, E19) and adulthood with reverse transcriptase-PCR. Potential mutation(s) in candidate gene SNAP29, whose expression pattern appeared to be unique, was screened in 44 patients and 220 normal controls with PCR-single strand conformation polymorphism (SSCP). Suspected positive regions were sequenced to verify the mutations.</p><p><b>RESULTS</b>Nine genes showed no expression throughout the whole development process; 18 genes with various expression levels showed continuous expression from the beginning of development; 6 genes only expressed for a short time, among which SNAP29 was selected for mutation screening. Upon sequencing, three mutations were identified from the 44 patients, including a G to A transition (GAG to AAG) in exon 2, and two A to G transitions (AGC to GGC) in exon 3.</p><p><b>CONCLUSION</b>Through systematic analysis of the expression of genes from chromosomal region 22q11.2, the SNAP29 gene was found to have a potential role in the development of genitourinary system. Two missense mutations were identified in three patients. These included one in exon 2 (featuring cryptorchidism), and the other in exon 3 (featuring cryptorchidism and hypospadia). Neither of the mutations was found in the normal controls. The results suggested that mutation(s) of gene(s) from chromosomal region 22q11.2 may play an important role in the genesis of genitourinary malformations.</p>
Subject(s)
Animals , Female , Humans , Male , Mice , DNA Mutational Analysis , Methods , Exons , Genetics , Membrane Glycoproteins , Membrane Proteins , Genetics , Platelet Glycoprotein GPIb-IX Complex , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Polymorphism, Single-Stranded Conformational , Qb-SNARE Proteins , Genetics , Qc-SNARE Proteins , Genetics , Urogenital Abnormalities , GeneticsABSTRACT
Clostridial neurotoxin inhibits neurotransmitter release by selective and specific intracellular proteolysis of synaptosomal associated protein of 25KDa [SNAP-25], synaptobrevin/VAMP-2 and syntaxin. SNAP-25 is one of the components that forms docking complex in synaptic ends. This protein is subtrate for botulinum neurotoxins types A,C, and E. Each of these toxin serotypes specifically cleaves SNAP-25 in a particular position and thereby block docking and synaptic vesicle membrane fusion and finally prevents neurotransmitter exocytosis and transition of neurotic signals. Recombinant production of SNAP-25 in the laboratory can be used as a subtrate for the detection of clostridium botulinum types A, and E neurotoxins. In order to use the protein as a subtrate for detection of different types of clostridium neurotoxins in-vitro the protein was produced by recombinant technique. The cDNA from SNAP-25 was synthesized from total RNA purified from frozen Rattus norvegicus brain. and amplified by RT-PCR The amplified fragment was cloned into pET32a expression vector. The identity of recombinant protein was confirmed by Western blot using specific antibody and finally the recombinant protein was purified through an affinity column chromatography [Ni-NTA]. The optimum conditions of expression of SNAP-25 were found to be IPTG[1mM] and incubation at 37°C for 5 hours. The recombinant protein was isolated and purified using Ni-NTA column with imidazole at a concentration of 25OmM. Using enterokinase to cut the fision at 37°C comparatively yielded better results than room temperature. The protein retained its structure during the purification process being suitable for cutting and further tests. The purified protein we obtained can be used as subtrate for detection of clostridium botulinum types A, and E toxins