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PubMed2005

Long-range DNase I hypersensitivity mapping reveals the imprinted Igf2r and Air promoters share cis-regulatory elements.

Epigenetic mechanisms restrict the expression of imprinted genes to one parental allele in diploid cells. At the Igf2r/Air imprinted cluster on mouse chromosome 17, paternal-specific expression of the Air noncoding RNA has been shown to silence three genes in cis: Igf2r, Slc22a2, and Slc22a3. By an unbiased mapping of DNase I hypersensitive sites (DHS) in a 192-kb region flanking Igf2r and Air, we identified 21 DHS, of which nine mapped to evolutionarily conserved sequences. Based on the hypothesis that silencing effects of Air would be directed towards cis regulatory elements used to activate genes, DHS are potential key players in the control of imprinted expression. However, in this 192-kb region only the two DHS mapping to the Igf2r and Air promoters show parental specificity. The remaining 19 DHS were present on both parental alleles and, thus, have the potential to activate Igf2r on the maternal allele and Air on the paternal allele. The possibility that the Igf2r and Air promoters share the same cis-acting regulatory elements, albeit on opposite parental chromosomes, was supported by the similar expression profiles of Igf2r and Air in vivo. These results refine our understanding of the onset of imprinted silencing at this cluster and indicate the Air noncoding RNA may specifically target silencing to the Igf2r promoter.

Animals↗
PubMed1988

Translation in mammalian cells of a gene linked to the poliovirus 5' noncoding region.

The central portion (region P) of the 742-nucleotide noncoding 5' end of poliovirus allows the RNA to initiate protein synthesis in the absence of the usual 5' 7-methylguanosine capping group. Poliovirus 5' noncoding region was fused to a reporter gene and transfected into cells. There was extensive augmentation of the expression of this gene by poliovirus-mediated inhibition of cap-dependent protein synthesis. That the construct initiated in a cap-independent manner was verified through in vitro experiments. Small lesions throughout region P blocked its initiation function, implying that a coherent functional unit, hundreds of nucleotides long, is responsible for cap-independent initiation by poliovirus RNA.

Acetyltransferases↗
PubMed1989

Structural and functional characterization of the unusually short long terminal repeats and their adjacent regions of a novel endogenous avian retrovirus.

We have cloned the long terminal repeats and their flanking regions from four different proviruses belonging to a large, highly conserved, novel family of avian endogenous retroviruses. This family, termed the endogenous avian retrovirus (EAV) family, is distinct from the previously characterized avian endogenous and exogenous retroviruses. We have analyzed the sequences of the long terminal repeats and their adjacent noncoding viral sequences, including the gag leader region and the 3' noncoding region, of several different members of the EAV family and have found that the regulatory region of these novel viruses contains several unique features. The LTRs of the EAV proviruses are extremely short (243 bp long) but contain all of the essential regulatory features of longer avian retrovirus LTRs. The gag leader region and the 3' noncoding region of the novel EAVs are only weakly related to those of other avian retroviruses. Northern blot hybridization analysis of RNA from Line-0 chicken embryos reveals several transcripts derived from the EAV proviruses. Primer extension analysis indicates that all transcripts initiated from 5' proviral LTRs are initiated at the predicted +1 position within the EAV LTRs. The relative shortness, sequence divergence from other known LTRs, and the retention of the transcriptional integrity of the EAV LTRs make these LTRs an interesting model system for LTR function and for study of the potential involvement of such highly conserved retroviral elements in development.

Animals↗
PubMed1995

The genome of echovirus 11.

Echoviruses are the largest enterovirus subgroup consisting of 32 serotypes. They are common human pathogens causing, for example, meningitis, encephalitis and exanthema, but in spite of their clinical importance, relatively little is known about their biology. To illuminate the molecular characteristics of echoviruses, we have completed the genomic sequence of serotype 11. The RNA genome is 7438 nucleotides in length and it codes for a 2195 amino acid long polyprotein. When compared to other sequenced enteroviruses, echovirus 11 (EV11) shows remarkable similarity with coxsackie B viruses (CBVs) and coxsackievirus A9 (CAV9). On the basis of amino acid sequence homology in the capsid region, CAV9 is the virus most closely related to EV11. These two viruses have an apparent insertion sequence located at the C-terminus of the VP1 polypeptide. EV11, however, lacks the RGD motif found in the corresponding region of CAV9. The organization of the 5' end noncoding region resembles that of other enteroviruses, but contains a 12 nucleotides long poly-U stretch not seen in any other enterovirus sequenced to date.

Amino Acid Sequence↗
PubMed1986

Comparison of mouse and human keratin 18: a component of intermediate filaments expressed prior to implantation.

Keratin 18 is a type-I keratin that is found in a variety of simple epithelial tissues. In mice, the corresponding protein, called Endo B, is expressed at the 4- to 8-cell stage of mouse development and may be one of the first intermediate-filament proteins synthesized after fertilization. A cDNA clone for keratin 18, designated pK18, was isolated from a human placental cDNA library by hybridization with the mouse Endo-B probe. It was characterized by hybridization selection of RNA, translation, immunoprecipitation, Northern blotting, and sequence analysis. Synthetic T7 polymerase transcripts of the cDNA were indistinguishable in size from keratin-18 mRNA, suggesting that pK18 represents a full-length copy of the RNA. The cDNA insert is 1,428 nucleotides long and contains a single open reading frame of 1,342 nucleotides coding for 429 amino acids. The deduced amino acid sequence is 89.7% identical with that of Endo B. The only extensive difference between the two sequences is due to 9 additional amino acids being present in the last half of the N-terminal domain of keratin 18. The 38-nucleotide-long 3' noncoding region of the cDNA is 75% identical with the corresponding portion of Endo B. The 5' noncoding regions are 59% identical. The expression of keratin-18 mRNA was found to vary more than tenfold when HeLa cells and BeWo trophoblastic cells were compared.

Amino Acid Sequence↗
PubMed2004

Retroposon mapping in molecular systematics.

Advances in genome sciences are demonstrating the dynamic nature of noncoding DNA regions, which are comprised largely of repetitive elements with no apparent function. Retroposons are one class of mobile genetic elements that amplify and move about the genome via a copy-and-paste mechanism that employs an RNA intermediate. Short and long interspersed elements (SINEs and LINEs, respectively) are types of retroposons of particular interest because of their active role in shaping the architecture of genomes and their diagnostic value as evolutionary markers for studies of phylogeny and population biology. Although the use of SINEs and LINEs for molecular systematic studies is proliferating, a comprehensive laboratory protocol that explicitly outlines how to isolate and characterize retroposons for systematic studies in a detailed, step-by-step fashion has been lacking. The present chapter addresses this gap in the literature by focusing on the strategy for isolating new SINEs from a genomic library, the screening process, the sequencing and characterization of clones into subfamilies, quantification of copy number in host taxa, and the critical diagnosis of phylogenetically informative SINE and LINE insertion patterns. Practical limits to the method are discussed in relation to sampling design, systematic character theory, and the empirical distribution of elements observed in eukaryotic lineages. Major steps in the experimental process are illustrated with case examples from a diversity of taxonomic groups and by published results in the molecular biology and systematics literature.

Animals↗
PubMed1988

Shifts in configuration of the 5'-noncoding region of a mouse messenger RNA under translational control.

Several major mRNA species of mouse and other mammalian cells occur both as small untranslated ribonucleoprotein particles and as functional molecules associated with ribosomes in polysomes. One of these, that codes for a 21-kDa polypeptide, was analyzed with respect to distribution of sites accessible to RNase T1 in the 5'-noncoding region. This region, which is about 100 nucleotides long, contains several sites that are highly sensitive to the enzyme, as well as many G residues not susceptible to cleavage. The distribution of highly sensitive sites was compared in the active and inactive states of the P21 mRNA present in cytoplasmic extracts by subjecting the extract to limited nuclease digestion followed by separation of partially fragmented polysomes from free messenger ribonucleoprotein particles. The mRNA in polysomes contained two highly sensitive sites, one near the 5' terminus and the other in the middle of the region, next to a sequence potentially capable of Shine-Dalgarno interaction. The untranslated molecules lacked the 5'-proximal site but had several highly accessible sites not present in the active molecules. The initiation AUG showed little accessibility both in polysomes and in messenger ribonucleoproteins. Both forms were quite different from the deproteinized mRNA with respect to distribution of nuclease-sensitive sites. Our results indicate that interaction of the mRNA with cytoplasmic factors strongly affects its conformation in the 5'-noncoding region and that a particular conformation may be important for effective interaction with ribosomal particles during polypeptide chain initiation.

Animals↗
PubMed1995

Molecular characterization of the 11th RNA segment from human group C rotavirus.

The complete nucleotide sequence of genome segment 11 from the noncultivatable, human group C rotavirus (Bristol strain) was determined. Comparison of the nucleotide sequence of the segment termini with the consensus 5' and 3' terminal noncoding sequences of the human group C rotavirus genome revealed characteristic 5' and 3' sequences. Human group C rotavirus genome segment 11 is 613 bp long and encodes a single open reading frame of 450 nucleotides (150 amino acids) starting at nucleotide 39 and terminating at nucleotide 489, leaving a long 3' untranslated region of 124 nucleotides. The predicted translation product has a calculated molecular weight of 17.7 kD and contains four potential N-linked glycosylation sites. No significant homologies to other viral proteins were found in database searches. Hydropathy analysis predicted the human group C rotavirus genome segment 11 translation product has a hydrophilic carboxy terminus (amino acids 54-150) and a hydrophobic amino terminus (amino acids 1-53) that can be further subdivided into three short hydrophobic sequences--H1, H2, and H3. These features are analogous to the integral membrane glycoprotein NSP4 encoded by group A rotavirus gene 10.

Amino Acid Sequence↗
PubMed1985

Trans-activator gene of human T-lymphotropic virus type III (HTLV-III).

Human T-lymphotropic virus type III (HTLV-III) encodes a trans-acting factor that activates the expression of genes linked to the HTLV-III long terminal repeat. By functional mapping of complementary DNA transcripts of viral messenger RNA's the major functional domain of the gene encoding this factor was localized to a region immediately before the env gene of the virus, a region previously thought to be noncoding. This newly identified gene consists of three exons, and its transcription into messenger RNA involves two splicing events bringing together sequences from the 5' part (287 base pairs), middle (268 base pairs), and 3'part (1258 base pairs) of the HTLV-III genome. A similar messenger RNA with a truncated second exon (70 base pairs) does not encode a trans-acting function. It is proposed that this second messenger RNA is the transcript of a gene (3'-orf) located after the env gene. Messenger RNA's were also identified for the env and gag-pol genes of HTLV-III.

Amino Acid Sequence↗
PubMedDate not supplied

Complete sequence of one of the mRNAs coding for the small subunit of ribulose bisphosphate carboxylase of Nicotiana sylvestris.

The combination of cDNA and RNA sequencing techniques has enabled determination of the complete sequence of one of the mRNAs coding for the precursor of the small subunit of ribulose bisphosphate carboxylase of Nicotiana sylvestris. In this 898-nucleotide-long mRNA, 540 nucleotides code for the entire 180-amino-acid-long precursor polypeptide consisting of the 57-amino acid-long transit peptide and the 123-amino-acid-long mature protein, while 60 and 195 nucleotides belong to the 5' and 3' noncoding flanking regions, respectively. The 5' end, which is very rich in AG residues, contains several direct and indirect repeated sequences, and a possible hairpin structure. The 3' end, terminated by a 103-nucleotide-long poly-A tail, is very rich in AU residues but does not contain the classical polyadenylation signal sequence.

Amino Acid Sequence↗
PubMed1989

Circumstances and mechanisms of inhibition of translation by secondary structure in eucaryotic mRNAs.

This paper describes in vitro experiments with two types of intramolecular duplex structures that inhibit translation in cis by preventing the formation of an initiation complex or by causing the complex to be abortive. One stem-loop structure (delta G = -30 kcal/mol) prevented mRNA from engaging 40S subunits when the hairpin occurred 12 nucleotides (nt) from the cap but had no deleterious effect when it was repositioned 52 nt from the cap. This result confirms prior in vivo evidence that the 40S subunit-factor complex, once bound to mRNA, has considerable ability to penetrate secondary structure. Consequently, translation is most sensitive to secondary structure at the entry site for ribosomes, i.e., the 5' end of the mRNA. The second stem-loop structure (hp7; delta G = -61 kcal/mol, located 72 nt from the cap) was too stable to be unwound by 40S ribosomes, hp7 did not prevent a 40S ribosomal subunit from binding but caused the 40S subunit to stall on the 5' side of the hairpin, exactly as the scanning model predicts. Control experiments revealed that 80S elongating ribosomes could disrupt duplex structures, such as hp7, that were too stable to be penetrated by the scanning 40S ribosome-factor complex. A third type of base-paired structure shown to inhibit translation in vivo involves a long-range interaction between the 5' and 3' noncoding sequences.

Animals↗
PubMed1989

A second locus for the 5S multigene family in Secale L.: sequence divergence in two lineages of the family.

The 5S RNA genes in Secale sp. are arranged as tandem arrays of a 460- and 480-bp repeating sequence. These size classes were initially discovered by restriction endonuclease analysis using BamHI and subsequently by DNA sequencing of cloned units. The length variation between short and long units originated from major deletion-insertion events in the noncoding spacer region of the 5S DNA repeat units. In situ hybridization with [3H]cRNA and biotin-labelled probes synthesized from both the short and long 5S DNA units of S. cereale localized the sites on chromosome 1R and a new site on a chromosome identified as 5R. We propose that the chromosome 1R locus, which has been mapped previously, be named 5SDna-R1 and the second locus, reported in the present paper, be referred to as 5SDna-R2. A preferential hybridization of a probe from the long unit to the 5SDna-R2 locus and of a probe from the short unit to the 5SDna-R1 locus is reported. The clustering of long units in the 5SDna-R2 locus was confirmed by restriction endonuclease digestion of DNA from rye chromosome 5R additions to wheat. Nucleotide sequence alignment of 5S DNA repeat units from a number of Secale species, using both phenetic and cladistic computer programmes, demonstrated that two clear lineages corresponding to the long and short units existed in this genus. The different Secale species could not be unambiguously differentiated using the 5S DNA sequences.

Base Sequence↗
PubMed1991

Sequence of the 5S rRNA gene and organization of ribosomal RNA operons in Streptomyces rimosus.

One Streptomyces rimosus ribosomal RNA gene set (rrnF) was cloned into pBR322 as 8.8 kilobase-pairs BamHI fragment and sequence of the 3' end of the operon with 5S rRNA gene was determined. 5S rRNA gene is 120 bp long and highly homologous with two other known 5S rRNA from Streptomyces. Remarkable sequence homology with S. ambofaciens rrnD operon exists in the 3' noncoding regions, including first transcription termination signal. S. rimosus rrnF operon contains second putative terminator which is absent in S. ambofaciens rrnD operon. tRNA genes were not found at the 3' end of rrnF operon. The number of rRNA operons in S. rimosus was defined by Southern hybridization analysis. S. rimosus possess six rRNA operons and all rRNA operons contain identical PstI fragment of 4.5 kb. rRNA genes in operons are separated by short intergenic regions and organized in the order 16S-23S-5S. Efforts to connect tRNA genes with S. rimosus rRNA operons were unsuccessful.

Base Sequence↗
PubMed1985

Transcription of three c-myc exons is enhanced in chicken bursal lymphoma cell lines.

The chicken c-myc gene, as defined by its homology to the v-myc gene of MC29 virus, is comprised of two exons. Using the techniques of runoff transcription, primer extension, and S1 nuclease protection, we demonstrate that there is a third c-myc exon of approximately equal to 345 base pairs (bp) located 0.7 kbp upstream of the 5' end of the v-myc homology. This first exon is transcribed and present in myc mRNA in normal chicken cells. We also examined RNA from five cell lines derived from avian leukosis virus-induced bursal lymphomas. In all these lines, the level of transcription of the 2.2- to 2.5-kbp myc mRNA is increased 30- to 60-fold over normal cells. The myc mRNA in four of these lines also contains increased levels of the first noncoding exon, and evidence is presented that the long terminal repeat (LTR) in the vicinity of c-myc is functioning as an enhancer of c-myc transcription rather than as a promoter in several of these cell lines. In two cell lines in which the viral LTR has integrated between the first and second exons in the proper orientation for downstream promotion of myc, the LTR does not exhibit promoter function. The pattern of c-myc transcription observed by others in a vast majority of avian leukosis virus-induced neoplasms is not observed in any of the five cell lines examined.

Animals↗
PubMed2007

Sequence determination and functional analysis of the Akabane virus (family Bunyaviridae) L RNA segment.

Akabane virus (AKAV) causes epizootic congenital deformities in cattle, sheep, and goats. Due to the lack of a complete genome sequence, the molecular biological properties of this virus are not known. We have cloned and sequenced the functional large (L) RNA segment of AKAV, and shown that it has polymerase activity using a minireplicon system with RNA polymerase I. The complete L RNA segment is 6868 nucleotides long and encodes an L protein of 2251 amino acids, which functions as an RNA-dependent RNA polymerase. A minireplicon reporter plasmid was constructed by flanking either the firefly luciferase or the green fluorescent protein gene in the antisense orientation with the 5'- and 3'-terminal noncoding regions of the small RNA segment. HmLu-1 cells were transfected with the reporter plasmid, and the L protein and nucleoprotein (N protein) expression plasmids. The reporter activity was upregulated in a dose-dependent manner with increasing concentration of either the L or N protein expression plasmid. Furthermore, the reporter activity could be downregulated by the AKAV NSs protein as well as by other orthobunyaviruses. These results show that the AKAV minireplicon system is a powerful tool for studying transcription and for rescuing infectious viruses from cloned cDNAs.

Animals↗
PubMed1994

Genome structure and variability of a virus causing hantavirus pulmonary syndrome.

A previously unrecognized hantavirus (family Bunyaviridae) has recently been detected and shown to be associated with a severe respiratory illness with high mortality, termed hantavirus pulmonary syndrome (HPS). This disease has now been identified throughout the western United States. We present nucleotide sequence characterization of the three RNA segments composing the HPS virus genome and address the question of the apparent emergence of this highly lethal virus. No evidence of genetic reassortment with previously recognized hantaviruses was found, each RNA segment being unique and approximately 30% different at the nucleotide level to the segments of the closest relative, Prospect Hill virus. These findings, together with the observed extensive genetic diversity of HPS viruses and examples of geographic clustering of distinct virus genotypes, suggest that HPS and associated virus have likely existed undetected for many years. The virus genome M segment was determined to be 3696 nucleotides in length and encode G1 and G2 proteins, 652 and 488 amino acids in length. The S segment was found to be 2059 nucleotides in length and to encode a nucleocapsid protein, 428 amino acids in length. S segment analysis also revealed an unusually long noncoding region with numerous repeats and evidence for a potential NSS protein encoded in an overlapping frame.

Amino Acid Sequence↗
PubMed1984

Molecular cloning and nucleotide sequence of cDNA coding for rat brain cholecystokinin precursor.

A mixture of 14-mer oligodeoxynucleotides was used for the screening of a cDNA clone coding for a cholecystokinin (CCK) precursor from a cDNA library for rat brain microsomal poly(A)RNA. The longest insert is 718 bp long which was verified to contain a nearly full-length cDNA sequence coding for rat CCK precursor, because the size of CCK mRNA was estimated to be about 850 bases long by Northern blotting analysis. Sequence analysis revealed 110 bp in the 5'-untranslated region, 345 bp in the amino acid coding region corresponding to the CCK precursor and 263 bp in the 3'-noncoding region which contains polyadenylation signal AUUAAA and the poly(A) sequence. The precursor may contain a 28 amino acid signal peptide and 12 additional amino acids at the carboxyl terminus.

Amino Acid Sequence↗
PubMed1996

Noncoding RNA for CR20, a cytokinin-repressed gene of cucumber.

The CR20 gene was identified as a cytokinin-repressed gene in excised cotyledons of cucumber. We determined the sequences of some CR20 cDNAs with different structures and sequenced genomic clones for CR20. This gene consisted of three exons, and there were at least three types of transcript, which seemed to be generated by alternative splicing of the second intron. None of the CR20 transcripts included a long open reading frame (ORF). We isolated a cDNA of Arabidopsis thaliana with cucumber CR20 cDNA as a probe. This cDNA for a gene designated AtCR20-1 also lacked a long ORF. A region of 180 nucleotides was conserved in the CR20 RNA of cucumber and the AtCR20-1 RNA of Arabidopsis, although the homology was relatively low when the entire sequences were compared. Each conserved region consisted of seven elements, and seems to form stable secondary structure. These suggest that CR20 RNA may function as an RNA that is not translated into a protein.

Alternative Splicing↗