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A Efstratiadis

Publications and source records attributed to A Efstratiadis.

At least 55 records · Page 3Linked to original sources
PubMed1989

Autonomous splicing and complementation of in vivo-assembled spliceosomes.

We have used an in vivo system generating assayable amounts of a specific pre-mRNA to study the relationship between splicing and an operationally defined nuclear matrix preparation (NM). When NM is prepared by extraction of DNase I-treated nuclei with an approximately physiological concentration of KCl (0.1 M), a portion of NM-associated precursor can be spliced in vitro in the presence of ATP and Mg2+ and in the absence of splicing extract ("autonomous splicing"). We propose that the autonomous reaction, which does not exhibit a temporal lag and is half-complete in 5 min, occurs in fully assembled, matrix-bound ribonucleoprotein complexes (in vivo spliceosomes). Extraction of the NM with concentrations of KCl greater than 0.4 M eliminates autonomous splicing but leaves behind preassembled complexes that can be complemented for splicing with HeLa cell nuclear extract. The splicing complementing factor, representing one or more activities present in the nuclear extract and also in the cytoplasmic S100 fraction, is relatively heat resistant, devoid of an RNA component, and does not bind to DEAE-Sepharose in 0.1 M KCl. It exists in the nucleus in two forms; bound to autonomous spliceosomes and free in the nucleoplasm. Biochemical features of the complementation reaction, and conditions for reversible uncoupling of the two splicing steps are described and discussed.

Adenosine Triphosphate↗
PubMed1988

A promoter of the rat insulin-like growth factor II gene consists of minimal control elements.

We have characterized the cis-control signals in one of the two promoters of the developmentally regulated rat insulin-like growth factor II gene (rIGF-II) by a combination of in-vivo transient expression, in-vitro transcription, footprinting, gel band-shifting and methylation-interference experiments, using a series of deletion mutant templates. Our results indicate that this simple (minimal) promoter (P2) consists of no more than 128 base-pairs, which include an ATA box and four proximal upstream GC boxes binding the general transcription factor Sp1. Three of the latter sites deviate from the known Sp1 consensus recognition sequence. The two types of cis-acting regulatory signals (GC/ATA motif) of the P2 promoter are inter-dependent and sufficient for transcription. A model for the operation of this type of minimal promoter is discussed. S1 nuclease-hypersensitive sites, localized by in-vitro mapping to the region of the P2 Sp1-binding sites, are also present in vivo and correlate with the transcriptional state of chromatin in the rIGF-II locus. We show that recognition sites for Sp1 binding are a subset of sequences that exhibit hypersensitivity to S1.

Animals↗
PubMed1988

Expression of the insulin-like growth factor II gene in the choroid plexus and the leptomeninges of the adult rat central nervous system.

The rat insulin-like growth factor II gene, encoding a fetal somatomedin, expresses a multitranscript family in embryonic/fetal tissues and in the adult brain and spinal cord. By performing in situ hybridization on tissue sections of adult brain and spinal cord, we have found that these transcripts are not expressed in neural or glial cells but are expressed in the epithelium of the choroid plexus of each cerebral ventricle and in the leptomeninges. We propose that the choroidal epithelial cells synthesize and secrete insulin-like growth factor II into the cerebrospinal fluid.

Animals↗
PubMed1988

Pattern of the insulin-like growth factor II gene expression during rat embryogenesis.

The rat insulin-like growth factor II (IGF-II) gene, encoding a fetal somatomedin, expresses a family of transcripts in embryonic/fetal tissues, and also in the adult choroid plexus and the leptomeninges. We have localized IGF-II gene transcripts in sections of rat embryos of embryonic days 10-16 by performing in situ hybridization. These transcripts are present in the head mesenchyme, formed from both the mesoderm and the cephalic portion of the neural crest, and also in the majority of other tissues of mesodermal origin, predominantly those derived from the somites and the lateral mesoderm. Intense labelling was detected in muscle cells, and their precursors, throughout the examined stages, whereas in chondrocytes the initial high level of hybridization declined substantially prior to ossification. IGF-II gene transcripts are also present in derivatives of other germ layers, but in restricted sites. Thus, from the derivatives of the endoderm, only the liver and the bronchial epithelium yielded hybridization signals. Ectoderm-derived tissues, including the central and peripheral nervous system, were negative for hybridization, with the exception of the choroid plexus, the newly forming pituitary rudiment and, to a lesser extent, the auditory placode. The pattern of IGF-II gene expression during embryogenesis overlaps significantly with the reported distribution of immunohistochemically detected TGF-beta 1. A paracrine/autocrine role for IGF-II in the developmental process is discussed.

Animals↗
PubMed1987

Minimal exon sequence requirements for efficient in vitro splicing of mono-intronic nuclear pre-mRNA.

Measurements of the in vitro splicing efficiency of deletion mutant RNA precursors containing the small intron of the rabbit beta-globin gene, which are truncated in the first or in the second exon, revealed that no more than approximately 20 nucleotides of either exon are necessary for efficient splicing. At least for the second exon, this minimal length requirement is globin sequence-independent. Reduction of the exon-2 length to 14 nucleotides resulted in very inefficient splicing, whereas further reduction to 5 nucleotides apparently abolished the second splicing step (3' cutting and ligation), whereas the first step (5' cutting and branching) still occurred. The splicing efficiency of a double-mutant substrate retaining approximately 20 nucleotides of each exon was reduced to 50%. A kinetic study indicated that in the reaction of this double-mutant substrate the second, but not the first, splicing step was delayed, in contrast to the reaction of the wild-type precursor. Duplication or triplication of the entire sequence of exon-1 did not affect the splicing efficiency, whereas elongation of this exon with approximately 100 nucleotides of 5'-flanking (nontranscribed) beta-globin sequence diminished the level of correct splicing with the simultaneous appearance of aberrant lariat forms. We conclude that for mono-intronic precursors in which there is only one choice of splice sites, most of the exon sequences are not mechanistically involved in the splicing process.

Animals↗
PubMed1987

Cloning vectors for expression of cDNA libraries in mammalian cells.

We have constructed a series of compound cloning vectors (lambda ZD vectors), each consisting of phage lambda arms carrying a modified version of the retroviral expression vector pZIP-neoSV (x)1. cDNA, inserted into a cloning site present in the retroviral vector component, is cloned with high efficiency using the lambda system. A cDNA library in plasmids is then released by homologous recombination between the retroviral long terminal repeats. Retroviral transduction is achieved by transient expression of the released library in a cell line containing a packaging mutant of Moloney murine leukemia virus, followed by cocultivation of these producers with recipient cells. Transcription of cDNAs in the recipient cells is driven by the strong long terminal repeat promoter, and the transcripts, even from truncated cDNAs, can be expressed because translational start sites have been provided in all three reading frames (tri-initiator). Sequences conferring a recognizable phenotype can be rescued by cell fusion. The functionality of the tri-initiator and the rescue of a rare cDNA have been successfully tested using model systems.

Animals↗
PubMed1987

Pre-mRNA splicing and the nuclear matrix.

We examined the relationship between pre-mRNA splicing and the nuclear matrix by using an in vivo system that we have developed. Plasmids containing the inducible herpesvirus tk gene promoter linked to an intron-containing segment of the rabbit beta-globin gene were transfected into HeLa cells, and then the promoter was transactivated by infection with a TK- virus. Northern analysis revealed that the globin pre-mRNA and all its splicing intermediates and products are associated with the nuclear matrix prepared from such transfected cells. When the nuclear matrix was incubated with a HeLa cell in vitro splicing extract in the presence of ATP, the amount of matrix-associated precursor progressively decreased without a temporal lag in the reaction, with a corresponding increase in free intron lariat. Thus, most of the events of the splicing process (endonucleolytic cuts and branching) occur in this in vitro complementation reaction. However, ligation of exons cannot be monitored in this system because of the abundance of preexisting mature mRNA. Since the matrix is not a self-splicing entity, whereas the in vitro splicing system cannot process efficiently deproteinized matrix RNA, we conclude from our in vitro complementation results (which can be reproduced by using micrococcal nuclease-treated splicing extract) that the nuclear matrix preparation retains parts of preassembled ribonucleoprotein complexes that have the potential to function when supplemented with soluble factors (presumably other than most of the small nuclear ribonucleoproteins known to participate in splicing) present in the HeLa cell extract.

Animals↗
PubMed1986

Rat insulin-like growth factor II gene. A single gene with two promoters expressing a multitranscript family.

We have characterized the single-copy rat gene encoding the protein precursor of insulin-like growth factor II (pre-pro-rIGF-II) that is located downstream from and in the same transcriptional orientation as the homologous insulin II gene (5'-insulin-IGF-II-3'). This gene consists of at least three coding exons and utilizes two promoters that generate alternate 5' non-coding exons. Multiple transcripts from both promoters appear primarily in fetal or neonatal tissues (in all of the developmental stages and tissues that we have examined), but they are extremely rare or undetectable in adult tissues, with the exception of the brain and the spinal cord. These transcripts, which exhibit characteristic developmental profiles in various tissues, differ both in the presence of one of the alternate 5' non-coding exons and in the length of their fourth exon. The possible occurrence of differential splicing or differential polyadenylation (or both) in this region is discussed.

Animals↗
PubMed1986

Sequence-dependent S1 nuclease hypersensitivity of a heteronomous DNA duplex.

Using cloned (dG-dA)n X (dC-dT)n DNA duplexes [GA)n) as models of homopurine-homopyrimidine S1-hypersensitive sites, we show that cleavage of the alternate (non-B, non-Z) DNA structure by S1 nuclease is length-dependent, in both supercoiled and linear forms, which are similar because of the identity of their nicking profiles. However, the length of flanking sequences, the presence of borders, and the DNA topology affect the equilibrium between the alternate structure and B-DNA. The B form of (GA)38 has a 10.4-base pair helical repeat, but the two phosphodiester backbones have different conformations (heteronomous DNA with a dinucleotide repeat unit). Extension experiments reveal that the alternate structure is also heteronomous, in agreement with the nicking patterns generated by S1 and mung bean nucleases and by venom phosphodiesterase. Sensitivity to the latter enzyme at pH 9.0 indicates that the alternate DNA does not appear only in the low pH of the S1 nuclease reaction. Moreover, Hoogsteen G-CH+ base-pairing does not seem to be a prerequisite for the appearance of sensitivity because S1 still recognizes the structure even when all Gs are methylated at N-7. This is consistent with the results of chemical probing of the structure using dimethyl sulfate and diethyl pyrocarbonate at various pH values, which show absence of protection at guanine N-7. However, diethyl pyrocarbonate treatment at low pH results in hyper-reactivity of A residues.

Base Sequence↗
PubMed1985

Developmental and tissue-specific expression of a family of transcripts related to rat insulin-like growth factor II mRNA.

We have constructed a cDNA library from the mRNA of a rat liver cell line (BRL-3A) and characterized cDNA clones encoding the protein precursor of the rat insulin-like growth factor II (pre-pro-rIGF-II). This precursor, inferred from the nucleotide sequence, consists of a signal peptide, the rIGF-II sequence, and a trailer polypeptide of unknown significance. The characterized cDNA sequence (1016 nt) is part of a 3.4 kb mRNA species. Northern analysis reveals that a probe containing the extreme 5' noncoding region hybridizes to a second RNA (1.6 kb), while a probe corresponding to the 5' noncoding region proximal to the coding region hybridizes to two other RNA species (1.75 and 1.1 kb). All four RNAs are differentially expressed in all of the neonatal tissues that were examined, while the 3.4 kb pre-pro-rIGF-II mRNA and the 1.1 kb transcript are absent from adult tissues.

Amino Acid Sequence↗
PubMed1985

Rat LINE1: the origin and evolution of a family of long interspersed middle repetitive DNA elements.

We present approximately 7.0 kb of composite DNA sequence of a long interspersed middle repetitive element (LINE1) present in high copy number in the rat genome. The family of these repeats, which includes transcribing members, is the rat homologue of the mouse MIF-Bam-R and human Kpn I LINEs. Sequence alignments between specimens from these three species define the length of a putative unidentified open reading frame, and document extensive recombination events that, in conjunction with retroposition, have generated this large family of pseudogenes and pseudogene fragments. Comparative mapping of truncated elements indicates that a specific endonucleolytic activity might be involved in illegitimate (nonhomologous) recombination events. Sequence divergence analyses provide insights into the origin and molecular evolution of these elements.

Amino Acid Sequence↗
PubMed1985

"Transactivation" control signals in the promoter of the herpesvirus thymidine kinase gene.

The herpes simplex virus thymidine kinase (TK) gene is transcriptionally activated in trans ("transactivated") by virus-encoded proteins during the infectious cycle. We show that TK plasmids introduced into a HeLa cell transient transcription assay system are also transactivated after infection with a TK- virus. Several aspects of this response are similar to regulation during the normal infectious cycle. Assay of TK promoter deletion and 5- to 10-base-pair substitution mutants in this system reveals that the transactivation response depends on the intactness of 109 base pairs of 5' gene flanking sequence. Differences between these results and analogous assays in the Xenopus oocyte system are discussed. A model for the putative binding of transactivator(s) to the promoter region is presented.

Animals↗
PubMed1985

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

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.

Animals↗
PubMed1984

S1-hypersensitive sites in eukaryotic promoter regions.

We have examined by fine mapping the S1 nuclease-hypersensitivity of the 5' flanking regions of the human beta-globin and rat preproinsulin II genes and of the SV40 origin/enhancer region. In all cases S1-hypersensitive sites are located in known or presumed promoter/regulatory regions. Though a consensus DNA sequence is not evident, all of these sites reside in predominantly homopurine-homopyrimidine stretches. The alternate (non-B) DNA structure which is revealed by the enzymatic probe is a sequence-dependent feature of a short stretch of DNA, which is retained upon transplantation into a foreign environment. The alternate structure exhibits S1-nicking patterns uniquely different from those associated with the presence of Z-DNA.

Animals↗
PubMed1984

Possible structures of homopurine-homopyrimidine S1-hypersensitive sites.

S1 nuclease hypersensitive sites in the 5' flanking regions of eukaryotic genes, present in small artificial supercoiled DNA circles, reside in homopurine-homopyrimidine stretches. The hierarchical behavior which these sites exhibit is consistent with the notion that they act as sinks of torsional free energy. By employing DMS as a single-strand-specific reagent, we show that these sites (despite their S1 sensitivity) are regions of duplex DNA. A simple thermodynamic treatment indicates that the high torsional stress in the small DNA circles is almost certain to be relieved by the formation of alternate DNA structures. The same treatment places some constraints on the types and sizes of the regions with alternate conformation. While no definitive structural conclusions can be drawn, left-handed helices seem most consistent with the extent and the pattern of sensitivity to S1 nuclease.

Base Sequence↗
PubMed1984

In vivo splicing products of the rabbit beta-globin pre-mRNA.

Analysis of the rabbit beta-globin transcripts present in the steady-state RNA population of fetal liver reveals that there is no strict order in the removal of the two introns from the pre-mRNA, though IVS1 seems to be preferentially eliminated first. The population of RNA molecules contains, among other splicing products, two forms of full-length IVS2 (and presumably of IVS1), released from exon sequences. One of these forms is linear, while the other has a lariat structure (circle with tail), which results from looping of the intron sequence to itself and formation of a covalent bond between the 5' end and an internal IVS2 position at a distance of 31 bases from the 3' splice site. This branching point maps at the penultimate position of the heptamer TGCTAAC. Branched Y-forms of IVS2, presumably resulting from nicking of the lariat loop (in vivo or during purification) are also detected. Comparison of in vivo RNA with in vitro splicing products reveals corresponding lariat forms. However, the in vivo linear intron species are absent from the in vitro products, which, in contrast, contain more than one intron conformer.

Animals↗
PubMed1984

Cell-specified expression of a selectable hybrid gene.

We have inserted into a retroviral vector the structural information encoding the selectable bacterial genes neo and gpt linked, respectively, to the promoters of the herpesvirus thymidine kinase gene and the rat preproinsulin II gene. We have used this recombinant retrovirus construction to transduce insulinoma cell lines that are positive or negative for insulin expression. Selection of the transductants for neo yielded resistant colonies from all of the cell lines, while selection for gpt yielded resistant transductants in relatively high frequency only from insulin-producing cells. The 5' end of the gpt transcripts maps to the authentic preproinsulin capping site of the construction.

Animals↗