Please use this identifier to cite or link to this item: https://scholarbank.nus.edu.sg/handle/10635/170572
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dc.titleCHARACTERISATION OF A NOVEL HUMAN GRP78 GENE AND TRANSCRIPTIONAL REGULATION OF THE HUMAN HSP70B' GENE
dc.contributor.authorPHANG SENG MENG
dc.date.accessioned2020-06-22T05:24:37Z
dc.date.available2020-06-22T05:24:37Z
dc.date.issued1994
dc.identifier.citationPHANG SENG MENG (1994). CHARACTERISATION OF A NOVEL HUMAN GRP78 GENE AND TRANSCRIPTIONAL REGULATION OF THE HUMAN HSP70B' GENE. ScholarBank@NUS Repository.
dc.identifier.urihttps://scholarbank.nus.edu.sg/handle/10635/170572
dc.description.abstractScreening of a human T-cell genomic library in A2001 for GRP78 genomic clones using its corresponding 3'-end 900 bp cDNA, GS, yielded six positive clones. These were divided into 2 classes based on BamH I restriction analysis. One clone from each class was further characterized by limited end sequencing. The analysis showed that one of clones, GR2, was identical to another reported pseudogene hU3-2B. The other clone, GR4, was different from the previously reported GRP78 genomic clones, hU28-1 and hU3-2B based on sequence and restriction analysis. Southern hybridisation of PstI-digested genomic DNA with sequence-specific oligonucleotide probes to GR4 and hU28-1 also supports that there are three GRP78 genes in the genome, hU28-1, GR4 and hU3-2B. Northerns and PCR-based southern hybridisation in a variety of cell lines and tissues suggest that hU28-1, but not GR4, is expressed. A search for ORFs for GR4 showed numerous nonsense codons dispersed along its amino acid sequence. These all indicate that GR4 is a pseudogene which apparently has distinctive features different from the processed pseudogene, hU3-2B. GR4 pseudogene may therefore not have originated in the same manner as hU3-2B. Previous work have mapped the GRP78 gene to chromosome 9. However at the time of the localisation it had not been established that there are three different GRP78 genes. This study showed that this GRP78 gene is the functional gene hU28-1. The pseudogene GR4 is on chromosome 4 whilst the other pseudogene hU3-2B is on another chromosome. The members of this gene family are all located on different chromosomes. The pseudogene GR4 was found to be polymorphic for a Pst I restriction site located 3.5 kb downsteam from an internal Pst I site located within the gene. This occurs at a frequency of 14/18 in an Asian population. Of the HSP70 gene family, the HSP70B' or HSPA6 represents a special member with no detectable expression under non heat-shock conditions. This study was centred specifically upon one genetic element which may be responsible for the expression pattern of the HSPA6 gene under non-stressed conditions. A repressor-like element, HRE, was identified by DNase I footprinting of the promoter using non heat-shock and heat-shock HeLa nuclear extract. Gel-retardation assay using oligonucleotides corresponding to HRE furthur indicated that the factor, HRF, which binds to this sequence occurs exclusively in the non heat-shock extract. A four base change in I-IRE sequences abolished binding to the HRF. When this change was introduced into the wildtype promoter linked to the CAT gene as reporter, a four-fold increase in expression under non heat-shock conditions was observed, suggesting that HRF-HRE complex may be partly responsible for the control of basal level of expression of HSP70B' gene in non-stressed conditions.
dc.sourceCCK BATCHLOAD 20200626
dc.typeThesis
dc.contributor.departmentINSTITUTE OF MOLECULAR & CELL BIOLOGY
dc.description.degreeMaster's
dc.description.degreeconferredMASTER OF SCIENCE
Appears in Collections:Master's Theses (Restricted)

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