We isolated the matrix attachment region-binding protein (MBP) DMBP-1 from
Genome project results show that only 5% to 10% of genome DNA sequences are coding regions in the genomic DNA of all eukaryotes. Most of the DNAs are noncoding and confer a large number of important regulatory functions, such as promoter, enhancer, and miRNA [
Matrix attachment region DNAs (MAR) are DNA sequences that bind to nuclear matrices after being digested by restriction enzymes. MARs consist of AT-rich sequences that extend from 200 bp to 2000 bp and contain various AT-rich and structural motifs, such as A-box (AATAAAYAAA), T-box (TTWTWTTWTT), base-unpairing regions, and intrinsically curved portions [
The mechanism of the MAR regulatory function is unclear. Experimental evidence that explains this function of MAR is lacking because the transacting factor that binds to MAR has not been discovered. We isolated one MAR-binding protein (MBP), namely, DMBP-1 (GenBank Accession nos. DQ124215, AAZ31075), through
In the present study, we screened the protein that can interact with DMBP-1 using yeast two-hybrid systems and the GST pull-down technology. We also provided a basis for the study of the MAR regulatory mechanism.
The cells of
The DNA (1623 bp) fragment coding for
The yeast strain AY190 was transformed with pGBKT-MBP through the lithium acetate method based on the instructions on the kit (Clonetech) to check for self-activation. The transformants were diluted at 1 : 10 and 1 : 1000. These transformants were then smeared on an SD/-Trp plate and cultured at 30°C for 72 h until single bacterial colonies appeared. The single colonies were picked and cultured in an SD/-Trp liquid medium overnight. The plasmids were extracted from the yeast and were identified through PCR by using the plasmid as a template.
One single colony (2 mm to 3 mm) was picked and cultured in a 50 mL SD/-Trp/Kan (20
The
The yeast cells of the Y190 strain that harbored pGBKT-MBP were transformed with 30
The single colonies (>2 mm diameter) were picked from the selective media SD/-Trp/-Leu/-His/-Ade. These single colonies were then transferred to a new SD/-Trp/-Leu/-His/-Ade/X-
A second round of selection on the same medium was performed, and the valid AD plasmids were removed. The colonies that activated both reporter genes in the Y190 strain were further analyzed. The AD-cDNA plasmid that encoded the interaction protein was isolated from the yeast cells using a yeast plasmid isolation kit (Omega, USA). The selected positive AD plasmids were transformed into
A partial cDNA that encodes a peptide that interacts with the DMBP-1 of
The primer was designed and synthesized for 5′-RACE of DMBP-2 based on the sequencing result of the 3′cDNA end. About 5
The full-length cDNA encoding DMBP-1 and DMBP-2 were amplified through PCR and subcloned into pGEX-4T-1 (Amersham Biosciences, USA), respectively. The recombinant proteins GST-DMBP-1, GST-DMBP-2, and protein GST were individually expressed in an
DMBP-1 gene was amplified through PCR technology using the plasmid as template and the corresponding primers and was identified through enzyme digestion and sequencing. The gene was then cloned into a pGBKT-7 vector to form the “bait” pGBKT-MBP plasmid.
The pGBKT-MBP and pGBKT-7 plasmids were transformed into yeasts Y187 and AH109, respectively. The results showed that these plasmids could only grow on an SD/-Trp/X-
The “three-leaf grass type” heterozygote was visible under microscope observation 20 h after yeast hybridization (Figure
(a) The diploid zygote of the yeast (400x); (b) primary screening of the clone (blue for positive clones).
The positive colonies were identified using the library plasmid universal primers (T7 and 3′AD). The length of the inserted DNA fragments was then determined (Figure
(a) PCR product of positive clones (AD Primer). M: Marker DL2000; 1–5: PCR product. (b) Additional yeast two-hybrid test for the positive clones. A: positive clones; B: negative control (pGBKT-7); C: negative control (pGBKT7-Lam).
The seven positive colonies obtained were sequenced and analyzed through
The 5′-upstream sequences of the selected cDNA fragments were determined using RACE technology. Only one gene (PA2) was successfully cloned. The other two were not cloned, possibly because the genes were induced under strong light. The new cloned gene was named DMBP-2 (GenBank no. JN006968).
The 823 bp DNA band was amplified through PCR. The sequencing result of this sequence showed that the cDNA of DMBP-2 possessed a 573 bp ORF from 68 bp to 640 bp of the sequence, which did not include 67 bp 5′UTR and 183 bp 3′UTR. The 3′UTR possessed a typical polyadenylation signal TGTAA at position 45 upstream of the poly (A)-tails (Figure
Nucleotide sequence of the gene encoding the DMBP-2 protein from the green unicellular alga
The DMBP-2 sequence was further confirmed through RT-PCR and sequencing. The results showed that the sequence was correct. We performed the sequence alignments through NCBI Blast (
The GST pull-down approach was used to validate the interactions between the newly identified DMBP-2 and DMBP-1. The DMBP-2 protein was mixed with glutathione agarose bound to either a GST protein or to GST-DMBP-1 fusion proteins. The unbound proteins were removed, and the remaining bound proteins were detected. The results showed that GST-DMBP-1 and the GST protein were bound to the glutathione beads (Figure
GST pull-down assay demonstrating the in vitro binding between DMBP-1 and DMBP-2. Purified His-DMBP-2 protein was added to GST. GST-DMBP-1 was immobilized on glutathione sepharose beads. His-DMBP-2 fusion protein at 10% was loaded as input. The interacting complexes were subsequently eluted and separated by SDS-PAGE (12%). (a) The immunoblot with monoclonal anti-GST antibody showed the presence of the GST-DMBP-1 fusion protein co-eluting with His-DMBP-2 from the glutathione sepharose beads. (b) The immunoblot with monoclonal anti-His antibody showed the presence of His-DMBP-2 coeluting with GST-DMBP-1 from glutathione sepharose beads. The presence of His-DMBP-2 in the input was also confirmed.
Some MAR-binding proteins have been isolated from different organisms in the past few years. These proteins are divided into several categories, such as MAR-binding filament-like protein, AT hook-containing MAR-binding protein, and nucleolar protein family. However, these proteins are isolated from higher plants and animals and have been demonstrated to regulate gene expression, influence cell development, induce cell apoptosis, and participate in chromosome assembly [
Two issues on the study of MAR-binding protein exist. First, the organisms are obtained from higher plants and animals and have tissue differentiations. The gene expression manner and the kinds of tissues are different. The MAR-binding proteins could also be different. Therefore, single-cell eukaryotes may be more suitable in studying the MAR-binding protein. Second, current studies mainly focus on a single isolated MAR-binding protein. Trans-acting proteins, which are protein clusters that contain different proteins, are ignored. Previous research has demonstrated that trans-acting proteins contain more than one protein and interact with each other to fulfill their regulatory functions. For example, the transcription factors include several kinds of proteins. Therefore, we propose the isolation of MBP from single-cell eukaryotes and the further study of the regulatory mechanism of MBP.
Yeast two-hybrid, which was first applied by Fields and Song [
Strict screening and hybridization were repeatedly performed. We then obtained six positive library plasmids, which possibly encoded the proteins that could interact with DMBP-1. The screened proteins include the nagariensis photosystem I reaction center subunit VI and the photosystem I P700 chlorophyll a apoprotein A1 ATP synthase CF0 C subunit. The full-length of one gene was cloned through 5′-RACE and possessed 573 bp ORF from 68 bp to 640 bp of the sequence. The sequence alignments show that the sequence is highly similar to that of the high light-stressed
We performed in vitro binding assays using GST pull-down assays to further confirm the specific and direct interactions between DMBP-1 and DMBP-2.
In summary, this study screened an MBP that interacts with DMBP-1 by establishing a link between DMBP-1 and DMBP-2. DMBP-1 was found to be a potential candidate in investigating the molecular mechanisms of MBP in regulatory functions.
The authors declare that they have no conflict of interest.
This work was supported by Grants from the National Natural Science Foundation of China (no. 30970055), the Natural Science Foundation of Department of Education, Henan Province, China (no. 12A310005), and Xinxiang Medical University, China (ZD2011-17).