 |
| About Bioline | All Journals | Testimonials | Membership | News |
|
||||||
|
||||||
Tsinghua Science and Technology, Vol. 6, No. 3, August 2001 pp. 265-268 Genomic Organization and Expression in E. coli of Zebrafish Terra* ZHAO Zhixing Institute of Cellular and Developmental Biology, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China *Supported by the National Natural Science Foundation of China (No.39970360
and No.30025020) and the National Key Basic Research and Development Program
(No.G19990756) Received: 2000-09-22 Code Number: ts01079 Abstract: Zebrafish terra encodes a transcription factor that is specifically expressed in developing somites. Previous studies suggested that this gene is involved in vertebrate somitogenesis. In this study, the genomic DNA of terra locus was isolated and its organization was investigated. The analysis showed that terra locus consists of 3 introns and occupies 3154 bp in the genome of zebrafish. The exon-intron junctions of the second and third introns conform to the GT-AG rule, while the first intron has the unusual junction sequences of GT-AC. An IPTG-inducible expression system was established to produce terra protein in bacterial cells. Overexpression of terra protein leads to the formation of inclusion bodies in the bacterial cells. The protein will be used to study its structure and function. Key words: zebrafish; terra; intron; exon; expression; bacterial cells Introduction The Terra is a zebrafish gene encoding a zinc-finger transcription factor[1]. The DNA binding domain of terra protein shares high homology with that of several sexual regulators, e.g., Doublesex in Drosophila[2] , Mab-3 in C. elegans[3] , and DMRT1 and DMRT2 in mammals[4-6]. However, the function of terra is different from those sexual regulators since Terra has a unique expression pattern. Expression of Terra is restricted to the presomitic mesoderm and newly formed somites, suggesting that it plays a role in somitogenesis rather than in sex determination in zebrafish. Human and mouse homologs of Terra have been found and mouse Terra is also specifically expressed in somites, indicating that terra is evolutionarily conserved[1]. This study characterizes the genomic structure of zebrafish Terra locus. In addition, a bacterial expression system was established for zebrafish terra, which made it possible to obtain sufficient amounts of terra protein for further functional studies. 1 Materials and Methods 1.1 Isolation of Terra locus-containing PAC clones A zebrafish genomic PAC library, constructed at the Boston University School of Medicine, was purchased from GenomeSystems, Inc. It contains 100 000 clones with an average insert size of 115 kb, which theoretically covers the zebrafish genome 3-4 times[7]. A 503-bp fragment of Terra cDNA, which contains a presumptive DNA binding domain, was obtained by EcoiRV digestion. This fragment was labeled with 32 P-dCTP and used as a probe to screen the library. Post-hybridization wash stringency was 1xSSC, 0.1% SDS, 60 °C . DNA isolation from the positive PAC clones followed the standard plasmid DNA isolation procedure. The positive clones were confirmed by sequencing using Primer1. 1.2 Identification of introns Ten primers (P1-P10) were synthesized, each containing 20-24 nucleotides. Positions and directions of these primers in cDNA are drawn schematically in Fig.1(a). Their sequences are as follows: P1: 5'-ATCAGGTTGTCTAAATGTCGC-3'; Primers were paired to amplify, by PCR, a specific region of the genomic DNA in one of the positive clones, PAC7. PCR fragments were run on 1% agarose gel to check their sizes. Fragments that might contain introns were subcloned into pT-Easy vector (Promega) and then sequenced to define exon-intron boundaries. 1.3 Generation of an expression construct Two primers, terra5' NcoI (5'-CATGCC[ZZ(Z]ATGGA-TCTGTCCGGCACGGAG[ZZ)]-3') and terra3' BamHI (5'-CGCGGATCCTTA[ZZ(Z]CTGAGATTTCCGATT-TAAAG[ZZ)] -3') (coding sequence was underlined), were used to amplify the open reading frame of Terra by PCR. The PCR product was digested with NcoI and BamHI, and then inserted into NcoI/BamHI sites of a bacterial expression vector pET15b to generate recombinant plasmid pET15-terra. The pET15-terra was amplified in the E. coli strain XL-1blue and the DNA was extracted using the standard procedure for plasmid DNA isolation. 1.4 Expression of terra in E. coli The pET15-terra DNA was transformed into an E. coli strain BL21 that was able to produce T7 RNA polymerase upon induction of IPTG. Four colonies were picked for DNA isolation and confirmed by restriction enzyme digestion. The pET15-terra/BL21 was cultured at 37 °C in LB with 100 mg/mL ampicillin. IPTG was added to the culture to a final concentration of 1 mmol/L when the cell density reached an OD600 of 0.5. Culturing continued to allow accumulation of terra protein for various durations. At different times, aliquots of the induced culture were removed and the proteins were checked on 15% SDS-PAGE gels. To investigate the form of terra protein present in the E. coli cells, cells from the induced culture were resuspended in 10 mmol/L NaH2PO4/NaH2PO4 (pH8.0), 10 mmol/L EDTA, 100 mmol/L NaCl, 300 mg/mL lysozyme, and disturbed in an ultrasonic generator. The treated sample was spun at 15 000 r/min at 4 °C for 20 min . The supernatant and the pellet were analyzed by SDS-PAGE. 2 Results and Discussion 2.1 Zebrafish Terra contains three introns A total of 12 positive PAC clones were identified by screening a zebrafish PAC library with a fragment derived from zebrafish Terra cDNA. One of the positive PAC clones, PAC7, was confirmed by sequencing to contain Terra locus and was then used as a template to identify introns. Ten primers which are complementary to specific regions of zebrafish Terra cDNA were synthesized (Fig.1(a)). These primers were combined in pairs to amplify appropriate regions of the Terra locus in PAC7 and the amplified products were analyzed on agarose gels. An amplified fragment was expected to contain one or more introns if its size was bigger than that of the corresponding cDNA region. For instance, the fragment amplified using P1 and P2 was about 500 bp while the distance between the first 5' nucleotides of P1 and P3 was only 214 bp on the cDNA (Fig.1(b)). This indicates that the amplified fragment should contain at least one intron. Subcloning and sequencing of this fragment confirmed that it contained a single intron of 280 bp, being the first intron of Terra locus. This intron was located in the 5' untranslated region. The fragment obtained with P2 and P6 was about 1300 bp in length, which was bigger than the expected length (470 bp). Sequencing revealed that this fragment contained two introns, i.e., the second and third introns of the Terra locus, which were 406 bp and 427 bp, respectively. The amplification with P5 and P9 or with P7 and P10 generated products of the expected size, suggesting that the region between P5 and P10 contains no more introns. The above results were also confirmed by PCR with the other primer pairs (Fig.1(b)). Therefore, zebrafish Terra locus consists of 4 exons and 3 introns (Fig.2(a)), which spans 3154 bp in the genome from the first 5' nucleotide of its cloned cDNA to the nucleotide immediately adjacent to the poly (A) tail. 2.2 Exon/intron junctions do not always conform to GT-AG or AT-AC rules Introns 2 and 3 of zebrafish Terra have GT dinucleotides at the 5' end and AG dinucleotides at the 3' end. They belong to the major class of introns which utilize the U2-dependent splicing system[8]. The first intron of Terra locus has unusual exon-intron junctions: GT and AC at its 5' and 3' ends, which conform neither to the GT-AG rule for the major intron class nor to the AT-AC rule for the minor AT-AC class of introns[9]. Whether GT-AC introns represent a new rare intron class remains unknown. Sequencing analysis revealed that introns 1, 2, and 3 have an AT content of 76.43%, 67.24%, and 67.68%, respectively, indicating that these introns are AT-rich. The intronic AT-richness is common in many organisms, including Tetrahymena, Drosophila melanogaster, Caenorhabditis elegans, vertebrates and plants. It has been demonstrated in plants and Drosophila that the intronic AT-richness affects the selection of splicing sites and the splicing efficiency[10-12]. 2.3 Terra protein can be overexpressed in E. coli To facilitate the study of the structure and function of zebrafish Terra, we intended to produce Terra protein in an E. coli expression system. The putative coding sequence of Terra was cloned into an expression plasmid vector pET-15b that contained the T7 promoter. The resultant recombinant plasmid pET-terra (Fig.3(a)) was introduced into strain BL21 that synthesizes RNA polymerase upon IPTG induction. As shown on SDS-PAGE gel (Fig.3(b)), the intensity of a protein band increased significantly 2 hours after induction. The size of this band matched the predicted 56 x 103 relative molecular mass of the terra protein, suggesting the success of terra expression in E. coli. It was noted that the amount of the induced terra protein did not substantially increase with extended culturing periods, and that the terra accounted for less than 10% of the total cellular proteins, suggesting that culturing and inducing conditions need to be optimized to maximize the terra production. Overexpression of a protein in prokaryotes often leads to the production of inclusion bodies that are insoluble aggregates of misfolded protein. To check whether the terra protein produced in E. coli was present in the form of inclusion bodies, the induced cells were collected and subjected to ultrasound sonication in lysis buffer and centrifugation. Proteins in the pellet and the supernatant were examined on SDS-PAGE gels. As shown in Fig.4, the expected protein is mainly in the pellet, and it is enriched when the pellet was washed and centrifugated again. The terra protein expressed in the bacterial cells is being purified and will be used to make a specific antibody by immunizing mice. The antibody against terra could be injected into zebrafish eggs, which may block activity of endogenous terra. References
Copyright 2001 - Tsinghua Science and Technology The following images related to this document are available:Photo images[ts01079f4.jpg] [ts01079f3.jpg] [ts01079f1.jpg] [ts01079f2.jpg] |
| |||||||||
,
HUA Zhengchun 
,
MENG Anming 
**
