End Flanking Sequences Research Articles

Identification of T-DNA Insertion Site and Flanking Sequence of a Genetically Modified Maize Event IE09S034 Using Next-Generation Sequencing Technology.

Molecular characteristics including information of insertion site, flanking sequence, and copy numbers are the base for the safety assessment and subsequent monitoring of genetically modified organisms (GMOs), which has to be revealed thoroughly in a case-by-case manner. Although both polymerase chain reaction (PCR)-based and next-generation sequencing (NGS)-based approaches are proven to be effective in the molecular characterization of most of GM events, they often fail to work with GM maize events, mainly due to the genome complexity. In this study, by using NGS, we successfully identified the 3' end T-DNA insertion site and flanking sequence of a GM maize event IE09S034, which were confirmed by PCR amplification and Sanger sequencing. Notably, insertions of unintended exogenous elements were revealed in this event although the single copy of target exogenous genes was also confirmed by digital PCR. The output of this study provides novel and important genetic evidence for the safety assessment and monitoring of GM maize event IE09S034.

Analysis of the promoter region of the gene LIP1 encoding triglyceride lipase from Fusarium graminearum

Triglyceride lipases catalyze the reversible degradation of glycerol esters with long-chain fatty acids into fatty acids and glycerol. In silico analysis of 5′-end flanking sequence of the gene LIP1 encoding a triglyceride lipase from the wheat head blight pathogen Fusarium graminearum revealed the presence of several cis-regulatory elements. To delineate the function of these regulatory elements, we constructed a series of deletion mutants in the LIP1 promoter region fused to the open reading frame of a green fluorescent protein (GFP) and assayed the promoter activity. Analysis of GFP expression levels in mutants indicated that a 563-bp promoter sequence was sufficient to drive the expression of LIP1 and regulatory elements responsible for the gene induction were located within the 563–372 bp region. To further investigate the regulatory elements, putative cis-acting elements spanned within the 563–372 bp region were mutated using a targeted mutagenesis approach. A CCAAT box, a CreA binding site, and a fatty acid responsive element (FARE) were identified and confirmed to be required for the basal expression of LIP1, glucose suppression and fatty acid induction, respectively.

Interlaboratory Trial Validation of an Event-Specific Qualitative Polymerase Chain Reaction-Based Detection Method for Genetically Modified RT73 Rapeseed

The qualitative event-specific polymerase chain reaction detection method of genetically modified (GM) RT73 rapeseed was developed based on the cloned 3' end flanking sequence of RT73 rapeseed integration. The specificity of the method for GM RT73 rapeseed was validated using several different GM rapeseed lines, GM maize lines, GM soybean line, non-GM rapeseed, and other non-GM crops. In this study, the developed method was validated through an interlaboratory study by 12 laboratories from 6 countries. The sensitivity of this method was evaluated using several mixed rapeseed meals with different GM RT73 rapeseed contents from 5.0 to 0.01% prepared by our laboratory. The evaluated results showed that all of the rapeseed endogenous reference high mobility group protein gene (HMG I/Y), figwort mosaic virus 35S (FMV 35S) promoter, and RT73 event-specific fragment could be detected from rapeseed samples at 0.1% (w/w) with a confidence level of more than 95%. All results from the 12 laboratories indicated that the developed method could be considered fit for the detection and identification of GM RT73 rapeseed.

Efficient Target Site Selection for an RNA-cleaving DNAzyme through Combinatorial Library Screening

Identification of accessible sites in targeted RNAs is a major limitation to the effectiveness of antisense oligonucleotides. A class of antisense oligodeoxynucleotides, known as the “10-23” DNA enzyme or DNAzyme, which is a small catalytic DNA, has been shown to efficiently cleave target RNA at purine-pyrimidine junctions in vitro. We have designed a strategy to identify accessible cleavage sites in the target RNA, which is hepatitis C virus nonstructural gene 3 (HCV NS3) RNA that encodes viral helicase and protease, from a pool of random DNAzyme library. A pool of DNAzymes of 58 nucleotides-length that possess randomized annealing arms, catalytic core sequence, and fixed 5'/3'-end flanking sequences was designed and screened for their ability to cleave the target RNA. The screening procedure, which includes binding of DNAzyme pool to the target RNA under inactive condition, selection and amplification of active DNAzymes, incubation of the selected DNAzymes with the target RNA, and target site identification on sequencing gels, identified 16 potential cleavage sites in the target RNA. Corresponding DNAzymes were constructed for the selected target sites and were tested for RNA-cleavage in terms of kinetics and accessibility. These selected DNAzymes were effective in cleaving the target RNA in the presence of <TEX>$Mg^{2+}$</TEX>. This strategy can be applicable to identify accessible sites in any target RNA for antisense oligonucleotides-based gene inactivation methods.

Blockade of murine erythroleukemia cell differentiation by hypomethylating agents causes accumulation of discrete small poly(A) − RNAs hybridized to 3′-end flanking sequences of β major globin gene

Induction of murine erythroleukemia (MEL) cell differentiation is accompanied by transcriptional activation of globin genes and biosynthesis of hemoglobin. In this study, we observed cytoplasmic accumulation of relatively small RNAs of different size (150–600 nt) hybridized to α 1 and β major globin DNA probes in MEL cells blocked to differentiate by hypomethylating agents (neplanocin A, 3-deazaneplanocin A and cycloleucine). These RNAs lack poly(A) tail and appear to be quite stable. Search within the 3′-end flanking sequences of β major globin gene revealed the presence of a B1 repeat element, several ATG initiation codons, a GATA-1 consensus sequence and sequences recognized by AP-1/NF-E2 and erythroid Krüppel-like factor (EKLF) transcription factors. These data taken together indicate that exposure of MEL cells to hypomethylating agents promotes accumulation of relatively small discrete RNA transcripts lacking poly(A) tail regardless of the presence or absence of inducer dimethylsulfoxide (DMSO). However, the relative steady-state level of small RNAs was comparatively higher in cells co-exposed to inducer and each one of the hypomethylating agents. Although the orientation of these RNAs has not been established as yet, the possibility these small poly(A) − RNAs which are induced by hypomethylating agents may be involved in the blockade of MEL cell differentiation program is discussed.

Conservation of RNase III processing pathways and specificity in hemiascomycetes.

Rnt1p, the only known Saccharomyces cerevisiae RNase III endonuclease, plays important functions in the processing of precursors of rRNAs (pre-rRNAs) and of a large number of small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs). While most eukaryotic RNases III, including the Schizosaccharomyces pombe enzyme Pac1p, cleave double-stranded RNA without sequence specificity, Rnt1p cleavage relies on the presence of terminal tetraloop structures that carry the consensus sequence AGNN. To search for the conservation of these processing signals, I have systematically analyzed predicted secondary structures of the 3' external transcribed spacer (ETS) sequences of the pre-rRNAs and of flanking sequences of snRNAs and snoRNAs from sequences available in 13 other Hemiascomycetes species. In most of these species, except in Yarrowia lipolytica, double-stranded RNA regions capped by terminal AGNN tetraloops can be found in the 3' ETS sequences of rRNA, in the 5'- or 3'-end flanking sequences of sn(o)RNAs, or in the intergenic spacers of polycistronic snoRNA transcription units. This analysis shows that RNase III processing signals and RNase III cleavage specificity are conserved in most Hemiascomycetes species but probably not in the evolutionarily more distant species Y. lipolytica.

CYP6B cytochrome p450 monooxygenases from Papilio canadensis and Papilio glaucus: potential contributions of sequence divergence to host plant associations.

Two groups of furanocoumarin-inducible cytochrome p450 genes, the CYP6B4 group and the CYP6B17 group, characterized in two closely related tiger swallowtails, Papilio glaucus and Papilio canadensis, are induced to different extents, with generally higher levels of CYP6B transcripts in P. glaucus. To investigate the evolutionary history of these CYP6B genes in the context of their association with furanocoumarin detoxification, we isolated thirteen CYP6B genes from these species. Each of these genes contains an intron at a conserved position (1334 nucleotides from the translation start site), which varies in length due to three insertion/deletions. The proximal 5' end flanking sequence from the transcription initiation site is highly conserved (91-98% nt identity). The sequence 5' to -640 is significantly variable due largely to the presence of three insertion/deletions. The sequence at the 3' end of this region contains a putative xenobiotic response element to xanthotoxin (XRE-xan), important for basal and xanthotoxin-inducible transcription of the P. polyxenes CYP6B1v3 gene, and multiple elements known to regulate vertebrate phase I and II promoters, including an XRE-AhR (Xenobiotic Response Element to Aryl hydrocarbon Receptor), an OCT-1 element (octamer protein binding site), an ARE (Antioxidant Response Element), an EcRE (Ecdysone Response Element), and an imperfect PXR (Pregnane X Receptor) responsive element (PRE). Our analyses of CYP6B genes in these two species indicate that these genes are in an early stage of divergence and that differential exposure of these two species to chemically distinct host plants resulting from geographical isolation has had functional impacts not only on the coding regions of these genes but also on their promoter regions. Thus, changes in p450 regulation as well as catalytic activity may play a role in the evolution of host plant associations in herbivorous insects.

Genomic Organization and Expression of a Human Gene for Myc-associated Zinc Finger Protein (MAZ)

We have cloned and characterized the genomic structure of the human gene for Myc-associated zinc finger protein (MAZ), which is located on chromosome 16p11.2. This gene is transcribed as an mRNA of 2.7 kilobases (kb) that encodes a 60-kDa MAZ protein. A 40-kb cosmid clone was isolated that includes the promoter, five exons, four introns, and one 3'-untranslated region. All exon-intron junction sequences conform to the GT/AG rule. The promoter region has features typical of a housekeeping gene: a high G + C content (88. 4%); a high frequency of CpG dinucleotides, in particular within the region 0.5 kb upstream of the site of initiation of translation; and the absence of canonical TATA and CAAT boxes. An S1 nuclease protection assay demonstrated the presence of multiple sites for initiation of transcription around a site 174 nucleotides (nt) upstream of the ATG codon and such expression was reflected by the promoter activity of a MAZ promoter/CAT (chloramphenicol acetyltransferase) reporter gene. Cis-acting positive and negative elements controlling basal transcription of the human MAZ gene were found from nucleotides (nt) -383 to -248 and nt -2500 to -948. Moreover, positive and negative autoregulatory elements were also identified in the regions from nt -248 to -189 and from nt -383 to -248 after co-transfection of HeLa cells with plasmids that carried the MAZ promoter/CAT construct and the MAZ-expression vector. Our results indicate that the 5'-end flanking sequences are responsible for the promoter activities of the MAZ gene.

A family of U1 pseudogenes in Bombyx mori may be derived from an ancestral pseudogene.

Seven EMBL-4 lambda clones containing U1 small nuclear RNA sequences were isolated from a Bombyx mori genomic library. Six of the seven represent unique sequences. The six unique U1 sequences exhibit fixed point 3'-end truncation. Five out of the six clones share immediate 3'-end flanking sequences while two share 5'-end flanking sequences. Fixed point 3'-end truncation and a hierarchy of shared to unique diagnostic mutations may suggest a family of U1 pseudogenes generated from a reverse-transcribed class II pseudogene in B. mori. An ancestral 'master' U1 pseudogene capable of RNA- and/or DNA-mediated transposition may give rise to generations of U1 pseudogenes that include the original pseudogene's flanking sequences. Identical 3'-end truncation in some of these U1 sequences can be explained by RNA self-priming due to intra-strand binding prior to reverse transcription.

Functional characterization of the 5'-regulatory region of the human thrombomodulin gene.

Thrombomodulin, a glycoprotein expressed in endothelial cells, has an important role in the blood coagulation system as a modulator. Functional characterization of the 5'-regulatory region of the human thrombomodulin gene was carried out to identify elements necessary for its expression. We used a series of dissected gene constructs containing the bacterial chloramphenicol acetyltransferase gene in transient transfection assays on human umbilical vein endothelial cells. The region extending from -290 to -33 of the 5' end flanking sequence is required for the full expression of this gene. Within this region, four potential Sp1 sites were found, and the sequences of Sp1 sites were mutated to identify their role in the promoter activity of the gene, showing that the two Sp1 sites at -207 and -141 are important for the full activity of the thrombomodulin promoter. Site-directed mutation analysis identified sequence elements GCAATC at -110 as a functioning CAAT box. Another three regions, -290 to -223, -99 to -68, and -67 to -33 have unidentified positively and negatively acting elements. A silencer element was located in the region spanning from -947 to -772 bases of the 5' end flanking region. These data indicate that the expression of the thrombomodulin gene is regulated by various elements which act positively or negatively.

Expression and methylation of the beta-subunit gene of prolyl 4-hydroxylase: in erythrocytes, tendon and cornea of chick embryos.

It has recently been demonstrated that the beta-subunit of prolyl 4-hydroxylase (E.C. 1.14.11.2) is the same gene product as protein disulfide isomerase (PDI) and cellular thyroid hormone binding protein (THP). Therefore, it is very likely that the beta-subunit of the prolyl 4-hydroxylase gene serves as a house keeping gene in most cell types. In the present study, we examined the distribution of the chicken beta-subunit of prolyl 4-hydroxylase/protein disulfide isomerase (CPH beta/PDI) in erythrocytes, corneas and tendons of 13-, 17-, and 19-day-old chick embryos by immunohistochemistry using antibodies against CPH beta/PDI. Our data indicate that erythrocytes do not express the CPH beta/PDI gene whereas tendon cells express CPH beta/PDI at all developmental stages examined. The basal cells of corneal epithelium express CPH beta/PDI, but the superficial cell layers of stratified corneas of 19-day-old chick embryos do not. The expression of the CPH beta/PDI gene is also confirmed by in situ hybridization with cDNA encoding CPH beta/PDI. The results indicate that the expression of CPH beta/PDI in cornea is probably developmentally regulated. It has been suggested that methylation of genomic DNA is one of many possible regulatory mechanisms for gene expression. In order to examine whether methylation of genomic DNA may play any role in the expression of the beta-subunit gene, genomic DNA was isolated from corneas, tendons, and erythrocytes of individual 13-, 17-, and 19-day-old chick embryos. DNA samples were digested with Sma I and Eco RI, or Pst I and Sma I and followed by either Msp I, Hpa II, or Hha I and were then subjected to Southern hybridization with 32P-labeled genomic DNA fragments of CPH beta/PDI. Our results indicate that the CPH beta/PDI gene is methylated at the Hha I site in the 4th exon in erythrocytes whereas the same sites in tendon and cornea are hypomethylated. Examination of 5'-end flanking sequences of exon 1 of the CPH beta/PDI gene with the methylation sensitive endonucleases, Hha I and Hpa II did not reveal any difference in erythrocyte, cornea and tendon cells. Thus, our results indicated that DNA methylation may not play an important role in the expression of CPH beta/PDI.

Functional characterization of the 5'-regulatory region of human factor IX gene.

Functional characterization of the 5'-regulatory region of the human factor IX gene was carried out by a series of gene dissection analyses. The region extending from -175 to -274 of the 5' end flanking sequence is required for the expression of this gene. Within this region, sequence elements AGCCACT at -238 and TCAAAT at -187 were assigned as the functional CAAT box and TATA box, respectively. The transcription initiation site was found to be at -150 for the factor IX chloramphenicol acetyltransferase chimeric gene. A negative regulatory (silencer) activity was located in the region spanning from 1.4 to 1.7 kilobases upstream of the promoter region. This region contains a short sequence element (ATCCTCTCC) known to have such activity. A strong promoter on the opposite strand was also located about 500 base pairs upstream of the promoter. The expression of factor IX gene was highly liver specific, as assessed by Northern blot analysis. Short sequence elements (TGGACC and CTTTGGACT) homologous to the known liver-specific elements were located in the vicinity of the defined promoter region.

Tissue-specific methylation patterns and expression of the human apolipoprotein AI gene.

To better understand the tissue-specific expression of the human apolipoprotein (apo)AI gene, we performed a detailed analysis of the pattern of methylation of the gene in various human adult and embryonic tissues and in tissues of transgenic mice harboring the human apo-AI gene. In addition, the gene was analyzed also in liver and intestine-derived human cell lines (HepG2 and Caco2, respectively). Using methyl-sensitive restriction enzymes (HpaII, HhaI, and SmaI) and the appropriate radioactive probes, we were able to determine separately the status of methylation of the 5'-end, the body of the gene, and 3'-end flanking sequences. The apo-AI gene in tissues that express the gene was undermethylated at the 5'-end. However, the 5'-end of the gene in sperm and in all adult tissues that do not express the gene was heavily methylated. The body of the gene which contains a CpG island and the 3'-end flanking sequences were, in general, hypomethylated except for specific sites that showed partial methylation. In contrast, while the gene showed tissue-specific expression already in a 12-week-old embryo, the 5'-end was invariably hypomethylated in all tissues of the embryo. A human apo-AI transgene has recently been shown to be active exclusively in the liver, while the endogenous gene is expressed in both liver and intestine (6). We show here that the 5'-end of the apo-AI transgene was methylated in all tissues of the mouse (including intestine) except liver. The results presented here demonstrate a clear correlation between hypomethylation of the 5'-end and activity of the apo-AI gene. However, the observed methylation pattern of the gene in embryonic tissues suggests that tissue-specific expression precedes formation of the tissue-specific methylation pattern.

Structure of the gene encoding the beta-subunit of chicken prolyl 4-hydroxylase

Prolyl 4-hydroxylase (EC 1.14.11.2) converts peptidyl proline to peptidyl hydroxyproline in procollagen polypeptides during collagen biosynthesis. The active enzyme is a tetramer which is composed of two pairs of non-identical subunits in a molecular form of α2β2. In addition to the tetrameric proly14-hydroxylase(α2β2), the free β-subunit is also found inside cells. Recently it was shown that the β-subunit of prolyl 4-hydroxylase is identical to the protein disulfide isomerase and cellular thyroid hormone-binding protein. We previously isolated and characterized cDNAs of the β-subunit of chicken proly14-hydroxylase. The cDNA of β-subunit was used to screen a chicken genomic DNA library constructed with the λEMBL-3 vector. Two clones,λgCPH β-22 and β-50, were isolated and characterized by restriction enzyme analysis, heteroduplex analysis, and nucleotide sequencing. The results showed that the 2.5-kb mRNA of the β-subunit is divided into eleven exons and that the gene is 9.0 kb long. The gene contains consensus sequence for TATA at -24 bp and four CAAT at -57, -157, -194 and -223 bp in the 5' end flanking sequence of the transcription start point. In addition, there are three GC boxes upstream from the TATA box and four GC boxes in the first intron. This is similar to the structure of the α1(I) collagen coding gene ( COLIA1). These elements may interact with nuclear factors and play important roles in expression regulation of the β-subunit gene as has been described in COLIA1.

Identification of a stable nuclear RNA complementary to the 3'-end flanking sequences of the mouse beta-major globin gene.

A stable nuclear RNA of approximately 1600 nucleotides (nt) isolated from dimethyl sulfoxide-induced Friend erythroleukemia cells has been characterized. This RNA has been shown to be homologous to a region of unique sequences situated 3' to the mouse beta-major globin gene between the poly(A) addition site and a BglII site located 1400 nt farther downstream. It is transcribed from the same DNA strand as the beta-major globin mRNA, and the amount of this RNA present in the cell is directly proportional to the level of beta-globin mRNA. Therefore, the 1600-nt RNA appears to be related to the large primary transcript of the beta-major globin gene. We feel that this RNA species is an unusually stable intermediate product of the early processing event which cleaves the primary transcript at the poly(A) addition site. The observed stability and discrete length which are contrary to the expected properties of such a processing intermediate may reflect peculiarities of the transformed state of the Friend erythroleukemia cells.

The actin gene in yeast Saccharomyces cerevisiae: 5' and 3' end mapping, flanking and putative regulatory sequences.

The 5' and 3' flanking regions of the yeast actin gene have been sequenced and the ends of the actin mRNA were determined by the single-strand nuclease mapping procedure. The mRNA starts with a pyrimidine residue 141 (or 140) nucleotides upstream from the initiation codon. The actin gene lacks a typical "TATA" box 30 base pairs upstream from the mRNA start site but it contains a region homologous to the canonical sequence 5'-GGCTCAATCT-3' which is found in several eukaryotic genes 70 to 80 bp upstream from the mRNA cap site. Judging from the S1 nuclease mapping, there are two populations of actin mRNA terminating 98 and 107 nucleotides downstream from the stop codon. The 3' termini are preceded by three AATAAA sequences found in most eukaryotic polyadenylated mRNAs.