Alu Insertion Element Inhibit The GFP Gene Expression

Objective: The research of the human genome reveals that non-coding DNAs occupy as much as 98% of the human genome and 50% of the non-coding DNAs are composed of repetitive elements. This fact has not been clearly explained. Over the last years, several lines of evidence demonstrated that the repetitive elements were associated with various biology phenomena, such as gene transposition, gene mutation, autoimmune diseases and primate evolution. Alu belongs to the SINE family (Short Interspersed Nuclear Elements) of repetitive elements. It is present in more than one million copies which altogether represent 10% of the whole genome mass. Recently, several lines of evidence showed that the presence of repetitive elements and especially of Alu element had a great influence on the human genome, in particular on its evolution. Although the research of repetitive elements had obtained greater progress, further work would be required fully to understand their function and mechanism on gene expression. In this research, a series of recombinant plasmids were constructed by inserting 1,2,4,8,14 tandem-repeated Alu sequence respectively into the downstream of GFP and inserting SV40 early mRNA polyA (240bp)in antisense orientation into GFP downstream and Alu14 upstream. Then we transfected these recombined vectors into HeLa cell and observed the expression of green fluorescent protein in 24 hours. We focused on the effects of downstream different -length Short Interspersed Elements on upstream gene expression and accumulated the experimental data for function of Alu.Method: Restriction sites of pEGFP-C1, which were not contained in Alu were determined by using analysis software. We used EcoRⅠand KpnⅠas Alu insert site, XbaⅠand NheⅠas Alu connected site because of its same compatible terminal. Firstly, NheⅠrestriction sites located in upstream of pEGFP-C1 MSC is deleted. Forward primer containing EcoRⅠ/XbaⅠand reverse primer containing KpnⅠ/NheⅠwere designed .Alu fragment of 283 bp was amplified from RP11-29107clone by PCR. To combine pEGFP-C1 with an Alu target on one plasmid, we created the following constructs. pEGFP-C1 and an Alu target were digested by EcoRⅠand KpnⅠto generate EcoR-Kpn fragments and these two fragments were combined by T4 DNA ligase. Then the product ligased was transformed into DH5a.Ecoli. After positive kanamycin resistant colony was picked up, the recombinant plasmid was identified by restriction. The resulting construct was named p-Alu1. We combined long fragment excised from p-Alu1 by HindⅢ- XbaⅠdigestion with short fragment excised from p-Alu1 by HindⅢ-NheⅠdigestion, and gained p-Alu2 recombinant plasmid. The construction of p-Alu4/p-Alu8 is just as above. Using T4 DNA Ligase, short fragment excised of p-Alu2 plasmid by HindⅢ-NheⅠdigestion was inserted into long fragment excised from p-Alu4 by HindⅢ- XbaⅠdigestion. Then p-Alu6 was gained.P-Alu14 recombinant plasmid was gained just as above. Finally, these five recombinant plasmids and p-polyAas-Alu14(reserved in our laboratory),pEGFP-C1 were transfected respectively into HeLa cells. After 24 hours, GFP expression was observed in fluorescence microscopy and counted.Results: 1 Five recombinant plasmids had successfully been constructed. Electrophoresis patterns of recombinant plasmids identified by restriction analysis were showed in Fig.4, Fig.5.Fig.6, Fig.7 is the result of sequencing of p-polyAas-Alu14.2 The Green Fluorescent Protein Transient expression fluorescence picture and counting result of HeLa cells transfected with recombinant plasmid in 24 hours (Tab.Fig.8 Fig.9).The mean of positive fluorescence cell are p- Alu117.4±0.7%,p-Alu2 13.7±1.31%,p- Alu4 10.2±0.41%,p-Alu8 5.6±0.27%,p-Alu14 0.07±0.16%,p-polyAas-Alu14 10.0±0.26%和pEGFP-C1 35.3±2.66%.Conclusions: 1 p-Alu1,p-Alu2,p-Alu4,p-Alu8 and p-Alu14 had successfully been constructed.2 Alu sequences inserted into GFP downstream inhibited the expression of GFP gene, which gradually were enhanced as it encountered more Alu sequences. 3 This effect of inhibitory was not due to more insertions.