Gene structure and transcription of IRF-2 in the mandarin fish Siniperca chuatsi with the finding of alternative transcripts and microsatellite in the coding region

The gene of interferon regulatory factor-2 (IRF-2) has been cloned from the mandarin fish (Siniperca chuatsi). The IRF-2 gene has 6,418 nucleotides (nt) and contains eight exons and seven introns, encoding two mRNAs. The two IRF-2 mRNAs each contained an open reading frame of 873 nt, which both translate into the same 291 amino acids but differed in their 5' untranslated region: one mRNA was transcribed initially from the exon 1 bypassing exon 2, while the other was transcribed from the exon 2. The microsatellites (CA repeats) could be found in the carboxyl terminal region of mandarin fish IRF-2, which result in the truncated form molecules. The microsatellites' polymorphism was investigated, and eight alleles were found in 16 individuals. The microsatellites were also examined in IRF-2 of several freshwater perciform fishes. The transcription of the IRF-2 in different tissues with or without poly inosine-cytidine stimulation was analyzed by real-time PCR, and the constitutive transcription of both molecules could be detected in all the tissues examined.

Genetic diversity of the HLA-G coding region in Amerindian populations from the Brazilian Amazon: a possible role of natural selection

HLA-G has an important role in the modulation of the maternal immune system during pregnancy, and evidence that balancing selection acts in the promoter and 3′UTR regions has been previously reported. To determine whether selection acts on the HLA-G coding region in the Amazon Rainforest, exons 2, 3 and 4 were analyzed in a sample of 142 Amerindians from nine villages of five isolated tribes that inhabit the Central Amazon. Six previously described single-nucleotide polymorphisms (SNPs) were identified and the Expectation-Maximization (EM) and PHASE algorithms were used to computationally reconstruct SNP haplotypes (HLA-G alleles). A new HLA-G allele, which originated in Amerindian populations by a crossing-over event between two widespread HLA-G alleles, was identified in 18 individuals. Neutrality tests evidenced that natural selection has a complex part in the HLA-G coding region. Although balancing selection is the type of selection that shapes variability at a local level (Native American populations), we have also shown that purifying selection may occur on a worldwide scale. Moreover, the balancing selection does not seem to act on the coding region as strongly as it acts on the flanking regulatory regions, and such coding signature may actually reflect a hitchhiking effect.Genes and Immunity advance online publication, 3 October 2013; doi:10.1038/gene.2013.47.

Analysis of Hepatitis C Virus Intrahost Diversity across the Coding Region by Ultradeep Pyrosequencing

Hepatitis C virus (HCV) is the leading cause of liver disease worldwide. In this study, we analyzed four treatment-naive patients infected with subtype 1a and performed Roche/454 pyrosequencing across the coding region. We report the presence of low-level drug resistance mutations that would most likely have been missed using conventional sequencing methods. The approach described here is broadly applicable to studies of viral diversity and could help to improve the efficacy of direct-acting antiviral agents (DAA) in the treatment of HCV-infected patients.

Trypanosoma cruzi mitochondrial maxicircles display species- and strain-specific variation and a conserved element in the non-coding region

Abstract Background The mitochondrial DNA of kinetoplastid flagellates is distinctive in the eukaryotic world due to its massive size, complex form and large sequence content. Comprised of catenated maxicircles that contain rRNA and protein-coding genes and thousands of heterogeneous minicircles encoding small guide RNAs, the kinetoplast network has evolved along with an extreme form of mRNA processing in the form of uridine insertion and deletion RNA editing. Many maxicircle-encoded mRNAs cannot be translated without this post-transcriptional sequence modification. Results We present the complete sequence and annotation of the Trypanosoma cruzi maxicircles for the CL Brener and Esmeraldo strains. Gene order is syntenic with Trypanosoma brucei and Leishmania tarentolae maxicircles. The non-coding components have strain-specific repetitive regions and a variable region that is unique for each strain with the exception of a conserved sequence element that may serve as an origin of replication, but shows no sequence identity with L. tarentolae or T. brucei. Alternative assemblies of the variable region demonstrate intra-strain heterogeneity of the maxicircle population. The extent of mRNA editing required for particular genes approximates that seen in T. brucei. Extensively edited genes were more divergent among the genera than non-edited and rRNA genes. Esmeraldo contains a unique 236-bp deletion that removes the 5'-ends of ND4 and CR4 and the intergenic region. Esmeraldo shows additional insertions and deletions outside of areas edited in other species in ND5, MURF1, and MURF2, while CL Brener has a distinct insertion in MURF2. Conclusion The CL Brener and Esmeraldo maxicircles represent two of three previously defined maxicircle clades and promise utility as taxonomic markers. Restoration of the disrupted reading frames might be accomplished by strain-specific RNA editing. Elements in the non-coding region may be important for replication, transcription, and anchoring of the maxicircle within the kinetoplast network.

Endothelium-specific replacement of the connexin43 coding region by a lacZ reporter gene

The murine gap junction protein connexin43 (Cx43) is expressed in blood vessels, with vastly different contribution by endothelial and smooth muscle cells. We have used the Cre recombinase under control of TIE2 transcriptional elements to inactivate a floxed Cx43 gene specifically in endothelial cells. Cre-mediated deletion led to replacement of the Cx43 coding region by a lacZ reporter gene. This allowed us to monitor the extent of deletion and to visualize the endothelial expression pattern of Cx43. We found widespread endothelial expression of the Cx43 gene during embryonic development, which became restricted largely to capillaries and small vessels in all adult organs examined. Mice lacking Cx43 in endothelium did not exhibit altered blood pressure, in contrast to mice deficient in Cx40. Our results show that lacZ activation after deletion of the target gene allows us to determine the extent of cell type-specific deletion after phenotypical investigation of the same animal.

Post-transcriptional Regulation of Mammalian Gene Expression in Non-coding Region of Target RNA

Tumor Suppressor Candidate 2 (TUSC2) is a novel tumor suppressor gene located in the human chromosome 3p21.3 region. TUSC2 mRNA transcripts could be detected on Northern blots in both normal lung and some lung cancer cell lines, but no endogenous TUSC2 protein could be detected in a majority of lung cancer cell lines. Mechanisms regulating TUSC2 protein expression and its inactivation in primary lung cancer cells are largely unknown. We investigated the role of the 5’- and 3’-untranslated regions (UTRs) of the TUSC2 gene in the regulation of TUSC2 protein expression. We found that two small upstream open-reading frames (uORFs) in the 5’UTR of TUSC2 could markedly inhibit the translational initiation of TUSC2 protein by interfering with the “scanning” of the ribosome initiation complexes. Site-specific stem-loop array reverse transcription-polymerase chain reaction (SLA-RT-PCR) verified several micoRNAs (miRNAs) targeted at 3’UTR and directed TUSC2 cleavage and degradation. In addition, we used the established let-7-targeted high mobility group A2 (Hmga2) mRNA as a model system to study the mechanism of regulation of target mRNA by miRNAs in mammalian cells under physiological conditions. There have been no evidence of direct link between mRNA downregulation and mRNA cleavages mediated by miRNAs. Here we showed that the endonucleolytic cleavages on mRNAs were initiated by mammalian miRNA in seed pairing style. Let-7 directed cleavage activities among the eight predicted potential target sites have varied efficiency, which are influenced by the positional and the structural contexts in the UTR. The 5’ cleaved RNA fragments were mostly oligouridylated at their 3’-termini and accumulated for delayed 5’–3’ degradation. RNA fragment oligouridylation played important roles in marking RNA fragments for delayed bulk degradation and in converting RNA degradation mode from 3’–5’ to 5’–3’ with cooperative efforts from both endonucleolytic and non-catalytic miRNA-induced silencing complex (miRISC). Our findings point to a mammalian miRNA-mediated mechanism for the regulation of mRNA that miRNA can decrease target mRNA through target mRNA cleavage and uridine addition

Mechanism of translationally coupled mRNA turnover mediated by c-fos protein-coding-region determinant

Proto-oncogene c-fos is a member of the class of early-response genes whose transient expression plays a crucial role in cell proliferation, differentiation, and apoptosis. Degradation of c- fos mRNA is an important mechanism for controlling c-fos expression. Rapid mRNA turnover mediated by the protein-coding-region determinant (mCRD) of the c-fos transcript illustrates a functional interplay between mRNA turnover and translation that coordinately influences the fate of cytoplasmic mRNA. It is suggested that mCRD communicates with the 3′ poly(A) tail via an mRNP complex comprising mCRD-associated proteins, which prevents deadenylation in the absence of translation. Ribosome transit as a result of translation is required to alter the conformation of the mRNP complex, thereby eliciting accelerated deadenylation and mRNA decay. To gain further insight into the mechanism of mCRD-mediated mRNA turnover, Unr was identified as an mCRD-binding protein, and its binding site within mCRD was characterized. Moreover, the functional role for Unr in mRNA decay was demonstrated. The result showed that elevation of Unr protein level in the cytoplasm led to inhibition of mRNA destabilization by mCRD. In addition, GST pull-down assay and immuno-precipitation analysis revealed that Unr interacted with PABP in an RNA-independent manner, which identified Unr as a novel PABP-interacting protein. Furthermore, the Unr interacting domain in PABP was characterized. In vivo mRNA decay experiments demonstrated a role for Unr-PABP interaction in mCRD-mediated mRNA decay. In conclusion, the findings of this study provide the first evidence that Unr plays a key role in mCRD-mediated mRNA decay. It is proposed that Unr is recruited by mCRD to initiate the formation of a dynamic mRNP complex for communicating with poly(A) tail through PABP. This unique mRNP complex may couple translation to mRNA decay, and perhaps to recruit the responsible nuclease for deadenylation. ^

Complex promoter and coding region β2-adrenergic receptor haplotypes alter receptor expression and predict in vivo responsiveness

The human β2-adrenergic receptor gene has multiple single-nucleotide polymorphisms (SNPs), but the relevance of chromosomally phased SNPs (haplotypes) is not known. The phylogeny and the in vitro and in vivo consequences of variations in the 5′ upstream and ORF were delineated in a multiethnic reference population and an asthmatic cohort. Thirteen SNPs were found organized into 12 haplotypes out of the theoretically possible 8,192 combinations. Deep divergence in the distribution of some haplotypes was noted in Caucasian, African-American, Asian, and Hispanic-Latino ethnic groups with >20-fold differences among the frequencies of the four major haplotypes. The relevance of the five most common β2-adrenergic receptor haplotype pairs was determined in vivo by assessing the bronchodilator response to β agonist in asthmatics. Mean responses by haplotype pair varied by >2-fold, and response was significantly related to the haplotype pair (P = 0.007) but not to individual SNPs. Expression vectors representing two of the haplotypes differing at eight of the SNP loci and associated with divergent in vivo responsiveness to agonist were used to transfect HEK293 cells. β2-adrenergic receptor mRNA levels and receptor density in cells transfected with the haplotype associated with the greater physiologic response were ≈50% greater than those transfected with the lower response haplotype. The results indicate that the unique interactions of multiple SNPs within a haplotype ultimately can affect biologic and therapeutic phenotype and that individual SNPs may have poor predictive power as pharmacogenetic loci.

Identification of the coding region for a second poly(A) polymerase in Escherichia coli.

We had earlier identified the pcnB locus as the gene for the major Escherichia coli poly(A) polymerase (PAP I). In this report, we describe the disruption and identification of a candidate gene for a second poly(A) polymerase (PAP II) by an experimental strategy which was based on the assumption that the viability of E. coli depends on the presence of either PAP I or PAP II. The coding region thus identified is the open reading frame f310, located at about 87 min on the E. coli chromosome. The following lines of evidence support f310 as the gene for PAP II: (i) the deduced peptide encoded by f310 has a molecular weight of 36,300, similar to the molecular weight of 35,000 estimated by gel filtration of PAP II; (ii) the deduced f310 product is a relatively hydrophobic polypeptide with a pI of 9.4, consistent with the properties of partially purified PAP II; (iii) overexpression of f310 leads to the formation of inclusion bodies whose solubilization and renaturation yields poly(A) polymerase activity that corresponds to a 35-kDa protein as shown by enzyme blotting; and (iv) expression of a f310 fusion construct with hexahistidine at the N-terminus of the coding region allowed purification of a poly(A) polymerase fraction whose major component is a 36-kDa protein. E. coli PAP II has no significant sequence homology either to PAP I or to the viral and eukaryotic poly(A) polymerases, suggesting that the bacterial poly(A) polymerases have evolved independently. An interesting feature of the PAP II sequence is the presence of sets of two paired cysteine and histidine residues that resemble the RNA binding motifs seen in some other proteins.

Methylation of coding region alone inhibits gene expression in plant protoplasts.

Derivatives of the cauliflower mosaic virus 35S promoter lacking CG and CNG methylation targets were constructed and used to direct transcription of reporter gene constructs in transiently transformed protoplasts. Such methylation-target-free (MTF) promoters, although weaker than the 35S promoter, retain significant activity despite mutation of the as-1 element. The effect of methylation on gene expression in MTF- and 35S-promoter driven constructs was examined. Even when the promoter region was free of methylation targets, reporter gene expression was markedly reduced when cytosine residues in CG dinucleotides were methylated in vitro prior to transformation. Mosaic methylation experiments, in which only specific parts of the plasmids were methylated, revealed that methylation of the coding region alone has a negative effect on reporter gene expression. Methylation nearer the 5' end of the coding region was more inhibitory, consistent with inhibition of transcription elongation.

Creation of a novel protein-coding region at the RNA level in black pine chloroplasts: the pattern of RNA editing in the gymnosperm chloroplast is different from that in angiosperms.

The phenomenon of RNA editing has been found to occur in chloroplasts of several angiosperm plants. Comparative analysis of the entire nucleotide sequence of a gymnosperm [Pinus thunbergii (black pine)] chloroplast genome allowed us to predict several potential editing sites in its transcripts. Forty-nine such sites from 14 genes/ORFs were analyzed by sequencing both cDNAs from the transcripts and the corresponding chloroplast DNA regions, and 26 RNA editing sites were identified in the transcripts from 12 genes/ORFs, indicating that chloroplast RNA editing is not restricted to angiosperms but occurs in the gymnosperm, too. All the RNA editing events are C-to-U conversions; however, many new codon substitutions and creation of stop codons that have not so far been reported in angiosperm chloroplasts were observed. The most striking is that two editing events result in the creation of an initiation and a stop codon within a single transcript, leading to the formation of a new reading frame of 33 codons. The predicted product is highly homologous to that deduced from the ycf7 gene (ORF31), which is conserved in the chloroplast genomes of many other plant species.

The 5' coding region of Paramecium surface antigen genes controls mutually exclusive transcription.

Paramecium tetraurelia stock 51 can express at least 11 different types of surface antigens, yet only a single type is expressed on the surface of an individual cell at any one time. The differential expression of stock 51 type A and B surface antigen genes (51A and 51B) is regulated at the level of transcription. Previously, we reported that nucleotide sequences upstream of position -26 (relative to the start of translation) in the 51A and 51B surface antigen genes are necessary for transcriptional activity but are not sufficient to direct differential transcriptional control. In this report we demonstrate that at least some of the critical elements necessary for differential transcription of the 51A and 51B genes lie within the 5' coding region. A hybrid gene that contains 51B upstream sequences (-475 to +1) attached to the ATG start codon of 51A is not cotranscribed with the 51B gene. In contrast, further substitution with 51B sequences (-1647 to +885) allows the chimeric gene to be coexpressed with 51B. A different hybrid gene containing a substitution of 51B sequence from -26 to +885 in the 51A gene is also coexpressed with 51B, revealing that the critical elements within the coding region of 51B do not require 51B upstream sequences for their effect. Coinjection of the 51A gene with the chimeric gene that contains 51B up to +885 showed that the same sequences that allow coexpression with 51B prevent cotranscription with 51A. Together, these results demonstrate that a region downstream of the transcriptional start site between nucleotide positions +1 and +885 (relative to translational start) is necessary to control differential transcriptional activity.

Highly organized structure in the non-coding region of the psbA minicircle from clade C Symbiodinium

The chloroplast genes of dinoflagellates are distributed among small, circular dsDNA molecules termed minicircles. In this paper, we describe the structure of the non-coding region of the psbA minicircle from Symbiodinium. DNA sequence was obtained from five Symbiodinium strains obtained from four different coral host species (Goniopora tenuidens, Heliofungia actiniformis, Leptastrea purpurea and Pocillopora damicornis), which had previously been determined to be closely related using LSU rDNA region D1/D2 sequence analysis. Eight distinct sequence blocks, consisting of four conserved cores interspersed with two metastable regions and flanked by two variable regions, occurred at similar positions in all strains. Inverted repeats (IRs) occurred in tandem or 'twin' formation within two of the four cores. The metastable regions also consisted of twin IRs and had modular behaviour, being either fully present or completely absent in the different strains. These twin IRs are similar in sequence to double-hairpin elements (DHEs) found in the mitochondrial genomes of some fungi, and may be mobile elements or may serve a functional role in recombination or replication. Within the central unit (consisting of the cores plus the metastable regions), all IRs contained perfect sequence inverses, implying they are highly evolved. IRs were also present outside the central unit but these were imperfect and possessed by individual strains only. A central adenine-rich sequence most closely resembled one in the centre of the non-coding part of Amphidinium operculatum minicircles, and is a potential origin of replication. Sequence polymorphism was extremely high in the variable regions, suggesting that these regions may be useful for distinguishing strains that cannot be differentiated using molecular markers currently available for Symbiodinium.

Sequence diversity in the coat protein coding region of the genome RNA of Johnsongrass mosaic virus in Australia

Sequence diversity in the coat protein coding region of Australian strains of Johnsongrass mosaic virus (JGMV) was investigated. Field isolates were sampled during a seven year period from Johnsongrass, sorghum and corn across the northern grain growing region. The 23 isolates were found to have greater than 94% nucleotide and amino acid sequence identity. The Australian isolates and two strains from the U.S.A. had about 90% nucleotide sequence identity and were between 19 and 30% different in the N-terminus of the coat protein. Two amino acid residues were found in the core region of the coat protein in isolates obtained from sorghum having the Krish gene for JGMV resistance that differed from those found in isolates from other hosts which did not have this single dominant resistance gene. These amino acid changes may have been responsible for overcoming the resistance conferred by the Krish gene for JGMV resistance in sorghum. The identification of these variable regions was essential for the development of durable pathogen-derived resistance to JGMV in sorghum.

Normal HPRT coding region in a male with gout due to HPRT deficiency (Brief Communication)

A deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT; EC 2.4.2.8) is associated with a spectrum of disease that ranges from gouty arthritis (OMIM 300323) to the more severe Lesch-Nyhan syndrome (OMIM 300322). To date, all cases of HPRT deficiency have shown a mutation within the HPRT cDNA. In the present study of an individual with gout due to HPRT deficiency, we found a normal HPRT cDNA sequence. This is the first study to provide an example of HPRT deficiency which appears to be due to a defect in the regulation of the gene. © 2005 Elsevier Inc. All rights reserved.