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Expression of the genetic information encoded in our genomes is usually regulated by proteins interacting with the DNA. In some cases, however, noncoding RNAs transcribed from DNA control elements cooperate with histone-modifying enzymes to regulate gene expression, as has recently been shown for noncoding RNA originating from Polycomb- and Trithorax-group response elements.
To analyze the molecular origin of an ER variant, the truncated ER product-1, transiently expressed at the proestrus in lactotrope cells, we generated a 2.5-kb sequence of a genomic region upstream and downstream the specific sequence truncated ER product-1. Genomic Southern blot analysis showed that truncated ER product-1 is spliced from a noncoding leader exon localized within the intron 4 of the ER alpha gene. Analysis of the promoter sequence revealed the presence of a major transcriptional start site, a canonical TATA box and putative cis regulatory elements for pituitary specific expression as well as an E-responsive element. In transient transfection, the truncated ER product-1 promoter was transcriptionally the most active in the lactotrope cell lines (MMQ). Analysis of truncated ER product-1 functionality showed that: 1) the protein inhibited ER alpha binding to the E-responsive element in electromobility shift assays, 2) inhibited the E2 binding to ER alpha in binding assays, 3) the truncated ER product-1/ER alpha complex antagonized the transcriptional activity elicited by E2, 4) nuclear localization of green fluorescent protein-ER alpha was altered in Chinese hamster ovary cell lines stably expressing truncated ER product-1. Collectively, these data demonstrated that the protein exerts full dominant negative activity against ER alpha. Moreover, truncated ER product-1/ER alpha complex also repressed the activity of all promoters tested to date, suggesting a general inhibitory effect toward transcription. In conclusion, the data suggest that truncated ER product-1 could regulate estrogen signaling via a specific promoter in lactotrope cells.
The BAX PCR for screening Listeria monocytogenes is a commercial PCR assay for specifically targeting L. monocytogenes, a foodborne pathogen that can contaminate a variety of foods and cause a potentially fatal disease, listeriosis, among high-risk populations. The high specificity (> 98%) of this PCR assay is achieved by targeting a species-specific genomic region (approximately 400 bp) presumably found only in L. monocytogenes. In this study, the identity of the BAX PCR-targeted genomic region was determined by using PCR cloning, DNA sequencing, and basic local alignment search tool (BLAST) analysis of the amplicon sequences of an L. monocytogenes serotype 1/2a strain. BLAST analysis identified the BAX PCR amplicon (GenBank accession no. AY364605) as a 423-bp genomic region between nucleotides 224,409 and 224,831 in the genome of L. monocytogenes (serotype 1/2a strain EGD-e), including a 145-bp noncoding region and a 278-bp partial coding sequence of a putative gene, lmo2234. The translated amino acid sequence (92 amino acids) of this partial coding region is highly conserved between L. monocytogenes and Listeria innocua (93% homology). Reverse-position-specific BLAST analysis identified a conserved domain in Lmo2234 that was similar (95.3% aligned, E value = 9E-18) to the consensus amino acid sequence of sugar phosphate isomerases/epimerases (National Center for Biotechnology Information conserved domain database accession no. COG 1082.1, IolE), indicating that Lmo2234 might be involved in bacterial carbohydrate transport and metabolism.
The neurotransmitter acetylcholine is synthesized by choline acetyltransferase (ChAT; EC 2.3.1.6). Since the expression of ChAT in the nervous system is restricted to cholinergic neurons, it serves as a specific marker for these neurons. In Alzheimer disease, ChAT activity is markedly reduced in the affected brain areas. Nerve growth factor can increase the ChAT activity of brain cholinergic neurons in vitro and in vivo. We have cloned the rat ChAT gene and identified one 5' noncoding exon and 14 exons that account for the entire coding sequence. The exon organization is compared with the protein domains conserved during evolution. These exons are distributed over at least 64 kilobases in the rat genome; the largest intron is at least 14 kilobases long. Within a 0.7-kilobase region immediately upstream of the confirmed sequence of the noncoding exon, TATA-like elements and numerous potential binding sites for transcription factors are found, including AP-1, Sp1, octamer-binding factor(s), CTF/NF-1, and the nuclear oncoprotein Myb.
Complementary DNA spanning the entire genome of the attenuated rabies virus strain SAD B19 which is used for oral immunization of foxes in Europe was cloned and sequenced. The viral genome comprises 11,928 nucleotides and encodes the five viral proteins N, NS, M, G, and L. Deduced protein sequences are highly similar to those of the pathogenic PV strain, homologies ranging from 90.6% for the M to 98.6% for the L protein. The five cistrons are separated by intergenic regions of 2, 5, 5, and 24 nucleotides, respectively. The G transcription stop/polyadenylation consensus signal in SAD B19 is destroyed by a deletion of three A residues. The strong conservation of both noncoding and coding nucleotide sequences indicates a high selective pressure on the primary structure of rabies virus genomic RNA.
We evaluated the formation and removal of (+-)-3 alpha,4 beta-dihydroxy-1 alpha,2 alpha-epoxy-1,2,3,4- tetrahydrobenzo[c]phenanthrene (BcPHDE)-DNA adducts in two Chinese hamster ovary (CHO) cell lines. One line of repair-proficient cells (MK42) carries a stable 150-fold amplification of the dihydrofolate reductase (DHFR) locus. The other line of repair-deficient cells (UV-5) is diploid for this gene and is defective in excision of bulky DNA lesions. Two methods were used to quantitate adduct levels in treated cells: Escherichia coli UvrABC excision nuclease cleavage and 32P-postlabeling. DNA repair was examined in the actively transcribed DHFR gene, in an inactive region located 25 kilobases downstream, and in the overall genome. Between 8 and 24 hr after BcPHDE exposure, preferential repair of the DHFR gene compared to the noncoding region was apparent in MK42 cells. This gene-specific repair was associated with adduct removal from the DHFR transcribed strand. However, UV-5 cells showed no lesion reduction from this strand of the gene. By both quantitation methods, regions accessible to repair in MK42 cells showed a 2-fold reduction in DNA adduct levels by 24 hr. That the decline in adducts reflects genomic repair was demonstrated by the constant damage level remaining in UV-5 cells. Since BcPHDE-induced mutations in DHFR apparently arise from adducted purines on the nontranscribed strand, results from the present study support the idea that a consequence of strand-specific repair is strand-biased mutations.
A novel cDNA encoding the nonselective type of endothelin (ET) receptor was isolated from a rat brain cDNA library. The cloned cDNA encoded a 442-amino acid protein with seven putative transmembrane domains. Nucleotide sequence analysis showed that the rat brain cDNA differed from the cloned rat lung nonselective ET receptor (ETB) cDNA by three extra nucleotides in its coding regions, which produced an encoded protein with four amino acid substitutions. In addition, both the 5' and 3' noncoding sequences of the rat brain cDNA were divergent from those of rat lung cDNA. Expression of the rat brain cDNA in COS-1 cells demonstrated that the encoded receptor displayed equal affinity toward the three ET isopeptides. However, Southern blot analysis indicated a single-copy gene for the rat ETB receptor. Further genomic cloning and sequence analysis demonstrated that rat brain cDNA encoded the authentic protein sequences of the rat ETB receptor. Moreover, the 5' noncoding sequences in rat brain cDNA that were divergent from those in rat lung cDNA were encoded by a distinct region, an upstream exon, in the rat ETB genome. All the findings suggest that rat brain cDNA represents an alternative transcript of the rat ETB gene. Preliminary Northern blot analysis indicated that the expression of this ETB cDNA sequence might be not only in the brain but also in other tissues, whereas its expression might be somehow tissue-specifically regulated.
Sequences critical for the transcription and replication functions of canine distemper virus (CDV) RNA polymerase were analyzed. The sequence was obtained from polymerase chain reaction (PCR) products using either c-DNA clones from a genomic library as template or in most instances genomic CDV RNA. Clones coding for the precise 3'- and 5'-ends of the CDV genome were sequenced and the results confirmed by additional PCR experiments. The virtual identity of terminal sequences and spacing at the two noncoding ends speak to the importance of these areas in replication and transcription. The sequence for each of the CDV gene boundaries was defined and all were compared to related viruses. This report completes the sequence determination of the CDV genome.
Transposable elements (TEs) have been identified in every organism in which they have been looked for. The sequencing of large genomes, such as the human genome and those of Drosophila, Arabidopsis, Caenorhabditis, has also shown that they are a major constituent of these genomes, accounting for 15% of the genome of Drosophila, 45% of the human genome, and more than 70% in some plants and amphibians. Compared with the 1% of genomic DNA dedicated to protein-coding sequences in the human genome, this has prompted various researchers to suggest that the TEs and the other repetitive sequences that constitute the so-called "noncoding DNA", are where the most stimulating discoveries will be made in the future (Bromham, 2002). We are therefore getting further and further from the original idea that this DNA was simply "junk DNA", that owed its presence in the genome entirely to its capacity for selfish transposition. Our understanding of the structures of TEs, their distribution along the genomes, their sequence and insertion polymorphisms within genomes, and within and between populations and species, their impact on genes and on the regulatory mechanisms of genetic expression, their effects on exon shuffling and other phenomena that reshape the genome, and their impact on genome size has increased dramatically in recent years. This leads to a more general picture of the impact of TEs on genomes, though many copies are still mainly selfish or junk DNA. In this review we focus mainly on discoveries made in Drosophila, but we also use information about other genomes when this helps to elucidate the general processes involved in the organization, plasticity, and evolution of genomes.
The portion of the pheromone 3 (Phr3)-encoding gene (phr3) of Euplotes octocarinatus, corresponding to secreted Phr3 was isolated using the polymerase chain reaction and oligodeoxyribonucleotide primers flanking the Phr3-encoding cDNA. Unexpectedly, the sequence analysis revealed that this gene is interrupted by two introns with lengths of 63 bp and 72 bp. These introns belong to the class of nuclear pre-mRNA introns and contain typical 5'- and 3'-consensus sequences, as well as unique features. Our findings constitute the first example of introns in any Euplotes species and the first case of multiple introns in hypotrichous ciliates. In ciliates such as Euplotes, the macronucleus is the transcriptionally active nucleus. It develops from a micronucleus in a process which involves chromosome breakage and the elimination of up to 95% of the micronuclear genome. The existence of the introns in the macronuclear version of phr3 shows that, in spite of this extensive elimination, some noncoding sequences are nevertheless retained in the macronucleus of hypotrichous ciliates. It was recently discovered by Meyer et al. [Proc. Natl. Acad. Sci. USA 88 (1991) 3758-3761] that the Phr3-encoding cDNA contains three in frame TGA triplets coding for cysteine. The genomic sequence of phr3 confirms this finding.
Fifty years after the publication of DNA structure, the whole human genome sequence will be officially finished. This achievement marks the beginning of the task to catalogue every human gene and identify each of their function expression patterns. Currently, researchers estimate that there are about 30,000 human genes and approximately 70% of these can be automatically predicted using a combination of ab initio and similarity-based programs. However, to experimentally investigate every gene's function, the research community requires a high-quality annotation of alternative splicing, pseudogenes, and promoter regions that can only be provided by manual intervention. Manual curation of the human genome will be a long-term project as experimental data are continually produced to confirm or refine the predictions, and new features such as noncoding RNAs and enhancers have not been fully identified. Such a highly curated human gene-set made publicly available will be a great asset for the experimental community and for future comparative genome projects.
Recent work in the Triticum-Aegilops complex demonstrates that allopolyploidization is associated with an array of changes in low-copy coding and noncoding sequences. Nevertheless, the behavior and fate of repetitive DNA elements that constitute the bulk of nuclear DNA of these plant species is less clear following allopolyploidy. To gain further insight into the genomic events that accompany allopolyploid formation, we investigated fluorescence in situ hybridization (FISH) patterns of a parental-specific, tandem DNA repeat (pGc1R-1) on three sets of newly synthesized amphiploids with different parental species. It was found that drastic physical elimination of pGc1R-1 copies occurred in all three amphiploids in early generations. DNA gel-blot analysis confirmed the FISH data and estimates indicated that approximately 70-90% of the copies of the pGc1R-1 repeat family were eliminated from the amphiploids by the second to third selfed generations. Thus, allopolyploidy in Triticum-Aegilops can be accompanied by rapid and extensive elimination of parental-specific repetitive DNA sequences, which presumably play a role in the initial stabilization of the nascent amphiploid plants.
A report is presented about studies on poliovirus type 3 isolates from vaccine-associated cases or contacts of cases of paralytic poliomyelitis, observed over a period of 30 years in East Germany (former GDR). In the viral isolates, some mutations were found in comparison to the Sabin vaccine type 3 strain, distributed over the whole genome. The significance of these mutations has been discussed, especially the mutation at position 472 in the 5' noncoding region found in all the isolates investigated. In five isolates, intertypic recombination between Sabin type 3 and Sabin type 1 vaccine strain occurred. Primary and secondary structures were analysed for the recombination sites.
We recently described the derivation of a dengue serotype 2 virus (DEN2mutF) that exhibited a host range-restricted phenotype; it was severely impaired for replication in cultured mosquito cells (C6/36 cells). DEN2mutF virus had selected mutations in genomic sequences predicted to form a 3' stem-loop structure (3'-SL) that is conserved among all flavivirus species. The 3'-SL constitutes the downstream terminal similar95 nucleotides of the 3' noncoding region in flavivirus RNA. Here we report the introduction of these same mutational changes into the analogous region of an infectious DNA derived from the genome of a human-virulent dengue serotype 1 virus (DEN1), strain Western Pacific (DEN1WP). The resulting DEN1 mutant (DEN1mutF) exhibited a host range-restricted phenotype similar to that of DEN2mutF virus. DEN1mutF virus was attenuated in a monkey model for dengue infection in which viremia is taken as a correlate of human virulence. In spite of the markedly reduced levels of viremia that it induced in monkeys compared to DEN1WP, DEN1mutF was highly immunogenic. In addition, DEN1mutF-immunized monkeys retained high levels of neutralizing antibodies in serum and were protected from challenge with high doses of the DEN1WP parent for as long as 17 months after the single immunizing dose. Phenotypic revertants of DEN1mutF and DEN2mutF were each detected after a total of 24 days in C6/36 cell cultures. Complete nucleotide sequence analysis of DEN1mutF RNA and that of a revertant virus, DEN1mutFRev, revealed that (i) the DEN1mutF genome contained no additional mutations upstream from the 3'-SL compared to the DEN1WP parent genome and (ii) the DEN1mutFRev genome contained de novo mutations, consistent with our previous hypothesis that the defect in DEN2mutF replication in C6/36 cells was at the level of RNA replication. A strategy for the development of a tetravalent dengue vaccine is discussed.
The X chromosomes of mammals and fruit flies exhibit unusual properties that have evolved to deal with the different dosages of X-linked genes in males (XY) and females (XX). The X chromosome dosage-compensation mechanisms discovered in these species are evolutionarily unrelated, but exhibit surprising parallels in their regulatory strategies. These features include the importance of noncoding RNAs, and epigenetic spreading of chromatin-modifying activities. Sex chromosomes have posed a fascinating puzzle for biologists. The dissimilar organization, gene content, and regulation of the X and Y chromosomes are thought to reflect selective forces acting on original pairs of identical chromosomes (1-3). The result in many organisms is a male-specific Y chromosome that has lost most of its original genetic content, and a difference in dosage of the X chromosome in males (XY) and females (XX).
Cytosine methylation is important for transposon silencing and epigenetic regulation of endogenous genes, although the extent to which this DNA modification functions to regulate the genome is still unknown. Here we report the first comprehensive DNA methylation map of an entire genome, at 35 base pair resolution, using the flowering plant Arabidopsis thaliana as a model. We find that pericentromeric heterochromatin, repetitive sequences, and regions producing small interfering RNAs are heavily methylated. Unexpectedly, over one-third of expressed genes contain methylation within transcribed regions, whereas only approximately 5% of genes show methylation within promoter regions. Interestingly, genes methylated in transcribed regions are highly expressed and constitutively active, whereas promoter-methylated genes show a greater degree of tissue-specific expression. Whole-genome tiling-array transcriptional profiling of DNA methyltransferase null mutants identified hundreds of genes and intergenic noncoding RNAs with altered expression levels, many of which may be epigenetically controlled by DNA methylation.
The human polyomavirus BK (BKV) has a proven oncogenic potential, but its contribution to tumorigenesis under natural conditions remains undetermined. As for other primate polyomaviruses, the approximately 5.2 kbp double-stranded circular genome of BKV has three functional regions: the coding regions for the two early (T, t antigens) and four late (agno, capsid proteins; VP1-3) genes separated by a noncoding control region (NCCR). The NCCR contains the origin of replication as well as a promoter/enhancer with a mosaic of cis-acting elements involved in the regulation of both early and late transcription. Since the original isolation of BKV in 1971, a number of other strains have been identified. Most strains reveal a strong sequence conservation in the protein coding regions of the genome, while the NCCR exhibits considerable variation between different BKV isolates. This variation is due to deletions, duplications, and rearrangements of a basic set of sequence blocks. Comparative studies have proven that the anatomy of the NCCR may determine the transcriptional activities governed by the promoter/enhancer, the host cell tropism and permissivity, as well as the oncogenic potential of a given BKV strain. In most cases, however, the NCCR sequence of new isolates was determined after the virus had been passaged several times in more or less arbitrarily chosen cell cultures, a process known to predispose for NCCR rearrangements. Following the development of the polymerase chain reaction (PCR), it has become feasible to obtain naturally occurring BKV NCCRs, and their sequences, in samples taken directly from infected human individuals. Hence, the biological significance of BKV NCCR variation may be studied without prior propagation of the virus in cell culture. Such variation has general interest, because the BKV NCCRs represent typical mammalian promoter/enhancers, with a large number of binding motifs for cellular transacting factors, which can be conveniently handled for experimental purposes. This communication reviews the naturally occurring BKV NCCR variants, isolated and sequenced directly from human samples, that have been reported so far. The sequences of the different NCCRs are compared and analyzed for the presence of proven and putative cellular transcription factor binding sites. Differences in biological properties between BKV variants are discussed in light of their aberrant NCCR anatomies and the potentially modifying influence of transacting factors.
The mu-opioid receptor (MOR) plays a mandatory role in the action of most opioid drugs, such as morphine, fentanyl, and heroin. It has been revealed that a deficiency in the MOR gene (Oprm1) or a difference in the 3' noncoding region of the gene markedly affects the sensitivity of mice to opioids. As the 3' noncoding region of the human OPRM1 gene had not yet been characterized, in the present study we conducted 3'-rapid amplification of cDNA ends (3'RACE)-PCR and identified the 3' end of the human MOR-1 mRNA, the most abundant transcript among OPRM1 gene transcripts. The poly(A) signal was located at 13612-13617 nucleotides downstream from the stop codon in the OPRM1 gene. Reverse transcription PCR analyses showed that the region from the stop codon to the poly(A) signal was transcribed. In the 3'UTR, we identified 33 AU-rich regions and more than 300 putative transcription factor-binding sites. Furthermore, we compared the 3' noncoding regions of the human and mouse OPRM1/Oprm1 genes and found apparent homology. In Northern blotting with mouse brain mRNAs, a same-size band was detected by a probe for the MOR-1 coding region and by a probe for a mouse genome region corresponding to the human MOR-1 3'UTR. Since 3'UTRs affect gene expression, the present characterization of the 3' noncoding region in the human OPRM1 gene should lead to a better understanding of the mechanisms underlying OPRM1 gene regulation and individual differences in sensitivity to opioids.