Showing papers on "Intron published in 1985"

The mitochondrial DNA molecular of Drosophila yakuba: nucleotide sequence, gene organization, and genetic code.

Abstract: The sequence of the 16,019 nucleotide-pair mitochondrial DNA (mtDNA) molecule ofDrosophila yakuba is presented. This molecule contains the genes for two rRNAs, 22 tRNAs, six identified proteins [cytochrome b, cytochrome c oxidase subunits I, II, and III (COI-III), and ATPase subunits 6 and 8] and seven presumptive proteins (URF1-6 and URF4L). Replication originates within a region of 1077 nucleotides that is 92.8% A+T and lacks any open reading frame larger than 123 nucleotides. An equivalent to the sequence found in all mammalian mtDNAs that is associated with initiation of second-strand DNA synthesis is not present inD. yakuba mtDNA. Introns are absent fromD. yakuba mitochondrial genes and there are few (0–31) intergenic nucleotides. The genes found inD. yakuba and mammalian mtDNAs are the same, but there are differences in their arrangement and in the relative proportions of the complementary strands of the molecule that serve as templates for transcription. Although theD. yakuba small and large mitochondrial rRNA genes are exceptionally low in G and C and are shorter than any other metazoan rRNA genes reported, they can be folded into secondary structures remarkably similar to the secondary structures proposed for mammalian mitochondrial rRNAs.D. yakuba mitochondrial tRNA genes, like their mammalian counterparts, are more variable in sequence than nonorganelle tRNAs. In mitochrondrial protein genes ATG, ATT, ATA, and in one case (COI) ATAA appear to be used as translation initiation codons. The only termination codon found in these genes is TAA. In theD. yakuba mitochondrial genetic code, AGA, ATA, and TGA specify serine, isoleucine, and tryptophan, respectively. Fifty-nine types of sense codon are used in theD. yakuba mitochondrial protein genes, but 93.8% of all codons end in A or T. Codon-anticodon interactions may include both G-A and C-A pairing in the wobble position. Evidence is summarized that supports the hypothesis that A and T nucleotides are favored at all locations in theD. yakuba mtDNA molecule where these nucleotides are compatible with function.

Comparative Anatomy of 16-S-like Ribosomal RNA

Abstract: Publisher Summary This chapter examines the range of the variation of secondary structure among the 16-S-like rRNAs. This brings into a larger structural context a recent detailed analysis of the individual helical elements and provides a basis for an accurate alignment of the corresponding regions of different primary structures. Computer-assisted comparative is used in the analysis of aligned sequences to describe the pattern of phylogenetic conservation for each nucleotide position in 16-S rRNA. A search for matching patterns among unpaired positions in the RNA chain then produces a list of candidates for potential base–base tertiary interactions. The completion of nucleotide sequences for 34 16-S-like rRNAs includes 4 eubacteria, 4 chloroplasts, 12 mitochondria, 4 archaebacteria, and 10 eukaryotes. Secondary structure models for these molecules have been developed in the course of refinement of the E. coli model, and have been used to arrive at improved sequence alignments for the 16-S-like rRNAs. Schematic drawings of (1) eubacterial, (2) archaebacterial, (3) eukaryotic cytoplasmic, (4) plant mitochondrial, (5) fungal mitochondrial, and (6) mammalian mitochondrial structures are shown in the chapter.

Nucleotide sequence of the gene for human factor IX (antihemophilic factor B).

Abstract: Two different human genomic DNA libraries were screened for the gene for blood coagulation factor IX by employing a cDNA for the human protein as a hybridization probe. Five overlapping lambda phages were identified that contained the gene for factor IX. The complete DNA sequence of about 38 kilobases for the gene and the adjacent 5' and 3' flanking regions was established by the dideoxy chain termination and chemical degradation methods. The gene contained about 33.5 kilobases of DNA, including seven introns and eight exons within the coding and 3' noncoding regions of the gene. The eight exons code for a prepro leader sequence and 415 amino acids that make up the mature protein circulating in plasma. The intervening sequences range in size from 188 to 9473 nucleotides and contain four Alu repetitive sequences, including one in intron A and three in intron F. A fifth Alu repetitive sequence was found immediately flanking the 3' end of the gene. A 50 base pair insert in intron A was found in a clone from one of the genomic libraries but was absent in clones from the other library. Intron A as well as the 3' noncoding region of the gene also contained alternating purine-pyrimidine sequences that provide potential left-handed helical DNA or Z-DNA structures for the gene. KpnI repetitive sequences were identified in intron D and the region flanking the 5' end of the gene. The 5' flanking region also contained a 1.9-kb HindIII subfamily repeat. The seven introns in the gene for factor IX were located in essentially the same position as the seven introns in the gene for human protein C, while the first three were found in positions identical with those in the gene for human prothrombin.

Human Lymphotoxin and tumor necrosis factor genes: structure, homology and chromosomal localization

Abstract: Human Tumor Necrosis Factor and Lymphotoxin are cytotoxic proteins which have similar biological activities and share 30 percent amino acid homology. The single copy genes which encode these proteins share several structural features: each gene is approximately three kilobase pairs in length and is interrupted by three introns. In addition, these genes are closely linked and have been mapped to human chromosome 6. However, only the last exons of both genes, which code for more than 80 percent of each secreted protein, are significantly homologous (56 percent).

The Origin and Evolution of Retroposons

Abstract: Publisher Summary This chapter describes the origin and evolution of retroposons. It deals with two of them which operate on very different timescales: (1) RNA splicing (particularly messenger RNA splicing), which is the excision of transcribed noncoding sequences from RNA during normal gene expression and (2) retroposition, which is the insertion of reverse-transcribed sequences from RNA back into the genome during evolution. RNA splicing was the first and most surprising discovery of eukaryotic molecular genetics. Retroposons are a class of dispersed sequences in DNA, which appear to have arisen during evolution by a particular mechanism of inserting RNA sequences into chromosomal DNA (retroposition). They include processed mRNA pseudogenes, snRNA pseudogenes, the highly repeated ALU sequences, and a variety of other sequences. They are entirely distinct from transposons and retroviruses. The chapter explains the splicing of ribosomal RNA, mitochondrial RNA, and chloroplast RNA.

The nucleotide sequence of the gene for human protein C.

Abstract: A human genomic DNA library was screened for the gene for protein C by using a cDNA probe coding for the human protein. Three different overlapping lambda Charon 4A phage were isolated that contain inserts for the gene for protein C. The complete sequence of the gene was determined by the dideoxy method and shown to span about 11 kilobases of DNA. The coding and 3' noncoding portion of the gene consists of eight exons and seven introns. The eight exons code for a preproleader sequence of 42 amino acids, a light chain of 155 amino acids, a connecting dipeptide of Lys-Arg, and a heavy chain of 262 amino acids. The preproleader sequence and the connecting dipeptide are removed during processing, resulting in the mature protein composed of a heavy and a light chain held together by a disulfide bond. The heavy chain also contains the catalytic region for the serine protease. Two Alu sequences and two homologous repeats of about 160 nucleotides were found in intron E. The seven introns in the gene for protein C are located in essentially the same positions in the amino acid sequence as the seven introns in the gene for human factor IX, while the first three introns in protein C are located in the same positions as the first three in the gene for human prothrombin.

A novel transcription property of SP6 and T7 RNA polymerases: dependence on template structure

Abstract: The in vitro synthesis of extraneous RNA sequences by SP6 and T7 RNA polymerases from specific DNA templates is described. Transcription of templates prepared by digestion with restriction enzymes that leave 3' protruding ends resulted in the production of significant amounts of long, template-sized RNA transcripts which hybridized to vector DNA. Sequences copied from the noncoding template strand were among the extraneous transcripts. The presence of these sequences in probe preparations were detected in Southern and RNase protection hybridization assays. In contrast, transcription of DNA templates with blunt or 5' protruding ends yielded few RNA products as extraneous sequences.

RNA-mediated gene duplication: the rat preproinsulin I gene is a functional retroposon.

Abstract: Rats and mice have two, equally expressed, nonallelic genes encoding preproinsulin (genes I and II). Cytological hybridization with metaphase chromosomes indicated that both genes reside on rat chromosome I but are approximately 100,000 kilobases apart. In mice the two genes reside on two different chromosomes. DNA sequence comparisons of the gene-flanking regions in rats and mice indicated that the preproinsulin gene I has lost one of the two introns present in gene II, is flanked by a long (41-base) direct repeat, and has a remnant of a polydeoxyadenylate acid tract preceding the downstream direct repeat. These structural features indicated that gene I was generated by an RNA-mediated duplication-transposition event involving a transcript of gene II which was initiated upstream from the normal capping site. Sequence divergence analysis indicated that the pair of the original gene and its retroposed, but functional, counterpart (which appeared about 35 million years ago) is maintained by strong negative selection operating primarily on the segments encoding the chains of the mature hormone, whereas the segments encoding the parts of the polypeptide that are eliminated during processing and also the introns and the flanking regions are evolving neutrally.

Secondary structure of the circular form of the Tetrahymena rRNA intervening sequence: a technique for RNA structure analysis using chemical probes and reverse transcriptase

Abstract: The structure of the intervening sequence (IVS) of the Tetrahymena rRNA precursor mediates cleavage-ligation reactions that result in pre-rRNA splicing and IVS cyclization. We have developed a method for RNA structure analysis and applied it to the circular form of the IVS RNA. The native RNA was treated with dimethyl sulfate or diethyl pyrocarbonate to modify bases not involved in secondary or tertiary interactions. The RNA was then used as a template for reverse transcription. Elongation of synthetic oligodeoxynucleotide primers was found to stop (or pause) one nucleotide prior to 1-methyladenosine, 3-methylcytidine, and 7-ethoxycarbonyladenosine residues. The detection of 1-methyladenosine is particularly useful for locating single-stranded regions. After chemical cleavage of the RNA, 7-methylguanosine also could be detected. In general, the sites of modification were consistent with a previous model of the secondary structure of the linear form of the IVS RNA, a model based on enzymatic cleavage data, free energy calculations, and phylogenetic comparison. Thus, IVS RNA autocyclization does not involve major rearrangements of the secondary structure, although there is evidence for a conformational change in one region of the molecule. The methods described here should be of general use for obtaining information about structure far from the ends of RNA molecules.

Mitochondrial class II introns encode proteins related to the reverse transcriptases of retroviruses.

Abstract: Organelle introns share several distinctive features that set them apart from their counterparts in nuclear-encoded pre-messenger RNAs (reviewed in ref 1): their termini do not obey the GUAG rule; the introns are 'structured' (members of the same family or 'class' can theoretically adopt very similar RNA secondary conformations and several of the postulated pairings have been confirmed by studies of splicing mutants and their revertants (see, for example, ref 4); many introns from both classes contain long open reading frames We report here that the proteins potentially encoded by four class II introns are related to several RNA-dependent polymerases of viral and transposable element origins

Nucleotide sequence of a B1 hordein gene and the identification of possible upstream regulatory elements in endosperm storage protein genes from barley, wheat and maize

Abstract: The B-hordeins are the major group of prolamin storage proteins in barley (Hordeum vulgare L.) and they are encoded by a small multigene family that is expressed specifically in the developing endosperm. We report the complete nucleotide sequence of a clone of one B-hordein gene (pBHR184). The cloned gene contains no introns and belongs to the B1 sub-family of B-hordein genes. Comparison of the 5'-flanking sequences of pBHR184 with those of related S-rich prolamin genes from wheat shows that several short sequences within 600 bp upstream of the translation initiation codon are strongly conserved. A sequence that is conserved at around -300 bp in the S-rich prolamins is also conserved at similar locations in genes encoding the two major classes of maize prolamin (the Z19 and Z21 zeins) and appears to be unique to prolamin genes. We discuss the possible role of this '-300 element' in the control of gene expression in the developing cereal endosperm.

An RNA processing activity that debranches RNA lariats

Abstract: The excised introns of pre-messenger RNA's (pre-mRNA's) and intron-containing splicing intermediates are in a lariat configuration in which the 5' end of the intron is covalently joined by a 2',5'-phosphodiester bond to a specific adenosine residue near the 3' end of the intron. A 2',5'-phosphodiesterase activity in HeLa cell extracts has been detected that debranches RNA lariats, converting them to linear RNA molecules by specific cleavage of the 2',5'-phosphodiester bond. This lariat debranching activity is distinct from previously reported 2',5'-phosphodiesterases with regard to its biochemical and substrate requirements as well as its stringent cleavage specificity. The debranching activity is observed only if the RNA lariats generated during in vitro processing are deproteinized and added back to the extract. These results suggest that during the normal in vitro splicing reaction the 2',5'-phosphodiester bond of RNA lariats is protected from cleavage by the lariat debranching activity.

Glucocorticoid receptor binding and activation of a heterologous promoter by dexamethasone by the first intron of the human growth hormone gene.

Abstract: In this study DNA-binding and gene transfer experiments were performed to examine a potential glucocorticoid regulatory element (GRE) in the human growth hormone gene. As assayed by nitrocellulose filter binding, only two regions of the human growth hormone gene, the 5'-flanking sequences and a fragment containing part of the first intron, were retained preferentially by purified glucocorticoid-receptor complexes. The relative binding by the transcribed sequences was three times greater than the relative binding by the 5'-flanking sequences, but less than the relative binding by a fragment containing the human metallothionein-IIA gene GRE. The intron, but not the 5'-flanking sequences, generated a "footprint" when the receptor complex was used to protect the segments against exonuclease III digestion; the protected sequence spanned nucleotides +86 to +115 in the first intron and contained a structure homologous in 14 of 16 nucleotides to a 16-nucleotide consensus GRE. The hexanucleotide 5'-TGTCCT-3', thought to be important for GRE activity, not only was found in this sequence and in the 5'-flanking region, but also was present twice in the 3' end of the gene that did not show specific receptor binding. The latter results suggest that the hexanucleotide alone is not sufficient to generate specific receptor binding tight enough to be assayed in this way. To test the biological activity of the intron binding site, a fragment containing these sequences was fused 5' to the human metallothionein-IIA gene promoter depleted of its GRE and linked to the structural sequences of the herpes simplex virus thymidine kinase (TK) gene. When this hybrid gene was transfected into Rat 2 TK- cells, its expression was induced threefold by the glucocorticoid dexamethasone, as assessed by transfection efficiency and RNA blotting analyses. Expression of the same gene without the human growth hormone gene segment was not affected by the steroid, whereas the wild-type human metallothionein-IIA gene promoter containing its GRE responded to the hormone by a sixfold increase in thymidine kinase mRNA. These results indicate that the human growth hormone gene contains a structure within its first intron that can function as a GRE.

Structure of the chromosomal gene for granulocyte-macrophage colony stimulating factor: comparison of the mouse and human genes.

Abstract: A cDNA clone that expresses granulocyte-macrophage colony stimulating factor (GM-CSF) activity in COS-7 cells has been isolated from a pcD library prepared from mRNA derived from concanavalin A-activated mouse helper T cell clones. Based on homology with the mouse GM-CSF cDNA sequence, the mouse GM-CSF gene was isolated. The human GM-CSF gene was also isolated based on homology with the human GM-CSF cDNA sequence. The nucleotide sequences determined for the genes and their flanking regions revealed that both the mouse and human GM-CSF genes are composed of three introns and four exons. The organization of the mouse and human GM-CSF genes are highly homologous and strong sequence homology between the two genes is found both in the coding and non-coding regions. A 'TATA'-like sequence was found 20-25 bp upstream from the transcription initiation site. In the 5'-flanking region, there is a highly homologous region extending 330 bp upstream of the putative TATA box. This sequence may play a role in regulation of expression of the GM-CSF gene. These structures are compared with those of different lymphokine genes and their regulatory regions.

Recombination between nonsegmented RNA genomes of murine coronaviruses.

Abstract: We have isolated a recombinant virus between the A59 and JHM strains of mouse hepatitis virus, which contain a single species of nonsegmented RNA genome. This recombinant was derived by mixed infection of DBT cells with temperature-sensitive mutants of A59 and JHM at nonpermissive temperature. Viruses recovered at this temperature were screened by oligonucleotide fingerprinting of their genomic RNAs. One recombinant virus, B1, was found to contain mostly A59-derived sequences, but the 3 kilobases at the 5' end of the genomic RNA was derived from JHM. Thus, the crossover point in the B1 genome is located within gene A, which codes for the viral RNA polymerases. The study of the intracellular RNA species of B1 virus revealed that probably all of the virus-specific subgenomic mRNA species contained the body sequences of strain A59 but the leader sequences of JHM. This result indicates that the JHM leader RNA, which differs from the A59 leader RNA, could be fused to the mRNAs of a different virus strain during RNA transcription. Furthermore, B1 virus-infected cells contain an additional subgenomic mRNA species which is transcribed from a new initiation site within gene C, suggesting that the leader RNA could determine the site of initiation for coronavirus mRNAs. These data represent a first report of RNA recombination between viruses, other than picornaviruses, which contain nonsegmented RNA genomes.

C μ -containing transcripts initiate heterogeneously within the IgH enhancer region and contain a novel 5′-nontranslatable exon

Abstract: Transcriptional competence of the immunoglobulin heavy-chain locus (IgH) is established at an early stage of lymphoid cell development, leading to the appearance of RNA components, previously called Cμ RNA1 or sterile-μ RNA2, which contain constant-region sequences but lack variable-region sequences. These components are of two types: those which initiate in the D region of alleles that have undergone DJH (diversity–joining region) rearrangement (Dμ transcripts) and those which initiate within the JH–Cμ intron (hereafter termed Iμ transcripts)3,4. In pre-B and early B cells, Dμ and lμ transcripts are nearly as abundant as the messenger RNA encoding μ heavy chain2,3,5. The Dμ transcripts are spliced into RNAs containing D, JH and Cμ sequences, and in some, but not all, cases these RNAs are translated into Dμ proteins4. To establish whether the Iμ transcripts have any translational potential and to elucidate the structure of their promoter region, we have determined their transcription initiation sites and their mode of splicing. As reported here, by using sequence analysis of cloned Iμ complementary DNAs, primer extension and S1, nuclease mapping, we have found that these transcripts have remarkable 5′ heterogeneity: ther e are more than five distinct start sites spanning a region of 44 nucleotides that is located downstream of an octanucleotide found in all variable-region promoters. Such imprecise initiation may result from the lack of a well-defined T A T AA motif and the unusual proximity of the octanucleotide to the enhancer region. Approximately 700 nucleotides downstream from these initiation sites, a cryptic splice site is used to create a nontranslatable exon (‘nontron’) which is joined to the Cμ1 domain. The properties of the nontron may be important for the mechanism of allelic exclusion.

The first intron of the human growth hormone gene contains a binding site for glucocorticoid receptor.

Abstract: Glucocorticoid receptor (GCR) protein stimulates transcription from a variety of cellular genes. We show here that GCR partially purified from rat liver binds specifically to a site within the first intron of the human growth hormone (hGH) gene, approximately 100 base pairs downstream from the start of hGH transcription. GCR binding is selectively inhibited by methylation of two short, symmetrically arranged clusters of guanine residues within this site. A cloned synthetic 24-base-pair deoxyoligonucleotide containing the predicted GCR binding sequence interacts specifically with GCR. The hGH binding site shares sequence homology with a GCR binding site upstream from the human metallothionein II gene and a subset of GCR binding sites from mouse mammary tumor virus. All of these binding sites for this eukaryotic transcriptional regulatory protein show remarkable similarity in overall geometry to the binding sites for several prokaryotic transcriptional regulatory proteins.