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PubMed1990

Translational regulation of complement protein C2 expression by differential utilization of the 5'-untranslated region of mRNA.

A recently isolated cDNA for human complement protein C2, C2HL5-3, has an unusually long 5'-untranslated region (UTR) containing four open reading frames upstream of the authentic initiation codon. Here we report that deletion of the 5'-UTR of C2HL5-3 resulted in a 10-fold enhancement in the translational efficiency of C2 in transient eukaryotic cellular assays. Elimination of the open reading frames in the 5'-UTR by site-directed mutagenesis of the initiation codons did not affect C2 synthesis in this assay system, indicating that other structural elements in this region are responsible for translational control. We also show that several C2 mRNAs of varying length in the 5'-UTR, including an alternative C2 mRNA expressing a considerably shorter 5'-UTR, are present in the liver and that the shorter C2 mRNA is the only C2 message found in U937 cells. These results suggest that tissue-specific utilization of alternative transcriptional start sites results in differential translational efficiencies of C2 and may provide insights into the tissue- and stimulus-specific regulation of eukaryotic gene expression.

Base Sequence↗
PubMed1994

Starting window, a distinct element in the cap-independent internal initiation of translation on picornaviral RNA.

Initiation of translation on picornaviral RNA templates occurs via cap-independent ribosome binding to a cis-acting element, internal ribosome entry site (IRES). Mapping of the starting point of translation relative to the IRES was attempted using Theiler's murine encephalomyelitis virus (TMEV) RNA as a model. The possibility that the starting point is determined by the conserved oligopyrimidine upstream of the initiator codon was studied. In contrast to poliovirus, neither the conserved oligopyrimidine nor an AUG at a fixed distance downstream of this oligopyrimidine are required for efficient translation of the TMEV RNA in Krebs-2 extracts or reticulocyte lysates or for viral infectivity; mutants lacking the oligopyrimidine/AUG tandem were stable upon passage in BHK-21 cells. A short template segment, the starting window, was defined, wherefrom ribosomes begin translation or downstream scanning depending, respectively, on the presence or absence of a good-context AUG within this window. Using a collection of the engineered TMEV mutant RNAs, the starting window was mapped to 16-17 nt downstream of the IRES and was found to be approximately a dozen nt long. The efficiency of translation initiation from an AUG linearly increased upon the 5'-->3' displacement of the initiator codon within the window. The competence of the starting window did not appear to depend markedly on its primary structure; however, it was completely inactivated ("closed") with concomitant dramatic inhibition of total protein synthesis upon conversion of the corresponding RNA segment into a double-stranded form.

Base Sequence↗
PubMed1988

Site-directed mutagenesis of potential protein-coding regions in expressible cloned cDNAs of cucumber mosaic viral satellites.

Site-directed mutagenesis was used to alter potential protein-coding regions (open reading frames or ORFs) within the cloned, expressible cDNAs of two satellites of cucumber mosaic virus (CMV), S-CARNA 5 and D-CARNA 5. RNA transcripts synthesized in vitro from the mutant and wild-type satellite cDNAs were tested for biological activity on tomato plants by coinfection with CMV RNAs 1, 2, and 3, and progeny CARNA 5s generated from such transcripts were isolated and sequenced. Two mutants of S-CARNA 5 were constructed in attempts to test whether the ORF (ORF IIB, beginning at nucleotide 135) responsible for synthesis in vitro of two small proteins (M. J. Avila-Rincon, C. W. Collmer, and J. M. Kaper (1986). Virology 152, 455-458) could be eliminated without loss of the satellite's biological activity. Biological tests with mutant transcripts that either lacked the AUG initiation codon or contained a premature translation termination codon were foiled by instability and/or reversion in the progeny. With progeny CARNA 5 of the former mutant, one of two altered nucleotides reverted, thus restoring the AUG but not the nucleotide immediately preceding it. In contrast, a mutation in the necrogenic D-CARNA 5 which altered the initiation codon of ORF I (nucleotides 11-94), whose predicted amino acid sequence is conserved in all necrogenic CMV satellites sequenced to date, was stable in biological testing and did not destroy necrogenicity. This was shown by nucleotide sequencing of the progeny CARNA 5 from necrotic test plants and by its direct comparison with wild-type D-CARNA 5 progeny in a tomato necrosis dilution assay. This experiment provides convincing evidence that a possible protein product of ORF I is not involved in the induction of tomato necrosis by D-CARNA 5.

Codon↗
PubMed2005

The HIV-1 leader RNA conformational switch regulates RNA dimerization but does not regulate mRNA translation.

The untranslated leader RNA is the most conserved part of the human immunodeficiency virus type I (HIV-1) genome. It contains many regulatory motifs that mediate a variety of steps in the viral life cycle. Previous work showed that the full-length leader RNA can adopt two alternative structures: a long distance interaction (LDI) and a branched multiple-hairpin (BMH) structure. The BMH structure exposes the dimer initiation site (DIS) hairpin, whereas this motif is occluded in the LDI structure. Consequently, these structures differ in their capacity to form RNA dimers in vitro. The BMH structure is dimerization-competent, due to DIS hairpin formation, but also presents the splice donor (SD) and RNA packaging (Psi) hairpins. In the LDI structure, an extended RNA packaging (Psi(E)) hairpin is folded, which includes the splice donor site and gag coding sequences. The gag initiation codon is engaged in a long distance base pairing interaction with sequences in the upstream U5 region in the BMH structure, thus forming the evolutionarily conserved U5-AUG duplex. Therefore, the LDI-BMH equilibrium may affect not only the process of RNA dimer formation but also translation initiation. In this study, we designed mutations in the 3'-terminal region of the leader RNA that alter the equilibrium of the LDI-BMH structures. The mutant leader RNAs are affected in RNA dimer formation, but not in their translation efficiency. These results indicate that the LDI-BMH status does not regulate HIV-1 RNA translation, despite the differential presentation of the gag initiation codon in both leader RNA structures.

Base Sequence↗
PubMed2002

The large subunit of initiation factor aIF2 is a close structural homologue of elongation factors.

The heterotrimeric factor e/aIF2 plays a central role in eukaryotic/archaeal initiation of translation. By delivering the initiator methionyl-tRNA to the ribosome, e/aIF2 ensures specificity of initiation codon selection. The three subunits of aIF2 from the hyperthermophilic archaeon Pyrococcus abyssi could be overproduced in Escherichia coli. The beta and gamma subunits each contain a tightly bound zinc. The large gamma subunit is shown to form the structural core for trimer assembly. The crystal structures of aIF2gamma, free or complexed to GDP-Mg(2+) or GDPNP-Mg(2+), were resolved at resolutions better than 2 A. aIF2gamma displays marked similarities to elongation factors. A distinctive feature of e/aIF2gamma is a subdomain containing a zinc-binding knuckle. Examination of the nucleotide-complexed aIF2gamma structures suggests mechanisms of action and tRNA binding properties similar to those of an elongation factor. Implications for the mechanism of translation initiation in both eukarya and archaea are discussed. In particular, positioning of the initiator tRNA in the ribosomal A site during the search for the initiation codon is envisaged.

Amino Acid Sequence↗
PubMed2004

GTP-dependent recognition of the methionine moiety on initiator tRNA by translation factor eIF2.

Eukaryotic translation initiation factor 2 (eIF2) is a G-protein that functions as a central switch in the initiation of protein synthesis. In its GTP-bound state it delivers the methionyl initiator tRNA (Met-tRNA(i)) to the small ribosomal subunit and releases it upon GTP hydrolysis following the recognition of the initiation codon. We have developed a complete thermodynamic framework for the assembly of the Saccharomyces cerevisiae eIF2.GTP.Met-tRNA(i) ternary complex and have determined the effect of the conversion of GTP to GDP on eIF2's affinity for Met-tRNA(i) in solution. In its GTP-bound state the factor forms a positive interaction with the methionine moiety on Met-tRNA(i) that is disrupted when GTP is replaced with GDP, while contacts between the factor and the body of the tRNA remain intact. This positive interaction with the methionine residue on the tRNA may serve to ensure that only charged initiator tRNA enters the initiation pathway. The toggling on and off of the factor's interaction with the methionine residue is likely to play an important role in the mechanism of initiator tRNA release upon initiation codon recognition. In addition, we show that the conserved base-pair A1:U72, which is known to be a critical identity element distinguishing initiator from elongator methionyl tRNA, is required for recognition of the methionine moiety by eIF2. Our data suggest that a role of this base-pair is to orient the methionine moiety on the initiator tRNA in its recognition pocket on eIF2.

Base Pairing↗
PubMed1991

Inhibition of translation initiation by antisense oligonucleotides via an RNase-H independent mechanism.

We have used alpha-oligomers as antisense oligonucleotides complementary to three different sequences of the rabbit beta-globin mRNA: a region adjacent to the cap site, a region spanning the AUG initiation codon or a sequence in the coding region. These alpha-oligonucleotides were synthesized either with a free 5' OH group or linked to an acridine derivative. The effect of these oligonucleotides on mRNA translation was investigated in cell-free extracts and in Xenopus oocytes. In rabbit reticulocyte lysate and in wheat germ extracts oligomers targeted to the cap site and the initiation codon reduced beta-globin synthesis in a dose-dependent manner, whereas the target mRNA remained intact. The anti-cap alpha-oligomer was even more efficient that its beta-counterpart in rabbit reticulocyte lysate. In contrast, only the alpha-oligomer, linked to the acridine derivative, complementary to the cap region displayed significant antisense properties in Xenopus oocytes. Therefore initiation of translation can be arrested by oligonucleotide/RNA hybrids which are not substrates for RNase-H.

Animals↗
PubMed2005

Identification and characterization of the promoter for the gene encoding human tripeptidyl-peptidase II.

Tripeptidyl-peptidase II (TPP II) is a ubiquitously expressed exopeptidase. The expression of this enzyme is increased, e.g. in some tumor cells, but the regulation of the expression of the gene has not been investigated previously. The gene encoding human TPP II (TPP2) is 82 kb and consists of 30 exons. An 8 kb NcoI fragment covering the 5'-flanking region of the TPP2 gene, including the initiation codon, was cloned into a luciferase-containing reporter vector. Human embryonic kidney cells (HEK-293 cells) and murine fibroblasts (NIH3T3 cells) were transiently transfected with the construct. Through sequential deletions and analysis of short PCR-fragments, the promoter could be localized to a 215 bp fragment upstream of the initiation codon. This region is GC-rich, lacks a TATA-box and contains two inverted CCAAT-boxes and a GC-box. Electrophoretic mobility shift assays showed that nuclear proteins bind to the promoter fragment. The 85 bp 5'-end of the promoter fragment is essential for transcriptional activation. Out of this a 44 bp fragment suffices to compete with binding of nuclear proteins to the 215 bp fragment. Supershift assays demonstrated that the CCAAT-binding factor (CBF; NF-Y) is involved in the formation of a complex with the 215 bp fragment. Although Sp1 binds to the promoter fragment in vitro, it was found to bind to the 3'-end of the 215 bp fragment which is not essential for transcription. The potential role of Sp1 in transcription of TPP2 therefore remains to be established.

Aminopeptidases↗
PubMed1992

Cloning and expression of gene 4 of bacteriophage T7 and creation and analysis of T7 mutants lacking the 4A primase/helicase or the 4B helicase.

T7 gene 4, which is required for DNA replication, specifies two proteins whose coding sequences overlap in the same reading frame: the 4A protein, a 566-amino acid primase/helicase, and the 4B protein, a 503-amino acid helicase whose initiation codon is the 64th codon of the 4A protein. To study better the individual functions of these two overlapping proteins, we made clones that express both 4A and 4B proteins, only 4B protein, or only what we refer to as the 4A' protein, in which methionine 64 is replaced by leucine, thereby eliminating the 4B initiation codon. These clones provide considerably more gene 4 protein for biochemical analysis than do infected cells. They can also be used to isolate and propagate T7 gene 4 deletion mutants, and we have made T7 mutants which lack all gene 4 coding sequences, or which express 4A' protein but no 4B protein, or 4B protein but no 4A protein. Analysis of these phage mutants shows that 4A' protein without any 4B protein can support essentially normal replication and growth, whereas 4B protein without any 4A protein supports little replication or growth. Apparently, the primase activity of the 4A protein is essential for replication, but the 4B protein is dispensable, presumably because the 4A protein also supplies helicase activity. The mutation at amino acid 64 of 4A' appears to have little effect on 4A function. The rate of replication during normal T7 infection appears to be limited by the amount of gene 4 protein, but too high a level of either 4A or 4B protein is inhibitory to growth.

Base Sequence↗
PubMed1998

Clinical similarities of hereditary progressive/dopa responsive dystonia caused by different types of mutations in the GTP cyclohydrolase I gene.

OBJECTIVE: Hereditary progressive dystonia with pronounced diurnal fluctuation [(HPD)/dopa responsive dystonia (DRD)] is a childhood onset dystonia which responds to levodopa. Various clinical signs and symptoms of HPD/DRD have been recognised to date. Mutations in the GTP cyclohydrolase I (GTP-CH-I) gene were recently identified as the cause of HPD/ DRD. In the present study, the GTP-CH-I gene and the clinical features of eight HPD/DRD patients from six families were analysed to determine the correlations between clinical expression and the mutations in the GTP-CH-I gene. METHODS: The exons, exon-intron junctions, and an indispensable part of the 5' flanking region of the GTP-CH-I gene were sequenced in the eight clinically diagnosed patients with HPD/DRD and their asymptomatic parents. RESULTS: Three independent mutations in the GTP-CH-I gene were found in three patients. One of the patients and her asymptomatic mother were heterozygous for a novel mutation at the initiation codon. The three patients with dissimilar GTP-CH-I mutations exhibited similar clinical features. The other five patients with normal sequences presented several features not manifested by the three patients with the mutations. No mutation was found in the 5' flanking region of any patients or their parents. CONCLUSIONS: A novel initiation codon mutation was found in a Japanese patient with HPD/DRD. The clinical manifestations common to the patients with HPD/ DRD with a mutated GTP-CH-I gene were also identified. Although focal manifestations of HPD/DRD associated with the mutations of this gene will be broadened, it is inferred that these clinical features are fundamental to HPD/DRD caused by mutations in this gene.

Adolescent↗
PubMed1989

Transcription of the chicken myb proto-oncogene starts within a CpG island.

The nucleotide sequence of an 8.2-kb DNA fragment from the 5' proximal part of the chicken myb proto-oncogene spanning 1761 nucleotides upstream and 6436 nucleotides downstream from a presumed c-myb initiation codon was determined. A 3.3-kb G + C-rich region found in this sequence had also other features characterizing CpG islands, i.e. no CpG underrepresentation and lack of CpG methylation. In haematopoietic tissues c-myb mRNA synthesis starts in two major regions of the CpG island, namely 98 to 108 and 143 to 145 nucleotides upstream from the c-myb initiation codon. These two regions are in or close to the 124-bp evolutionarily conserved element located in the middle part of the CpG island. No alternative splicing of the 5' end of c-myb mRNA suggested earlier (1,2) was observed. The c-myb promoter contains neither TATA nor CAAT box-like structures at the usual positions. Instead, numerous potential Sp1 factor binding sites were found both upstream and downstream from the transcription initiation sites. Moreover, consensus v-myb protein DNA-binding sites were revealed in the promoter region and in sequences downstream from it.

Animals↗
PubMed1998

Characterization and transcriptional regulation of the Synechocystis PCC 6803 petH gene, encoding ferredoxin-NADP+ oxidoreductase: involvement of a novel type of divergent operator.

The petH gene, encoding ferredoxin-NADP+ oxidoreductase (FNR), has been characterised in the unicellular cyanobacterium Synechocystis PCC 6803. Its product, FNR, was heterologously produced and functionally characterized. The start-site of the monocystronic petH transcript was mapped 523 bp upstream of the predicted PetH initiation codon, resulting in an unusually large 5'-untranslated region. The 5' end of the petH transcript is situated within the open reading frame of phosphoribulokinase (encoded by prk), which is transcribed in opposite orientation with respect to petH. The transcription start site of the prk transcript was mapped 219 bp upstream of the initiation codon, resulting in a 223 bp antisense region between both transcripts. Under many conditions the expression of both genes (i.e. petH and prk) is co-regulated symmetrically at the transcriptional level, as was concluded from both northern hybridization experiments and from primer extension analyses; it became uncoupled, however, when specifically petH expression was stimulated, independent of prk expression, by stressing the Synechocystis cells with high salt concentrations. A model for a new type of bidirectional operator, regulating the expression of petH and prk, is proposed.

Bacterial Proteins↗
PubMed1999

Fine structure of the human translocation protein 1 (HTP1/TLOC1) gene.

We characterized the genomic region corresponding to the human translocation protein 1 (HTP1/TLOC1) cDNA previously reported. An experiment using rapid amplification of cDNA ends revealed that the transcription initiation site was at -12 bp upstream from the translation initiation codon ATG. Using direct-sequencing PCR, we determined precise intron/exon boundaries and intron-exon composition of the gene. The gene region spanned approximately 28 kb and was composed of eight exons and seven introns. The lengths of exons and introns range from 48 to > 1707 bp and from 0.25 to 8.2 kb, respectively. The translation initiation codon and the termination codon were located in exons 1 and 8, respectively. The nucleotide sequences of the introns were also determined in the region around the intron/exon boundaries for 63 to 442 bp. All of the sequences around the intron/exon boundaries were consistent with the 5' and 3' consensus sequences for splice junctions of transcribed genes. Putative lariat sequences were identified between -28 and -64 nucleotides from the 3' splice junction for all seven introns. DNA walking experiments revealed a promoter region of 600 bp. The promoter region did not contain an apparent TATA box or a CAT box but did contain Evi-1, GATA, v-Myb, MZF1, and AP-1 binding sites--factors known as regulatory factors on expression of the gene in blood cells. Therefore, this gene may be one such gene.

Base Sequence↗
PubMed1998

Molecular cloning and functional analysis of the CD33 promoter.

CD33 is a leucocyte differentiation antigen restricted to myeloid cells in blood and bone marrow. Two mRNA transcripts encoding CD33 are observed in leukaemic cell lines. The smaller transcript of 1.5 kb is comparable in size to the isolated CD33 cDNA but the origin of the larger 1.8 kb transcript is unknown. To study the regulation of human CD33 expression, a 5' genomic clone from the CD33 gene was isolated and studied for promoter activity. The clone, although lacking a TATAA box, exhibits other sequences characteristic of a promoter. Two transcriptional start sites were identified, 414 and 527 nucleotides 5' of the ATG initiation codon, suggesting that these sites are used to generate the 1.8 kb transcript observed in CD33+ cell lines. The CD33 genomic sequence directed high expression of a luciferase reporter gene in myeloid cell lines. Using deletion mutants of the promoter sequence, maximal expression was localized to the first 220 bp 5' of the ATG initiation codon. Site-directed mutagenesis of an Sp-1 and PU.1 binding site within this region showed that the PU.1, but not Sp-1, was critical for CD33 expression in myeloid lines. Given the restricted expression of CD33 on haemopoietic cells, the identification of the CD33 promoter may be useful for the study of transcription factors that regulate gene expression during early myeloid differentiation.

Antigens, CD↗
PubMed1979

Synthesis of simian virus 40 t antigen in Escherichia coli.

Plasmids are constructed by using recombination in vitro according to Roberts, T.M., Kacich, R. & Ptashne, M. (1979) Proc. Natl. Acad. Sci. USA 76, 760-764 in which the t antigen gene of simian virus 40 is fused to a promoter of the Escherichia coli lac operon. In the fusions, transcription commences at the lac promoter, and, in some of the fusions, translation begins at the ATG initiator codon of the t gene. This translation is directed most efficiently by those plasmids in which the lac sequences abut the t gene such that a hybrid ribosome binding is encoded. In this case, the Shine-Dalgarno sequence is of lac origin but the ATG derives from the t gene. translation from this initiator codon is greatly decreased if the lac sequences are separated from the ATG by 17 base pairs and is abolished if the AT of this triplet is deleted. Cells bearing the productive fusions synthesize a 20,000-dalton protein with t antigenic determinants. This protein has an isoelectric point(s) indistinguishable from that of t antigen isolated from simian virus 40-transformed cells. Moreover, a partial sequence of the amino-terminal region of the bacterial product is that predicted for authentic t antigen. We conclude that these bacteria are for authentic t antigen. We conclude that these bacteria are producing a protein, the sequence of which is identical to that of authentic t antigen unfused to other polypeptides.

Amino Acid Sequence↗
PubMed1992

The anticodon and discriminator base are major determinants of cysteine tRNA identity in vivo.

Mutants of the Escherichia coli initiator tRNA (tRNA(fMet)) have been used to examine the role of the anticodon and discriminator base in in vivo aminoacylation of tRNAs by cysteinyl-tRNA synthetase. Substitution of the methionine anticodon CAU with the cysteine anticodon GCA was found to allow initiation of protein synthesis by the mutant tRNA from a complementary initiation codon in a reporter protein. Sequencing of the protein revealed that cysteine comprised about half of the amino acid at the N terminus. An additional mutation, converting the discriminator base of tRNA(GCAfMet) from A73 to the base present in tRNA(Cys) (U73), resulted in a 6-fold increase in the amount of protein produced and insertion of greater than or equal to 90% cysteine in response to the complementary initiation codon. Substitution of C73 or G73 at the discriminator position led to insertion of little or no cysteine, indicating the importance of U73 for recognition of the tRNA by cysteinyl-tRNA synthetase. Single base changes in the anticodon of tRNA(GCAfMet) containing U73 from GCA to UCA, GUA, GCC, and GCG (changes underlined) eliminated or dramatically reduced cysteine insertion by the mutant initiator tRNA indicating that all three cysteine anticodon bases are essential for specific aminoacylation of the tRNA with cysteine in vivo.

Acylation↗
PubMed2003

Characterization, expression pattern and chromosomal localization of the spermatogenesis associated 6 gene (Spata6).

We report the cloning and characterization of the spermatogenesis associated 6 gene (Spata6) encoding a predicted protein of 488 amino acids. It exhibits similarity with the motor domain of kinesin related proteins and with the Caenorhabditis elegans neural calcium sensor protein (NCS-2). The gene encodes three mRNAs of approximately 2.6, approximately 1.8 and approximately 1.2 kb. The expression of the 2.6 kb mRNA is detected at low levels in testis, ovary, thymus and placenta, while the 1.8 and 1.2 kb transcripts are exclusively expressed in testis. The 1.8 and 1.2 kb transcripts are specifically expressed in haploid germ cells. Data from in situ hybridization experiments suggested that mRNA expression of Spata6 in spermatids is higher than in spermatocytes and spermatogonia. RT-PCR analysis and whole mount in situ hybridization demonstrate that the Spata6 transcript is expressed during embryonic development and is localized in neural tube, somites and limb buds of mouse embryo. The Spata6 gene consists of 15 exons ranging in size between 40 and 596 bp. The 2.6 and 1.8 kb transcripts have different 5' untranslated sequences but have the same translational initiation site and therefore may encode the same protein with a predicted molecular weight of 49.7 kDa. The 1.2 kb transcript is derived from a proximal promoter between exons 7 and 8, and contains a translation initiation codon AUG, which is in frame with initiator AUG codon of the 2.6 and 1.8 kb transcripts. Therefore, the 1.2 kb transcript may code for a truncated protein of 32 kDa. Western blot analysis with the antiserum raised against a synthetic peptide from the C-terminal of the deduced Spata6 protein detects only a single protein of 53 kDa in all tissues studied. The Spata6 gene was localized to chromosome 5, region q34-35 in the rat and to chromosome 1, region p32-35 in the human. In an effort to determine the function of Spata6, we inactivated the mouse gene in embryonic stem cells through homologous recombination. Although the heterozygous mutant cells were able to generate low coat colour chimeric mice, all chimeras did not transmit the targeted allele to their progeny suggesting that a high contribution of Spata6(+/-) cells lead to the lethality of the chimeric embryos.

Amino Acid Sequence↗
PubMed1995

A novel screening system for construction and selection of active ribozymes using DHFR (dihydrofolate reductase) gene as selection marker.

Ribozyme is thought to be a powerful tool for suppression of specific gene expression, but there are many failure cases in their applications in vivo. One reason may be that there are many cellular proteins in vivo which may inhibit the catalytic activity of ribozymes. Such being the case, many researchers have made efforts to increase the activity of ribozymes. Here we have developed a new screening system for active ribozymes using DHFR (dihydrofolate reductase) gene as a selection marker. In our system, we set the ribozyme target site not to the DHFR gene itself but to the interspace between two ATG codons. One of which is the original initiation codon, ATG, for DHFR gene itself, and the second one locates upstream of the former. The upstream ATG has an ability to cause frame shift for DHFR gene. Since the upstream ATG is preferentially used during translation, unless it is removed, production of DHFR is negligible. By setting the ribozyme target site between the two ATGs, we can make this screening system as positive selection. To our best knowledge, this is the first successful screening system in vivo.

Base Sequence↗