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PubMed1984

Characterization of rat ribosomal DNA. The highly repetitive sequences that flank the ribosomal RNA transcription unit are homologous and contain RNA polymerase III transcription initiation sites.

The non-transcribed spacers (NTS) of the ribosomal genes of a number of organisms have been studied and were found to contain repetitive sequences. In these studies with plasmid subclones of NTS, designated p3.4, p2.6 and p1.7, which come from both 5' and 3' flanking regions of the rat ribosomal genes, respectively, it has been determined that these sequences are found elsewhere within the genome. Southern hybridization analysis has demonstrated that the 5' and 3' NTS subclones cross-hybridize, and that the cross-hybridizing regions are synonymous with the highly repetitive regions. Sequences homologous to the rat NTS were specifically localized to both 5' and 3' flanking regions as well as to a number of the introns of cloned genes including rat serum albumin, rat alpha-fetoprotein, rat casein and human serum albumin. No hybridization was detected of the 5' NTS subclone to the human Alu sequence clone, Blur 8, or to the rodent equivalent, a clone containing Chinese hamster ovary type I and II Alu sequences. However, as reported for type II Alu sequences, the subcloned rat NTS sequences contain RNA polymerase III initiation sites and also hybridize to a number of small RNAs, but not 4.5 S or 7 S RNA. Sequence analysis of two distinct repetitive regions in p1.7 has revealed a region of alternating purine-pyrimidine nucleotides, potentially of Z DNA, and stretches of repetitive sequences. The possible roles for these repetitive sequences in recombination and in maintaining a hierarchical structure for the ribosomal genes are discussed.

Animals↗
PubMed2000

Multiple amino acid substitutions allow DNA polymerases to synthesize RNA.

DNA and RNA polymerase exhibit similarities in structures and catalytic mechanisms, suggesting that both classes of enzymes are evolutionarily related. To probe the biochemical and structure-function relationship between the two classes of polymerases, a large library (200,000 members) of mutant Thermus aquaticus DNA polymerase I (Taq pol I) was created containing random substitutions within a portion of the dNTP binding site (motif A; amino acids 605-617), and a fraction of all selected active Taq pol I (291 of 8000) was tested for the ability to incorporate successive ribonucleotides; 23 unique mutants that added rNTPs into a growing polynucleotide chain were identified and sequenced. These mutants, each containing one to four substitutions, incorporate ribonucleotides at a efficiency approaching 10(3)-fold greater than that of wild type Taq pol I. Several mutants added successive ribonucleotides and thus can catalyze the synthesis of RNA. Sequence analysis of these mutants demonstrates that at least two amino acid residues are involved in excluding ribonucleotides from the active site. Interestingly, wild type DNA polymerases from several distinct families selectively discriminate against rUTP. This study suggests that current DNA and RNA polymerases could have evolved by divergent evolution from an ancestor that shared a common mechanism for polynucleotide synthesis.

Amino Acid Substitution↗
PubMed2003

Pair hidden Markov models on tree structures.

MOTIVATION: Computationally identifying non-coding RNA regions on the genome has much scope for investigation and is essentially harder than gene-finding problems for protein-coding regions. Since comparative sequence analysis is effective for non-coding RNA detection, efficient computational methods are expected for structural alignments of RNA sequences. On the other hand, Hidden Markov Models (HMMs) have played important roles for modeling and analysing biological sequences. Especially, the concept of Pair HMMs (PHMMs) have been examined extensively as mathematical models for alignments and gene finding. RESULTS: We propose the pair HMMs on tree structures (PHMMTSs), which is an extension of PHMMs defined on alignments of trees and provides a unifying framework and an automata-theoretic model for alignments of trees, structural alignments and pair stochastic context-free grammars. By structural alignment, we mean a pairwise alignment to align an unfolded RNA sequence into an RNA sequence of known secondary structure. First, we extend the notion of PHMMs defined on alignments of 'linear' sequences to pair stochastic tree automata, called PHMMTSs, defined on alignments of 'trees'. The PHMMTSs provide various types of alignments of trees such as affine-gap alignments of trees and an automata-theoretic model for alignment of trees. Second, based on the observation that a secondary structure of RNA can be represented by a tree, we apply PHMMTSs to the problem of structural alignments of RNAs. We modify PHMMTSs so that it takes as input a pair of a 'linear' sequence and a 'tree' representing a secondary structure of RNA to produce a structural alignment. Further, the PHMMTSs with input of a pair of two linear sequences is mathematically equal to the pair stochastic context-free grammars. We demonstrate some computational experiments to show the effectiveness of our method for structural alignments, and discuss a complexity issue of PHMMTSs.

Algorithms↗
PubMed1990

A transpositionally and transcriptionally competent Alu subfamily.

DNA base sequence comparisons indicate that a subfamily of recently transposed human Alu repeats are distinguished from most Alu repeats by diagnostic sequence differences. Using an oligonucleotide hybridization probe that incorporates these sequence features, we found that there was an expansion of this Alu subfamily following the divergence of humans and African apes. This oligonucleotide was used to select human genomic clones containing representatives of this subfamily. One representative member of this subfamily was evidently absent from the corresponding chimpanzee locus and was associated with a restriction fragment length polymorphism in the human genome. This apparently polymorphic member had all the diagnostic sequence features that initially predicted the existence of a newly expanding Alu subfamily. A transpositionally active sequence variant should also be transcriptionally active in at least some cell types or tissues. Northern (RNA) blot hybridization, primer extension, and RNA sequence analysis demonstrated the existence of different-length polyadenylated and nonpolyadenylated transcripts corresponding to this subfamily. Evidence for 3' processing and subcellular localization of these transcripts is discussed. Most of the nearly one million human Alu repeats are pseudogenes with respect to coding for either an RNA product or new family members; a select and identifiable subset of Alu repeats serve as transcriptionally and transpositionally competent source genes.

Base Sequence↗
PubMed2005

[Origin of internal genes of two strains of swine influenza A (H1N1) virus].

BACKGROUND: Through the analysis of internal genes of viruses to understand whether the internal genes of two strains of swine influenza A(H1N1) virus contain the gene segment deriving from avian influenza A viruses, and whether the reassortment of the internal genes occurred between swine H1N1 and swine H9N2 viruses. METHODS: Viruses were passaged in embryonated hen eggs and virion RNA was extracted from allantoic fluids and reverse transcribed to synthesize cDNA. cDNA was amplified by PCR and the PCR product was purified with a purification kit. Afterwards, RNA sequence analysis was performed by dideoxynucleotide chain termination and a cloning method. Finally, phylogenetic analysis of the sequencing data was performed with MegAlign (version 1.03). RESULTS: All the six, but PB2 gene segment internal gene segments were the same between A/swine/Beijing/1/2002(H1N1) and A/Swine/Beijing/3/2002(H1N1) viruses. Whereas all the six internal gene segments in two trains of swine (H1N1) virus were similar to those of swine H1N1 viruses, but different from those of classical strain of swine (H1N1) virus. CONCLUSION: Two strains of Beijing swine H1N1 influenza A virus were not reassortant, All the six internal gene segments were closely related to swine influenza A (H1N1) viruses.

Animals↗
PubMed1995

Isolation and characterization of a cDNA encoding for preprosomatostatin containing [Tyr7, Gly10]-somatostatin-14 from the endocrine pancreas of rainbow trout, Oncorhynchus mykiss.

Somatostatins are a diverse group of peptides known to influence various aspects of growth and metabolism of vertebrates. In order to further our understanding of the physiological roles of somatostatins in fish, we initiated an analysis of somatostatin gene structure and expression in rainbow trout pancreas. Using rapid amplification of cDNA ends polymerase chain reaction, we have isolated, cloned, and sequenced a novel cDNA derived from pancreatic total RNA. Sequence analysis revealed a 624-bp cDNA containing the complete 5'-untranslated region with a single initiation site 107 bases from the most 5' end and a single putative polyadenylation site 13 bases from the most 3' end that was terminated with a polyadenylated tail. The deduced protein is a 115-amino-acid preprosomatostatin molecule with [Tyr7,Gly10]-somatostatin-14 at the C-terminus of the coding region, making the rainbow trout precursor a member of the preprosomatostatin II family. Based on the location of putative cleavage sites, we propose that rainbow trout pancreatic preprosomastatin II is processed to yield a 28-amino-acid and/or, possibly, a 14-amino-acid somatostatin II molecule. The results also suggest that there has been limited conservation of the preprosomatostatin II gene family among teleosts.

Amino Acid Sequence↗
PubMed2025

In Vivo CRISPR Activation Screening Reveals Chromosome 1q Genes VPS72, GBA1, and MRPL9 Drive Hepatocellular Carcinoma.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) frequently undergoes regional chromosomal amplification, resulting in elevated gene expression levels. We aimed to elucidate the role of these poorly understood genetic changes by using CRISPR activation (CRISPRa) screening in mouse livers to identify which genes within these amplified loci are cancer driver genes. METHODS: We used data from The Cancer Genome Atlas to identify that frequently copy number-amplified and up-regulated genes all reside on human chromosomes 1q and 8q. We generated CRISPRa screening transposons that contain oncogenic Myc to drive tumor formation. We conducted CRISPRa screens in vivo in the liver to identify tumor driver genes. We extensively validated the findings in separate mice and performed RNA sequencing analysis to explore mechanisms driving tumorigenesis. RESULTS: We targeted genes that frequently undergo amplification in human HCC using an in vivo CRISPRa screening system in mice, which induced extensive liver tumorigenesis. Human chromosome 1q genes Zbtb7b, Vps72, Gba1, and Mrpl9 emerged as drivers of liver tumorigenesis. In human HCC there is a trend in correlation between levels of MRPL9, VPS72, or GBA1 and poor survival. In validation assays, activation of Vps72, Gba1, or Mrpl9 resulted in extensive liver tumorigenesis and decreased survival in mice. RNA sequencing revealed different mechanisms driving HCC, with Mrpl9 activation altering genes functionally related to mitochondrial function, Vps72 levels altering phospholipid metabolism, and Gba1 activation enhancing endosomal-lysosomal activity, all leading to promotion of cellular proliferation. Analysis of human tumor tissues with high levels of MRPL9, VPS72, or GBA1 revealed congruent results, indicating conserved mechanisms driving HCC. CONCLUSIONS: This study reveals chromosome 1q genes Vps72, Gba1, and Mrpl9 as drivers of HCC. Future efforts to prevent or treat HCC can focus on these new driver genes.

Animals↗
PubMed2003

Exocellular cyclic dipeptides from a Ruegeria strain associated with cell cultures of Suberites domuncula.

From cell cultures of Suberites domuncula was isolated a bacterial strain, SDC-1, which was identified by 16S ribosomal RNA sequence analysis as an alpha-Proteobacterium of the genus Ruegeria. The occurrence of the strain in sponge cell culture could be explained by its resistance to the antibiotics used in the isolation of sponge cell cultures or by the preservation of SDC-1 by host sponge cells. The fatty acid composition of SDC-1 is characterized by branched C-12 methyl fatty acids. Two new and 8 known cyclic dipeptides were isolated and characterized from the fermentation broth of SDC-1. Cyclodipeptides are one of the families of cell-cell signaling compounds and may have some role to play in sponge-bacteria interactions.

Animals↗
PubMed2002

Transcriptional analysis of a novel cluster of LY-6 family members in the human and mouse major histocompatibility complex: five genes with many splice forms.

Lymphocyte antigen-6 (LY-6) superfamily members are cysteine-rich, generally GPI-anchored cell surface proteins, which have definite or putative immune related roles. A cluster of five potential LY-6 superfamily members is located in the human and mouse major histocompatibility complex class III region. Comparative analysis of their genomic and cDNA sequences allowed us to carry out detailed annotations of these genes. We analyzed their mRNA expression patterns by RT-PCR performed on human and mouse cell line and tissue RNA. Sequence analysis of the transcripts revealed splice variants of all these genes in humans, and all but one in mouse. These splice forms retained introns or intron fragments, mainly generating premature stop codons, such that the only potentially functional mRNA was the predicted form. In some cases, the mis-spliced form was the most abundant form, suggesting a control mechanism for gene expression. Each gene showed mRNA expression differences between human and mouse.

Alternative Splicing↗
PubMed1999

Prevalence and characterization of hepatitis C virus in hemodialysis patients.

OBJECT: Chronic hepatitis C virus (HCV) infection is common in hemodialysis (HD) patients. In the present study, the prevalence and properties of HCV in HD patients were analyzed. METHODS AND RESULTS: Of 125 HD patients, 34 (27%) were positive for antibody to HCV, and HCV-RNA was detected in 23 (68%) of the 34 patients using reverse transcription polymerase chain reaction. The HCV-RNA sequence analysis did not identify the alterations specific to HD patients with HCV, although one patient had a variant virus containing the deletion of the core gene sequence. When serial changes in the levels of HCV-RNA were evaluated in 15 patients by a branched DNA assay, the values decreased immediately after HD procedure, but returned to the baseline values 2 days after the procedure. CONCLUSION: These results indicate that HCV in HD patients is replication-competent, although a transient reduction in the levels of HCV-RNA occurs during HD.

Adult↗
PubMed1993

Concurrent sequence analysis of 5' and 3' RNA termini by intramolecular circularization reveals 5' nontemplated bases and 3' terminal heterogeneity for lymphocytic choriomeningitis virus mRNAs.

We have used a technique of RNA circularization coupled with polymerase chain reaction amplification for simultaneous analysis of the 5' and 3' termini of subgenomic mRNAs derived from the S RNA of lymphocytic choriomeningitis virus during an acute infection of BHK cells. These mRNAs possess 1 to 7 nontemplated nucleotides of apparently random sequence at their 5' ends. The predominant mRNA species have 4 or 5 nontemplated nucleotides. The 5' termini of the mRNAs also have properties consistent with the presence of a 5' cap structure. The 3' termini of the mRNAs lack poly(A) tails, and we have shown that transcription termination occurs at heterogeneous positions within the intergenic region of the S RNA. The identification of several distinct termini in the vicinity of a putative stem-loop structure in the RNA templates suggests that transcription termination may be mediated by a structural signal rather than a precise sequence signal.

Animals↗
PubMed2004

Developmental regulation of EVF-1, a novel non-coding RNA transcribed upstream of the mouse Dlx6 gene.

We previously reported that sonic hedgehog (Shh) induces the differentiation of rat ventral forebrain neurons expressing a novel marker, EVF-1 [Development 125 (1998) 5079]. In this report, we show that EVF-1 is a novel, developmentally regulated, non-coding RNA, with no homology to other known non-coding RNA sequences. Sequence analysis, in vitro translation, and comparison of the rat and mouse EVF-1 sequences suggest that EVF-1 contains no protein coding regions. Chromosomal location indicates that EVF-1 maps adjacent to the Dlx6 gene on mouse chromosome 6. RNA in situ hybridization of the embryonic rat forebrain shows that EVF-1 is expressed by immature neurons in the subventricular zone and its expression decreases during forebrain development. Whole mount in situ hybridization shows that EVF-1 is expressed at high levels in the branchial arches, ventral forebrain, olfactory bulb, and limbs. EVF-1 expression is linked to Shh and the Dlx family of proteins, genes with a demonstrated importance to ventral forebrain and craniofacial development.

Animals↗
PubMed2006

Hereditary haemorrhagic telangiectasia: mutation detection, test sensitivity and novel mutations.

BACKGROUND: Hereditary haemorrhagic telangiectasia (HHT) is a genetic disorder present in 1 in 8000 people and associated with arteriovenous malformations. Genetic testing can identify individuals at risk of developing the disease and is a useful diagnostic tool. OBJECTIVE: To present a strategy for mutation detection in families clinically diagnosed with HHT. METHODS: An optimised strategy for detecting mutations that predispose to HHT is presented. The strategy includes quantitative multiplex polymerase chain reaction, sequence analysis, RNA analysis, validation of missense mutations by amino acid conservation analysis for the ENG (endoglin) and ACVRL1 (ALK1) genes, and analysis of an ACVRL1 protein structural model. If no causative ENG or ACVRL1 mutation is found, proband samples are referred for sequence analysis of MADH4 (associated with a combined syndrome of juvenile polyposis and HHT). RESULTS: Data obtained over the past eight years were summarised and 16 novel mutations described. Mutations were identified in 155 of 194 families with a confirmed clinical diagnosis (80% sensitivity). Of 155 mutations identified, 94 were in ENG (61%), 58 in ACVRL1 (37%), and three in MADH4 (2%). CONCLUSIONS: For most missense variants of ENG and ACVRL1 reported to date, study of amino acid conservation showed good concordance between prediction of altered protein function and disease occurrence. The 39 families (20%) yet to be resolved may carry ENG, ACVRL1, or MADH4 mutations too complex or difficult to detect, or mutations in genes yet to be identified.

Activin Receptors, Type II↗
PubMed1978

Nucleotide sequence of the DNA encoding the 5'-terminal sequences of simian virus 40 late mRNA.

We have used a combination of techniques of DNA and RNA sequence analysis to determine the nucleotide sequence of the portion of simian virus 40 DNA preceding and encoding the 5' end of mRNA for the structural protein VP2 of simian virus 40. Comparison of the sequence with those found in polyadenylated RNA in the cytoplasm of infected cells RNA shows that the transcript of sequences preceding the structural gene is more abundant than the transcript containing the codons for the protein. Between the abundant transcript of sequences preceding the coding region and the less abundant transcript of the coding region there is a short sequence whose transcript is not detected.

Base Sequence↗
PubMed1985

Structure and expression of the M2 genomic segment of a type 2 killer virus of yeast.

The M2 double-stranded (ds) RNA species encodes toxin and resistance functions in Saccharomyces cerevisiae strains with the K2 killer specificity. RNA sequence analysis reveals the presence of a large open reading frame on the larger heat-cleavage product of M2 dsRNA, which is translated in vitro to yield a 28 kd polypeptide as a major product. The postulated translation initiator AUG triplet is located within a stem and loop structure near the 5' terminus of the positive strand, which also contains plausible 18S and 5.8S ribosomal RNA binding sites. These features may serve to regulate the translation of the K2 toxin precursor. The M1 (from type 1 yeast killers) and M2 dsRNA species lack extensive sequence homology, although specific features are shared, which may represent structural elements required for gene expression and replication.

Amino Acid Sequence↗
PubMed1995

A cis-acting viral protein is not required for the replication of a coronavirus defective-interfering RNA.

Mouse hepatitis virus (MHV), a coronavirus, generates defective-interfering (DI) RNAs of different sizes during passages at high multiplicities of infection. All MHV DI RNAs characterized so far contain an open reading frame (ORF) encoding a fused viral protein; in addition, DI RNAs with a long ORF have a competitive advantage over those with a shorter ORF. These findings suggest that DI RNA replication may require an ORF encoding a cis-acting viral protein. In this study, we used a naturally occurring DI RNA and inserted a 12-nucleotide (nt) amber-mutation linker at various positions to truncate the ORF. Most of the mutants replicated as well as the wild-type DI RNA, irrespective of the presence or absence and the length of the ORF in the RNA. Sequence analysis showed that all of the mutants retained the insertional mutations even after two viral passages in tissue culture, establishing that the mutant DI RNAs replicated. We have further introduced two 3-nucleotide substitutions of the first two AUG codons of the ORF, thus completely closing the ORF. This DI RNA replicated as well as the wild-type DI, but, after a single passage, the majority of the mutant RNAs was replaced by recombinant RNAs which contain a restored functional ORF. However, an additional insertion of a 12-nt amber-mutation linker downstream of the AUG substitutions prevented recombination, and the DI RNA still replicated. These data indicate that DI RNA replication does not require a DI-specific ORF encoding cis-acting viral proteins and that a 12-nucleotide insertion could prevent or delay the occurrence of RNA recombination, suggesting the importance of direct or indirect RNA alignment in homologous RNA recombination.

Animals↗
PubMed2004

Secondary structure and function of the 5'-proximal region of the equine arteritis virus RNA genome.

Nidoviruses produce an extensive 3'-coterminal nested set of subgenomic mRNAs, which are used to express their structural proteins. In addition, arterivirus and coronavirus mRNAs contain a common 5' leader sequence, derived from the genomic 5' end. The joining of this leader sequence to different segments (mRNA bodies) from the genomic 3'-proximal region presumably involves a unique mechanism of discontinuous minus-strand RNA synthesis. Key elements in this process are the so-called transcription-regulating sequences (TRSs), which determine a base-pairing interaction between sense and antisense viral RNA that is essential for leader-to-body joining. To identify RNA structures in the 5'-proximal region of the equine arteritis virus genome that may be involved in subgenomic mRNA synthesis, a detailed secondary RNA structure model was established using bioinformatics, phylogenetic analysis, and RNA structure probing. According to this structure model, the leader TRS is located in the loop of a prominent hairpin (leader TRS hairpin; LTH). The importance of the LTH was supported by the results of a mutagenesis study using an EAV molecular clone. Besides evidence for a direct role of the LTH in subgenomic RNA synthesis, indications for a role of the LTH region in genome replication and/or translation were obtained. Similar LTH structures could be predicted for the 5'-proximal region of all arterivirus genomes and, interestingly, also for most coronaviruses. Thus, we postulate that the LTH is a key structural element in the discontinuous subgenomic RNA synthesis and is likely critical for leader TRS function.

Animals↗
PubMed2003

Relationship between nosocomial Acinetobacter species occurring in two geographical areas (Greece and the UK).

Fifty-two isolates of Acinetobacter spp. obtained from three Greek and one UK hospital, were studied using partial 16 S ribosomal DNA sequence analysis, repetitive extragenic palindromic sequence-based polymerase chain reaction (REP-PCR) mediated fingerprinting and DNA macro-restriction analysis. The aim was twofold: first, to discern the major differences in the population of Acinetobacter spp. between the two countries. Second, to compare a simple PCR-based typing scheme with pulsed-field gel electrophoresis (PFGE). The multi-resistant Greek isolates were within DNA groups 2 and TU13, and clustered into three types both by REP-PCR and PFGE. By contrast, the more susceptible Oxford isolates were heterogeneous on 16 S RNA sequence analysis and distinguishable on typing. The need for studies that elucidate the phylogeny of Acinetobacter spp. inside and outside hospitals are important, as this will help clarify the relationship between organisms that are increasingly recognized as causes of severe infections.

Acinetobacter Infections↗