Regulatory Genes - Lab Report Example

How Regulatory Genes Direct Vertebrate Development Introduction The Lhx3, a homeodomain transcription factor is known to be important in the formation of pituitary gland and anterior pituitary secreting cell type’s specification (Parker et al., 2000). There are two forms of LHX3 mutations that have been identified in humans; they include, firstly, an intragenic deletion that results to truncated proteins that have no DNA binding domain. Secondly, a missense mutation that involves changing of tyrosine to cysteine (Parker et al., 2000). In order to determine if mutations in the LXH3 gene have an effect on the LHX3 ability to bind to DNA promoter and reporter genes, the plasmids containing wild type and mutant type were constructed then later cultured with human embryonic kidney 293T cells. Half of each cell type was cultured with a plasmid that had alpha GSU promoter and luciferase gene. The remaining culture cells were cultured with plasmids that contained a synthesized luciferase reporter gene. The first objective of the experiment was to find out if mutations in the LXH3 gene can interfere with LHX3a and LHX3b’s ability to binding to DNA promoters and reporter genes. It is known that LHX3a has the ability of binding to alpha GSU promoter but LHX3b doesn’t. In case mutant type or wild type LHX3 isoforms activate the alpha GSU promoter after binding on it, there will be a transcription of luciferase gene and its activity can be detected as well as measured in the cells.LHX3 is known to have the ability of activating transcription from the anterior pituitary hormone gene promoters, and it can either be in synergy with other regulatory proteins or alone(Sloop e al.,1999).Two LHX3LHX3b and LHX3a isoforms in humans are seen to be generated from LHX3 gene(Schmitt et al.,2000) Similarly, if the mutant or wild type binds to the LHX3 binding sites to activate the luciferase gene transcription, its activity can be measured to show the functioning of the LHX3 isoforms. The second objective of the experiment was to examine the effect of the mutations on the abilities of the LHX3 isoforms to bind with the protein partners. In this case, it is as well known that LHX3 plays a role in a cell by binding selected proteins referred to as protein partners and they include NLI and Pit-1.Mutations in the LHX3 gene have been seen to be related with novel,severe form of CPHD(Netchine et al.,2000). The hypothesis tested for the first experiment is mutations in the LXH3 gene can interfere with LHX3a and LHX3b’s ability to bind to DNA promoters and reporter genes. The hypothesis of the second experiment is mutations in the LHX3 gene can have an effect on the LHX3b’s and LHX3a abilities of binding to protein partners including NLI and Pit-1. Method For the first experiment, the plasmids containing the mutant and wild type LHX3 and LHX3b isoforms genes were constructed and then cultured with human embryonic kidney 293T cells. The controls were set up by culturing the cells with empty DNA vector plasmids. In this case, the plasmids were taken up by the cells to express the genes encoded to these plasmids. There were seven cells of different types that contained the plasmids, they included Control cells, LHX3bY116C(point mutation in LHX3b),LHX3 3a(wild type),LHX3aY111C(point mutation in LHX3a),LHX3a” HD(deletion in the LHX3a), LHX3bHD(deletion in LHX3b)HLX3b(wild type).Half of the cells in every cell type was taken then cultured with a plasmid with alpha GSU promoter and luciferase gene.LHX3a is known to bind to the alpha GSU promoter but LHX3b doesn’t. The remaining cells from the 7 cell types were cultured with plasmids that had synthesized luciferase reporter gene which is preceded by 3 LHX3 binding sites. Similarly, if the mutant or wild type binds to the LHX3 binding sites to activate the luciferase gene transcription, its activity could be measured to demonstrate the functioning of the LHX3 isoforms. The results of the alpha GSU reporter gene and promoter activation by LHX3 isoforms were recorded. For the second experiment, the mutant and wild type cDNA were constructed. The cDNAs and GST genes were inserted into an expression vector. This stage was carried out to synthesize fusion proteins of LHX3a, LHX3bY116C, LHX3aY111C and LHX3b all fused to GST. Glutathione S-transferase (GST) is known to be a protein which aids in the purification and isolation of proteins of interest. The controls were set up by isolating the GST proteins only. The fusion proteins were later separated with the use of electrophoresis and the proteins were used as a substrate for the binding assay. Purified radio-labeled Pit-1 and NIL were later added to the gel that had the mutant and wild type fusion proteins .In this case, if there was binding sites(No mutation effect),the Pit-1 and NIL proteins could bind to the proteins(LHX3).The presence of the bound Pit-1 and NIL were visualized with fluorography. The binding of Pit-1 and NIL were then quantified through scintillation counting (electrophoresis gel band intensity measurement).The results of the protein-binding experiment were later recording as seen In the next section Results The first experiment aimed at determining if mutations in the LXH3 gene can interfere with LHX3a and LHX3b’s ability to binding to DNA promoters and reporter genes. As indicated in figure 1, both mutation types affected the activation of alpha GSU gene (red bars). The truncated mutations with no DNA binding domain were not able to activate the reporter gene, while on the other hand, point mutations with altered amino acid in the LHX3 protein interaction domain, had reduced activation (blue bars) Figure 1. LHX3a and LHX3b’s abilities of binding to DNA promoters and reporter genes The second experiment aimed at determining if mutations in the LHX3 gene can have an effect on the LHX3b’s and LHX3a abilities of binding to protein partners including NLI and Pit-1, in regard to protein interaction (figure 2), the LHX3 binding with protein partners, Pit-1 (blue bars) and NLI (red bars) was seen to be reduced but not eliminated by point mutations. Figure 2. LHX3b’s and LHX3a abilities of binding to protein partners including NLI and Pit-1 Discussion In regard to protein-protein interaction, there was no mutation that resulted to more than 50% decrease in the NLI binding. All LHX3 proteins (mutant and wild type isoforms proteins) indicated a substantial binding to Pit-1 and NLI. For experiment one, the deletion mutation had an effect on the LHX3a gene ability to activate both the alpha GSU promoter luciferase gene and reporter gene. Point mutation of the LHX3a gene seemed to have little effect on its capacity of activating the reporter gene than to the alpha GSU promoter. LHX3b doesn’t activate alpha GSU promoter. In this case, it was expected that the mutations not to have any effect on the activation ability of LHX3b’s to the promoter but according to the experiment results it is observed that it had an effect on the promoter. The alpha GSU gene encodes a common subunit of pituitary hormones TSH, FSH and LH. Basing on this, the function of LHX3a can be predicted. The results support the hypothesis and it can be concluded that mutations in the LHX3a genes have an effect on the activation ability and interaction with the NIL and Pit-1 proteins of the LHX3 isoforms. References Netchine, I, Sobrier, M.L., Krude, H., Schnabel, D., Maghnie, M., Marcos, E., Duriez, B., Cacheux, V., Moers, A., Goossens, M., Gruters, A.,Amselem, S. (2000). Mutations in LHX3 result in a new syndrome revealed by combined pituitary hormone de®ciency. Nat. Genet. 25, 182±186. Parker,G.E., Sandoval, R.M., Feister, H.A., Bidwell, J.P., Rhodes, S.J. (2000). The homeodomain coordinates nuclear entry of the Lhx3 neuroendocrine transcription factor and association with the nuclear matrix. J. Biol. Chem. 275, 23891±23898. Schmitt,S.,Biason-Lauber, A., Betts, D., Schoenle, E.J. (2000). Genomic structure, chromosomal localization, and expression pattern of the human LIM-homeobox3 (LHX 3) gene. Biochem. Biophys. Res. Commun. 274, 49±56. Sloop, K.W., Meier,B.C., Bridwell, J.L., Parker, G.E., Schiller, A.M., Rhodes, S.J. (1999). Differential activation of pituitary hormone genes by human Lhx3 isoforms with distinct DNA binding properties. Mol. Endocrinol. 13, 2212±2225. Read More