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S Zeitlin

Publications and source records attributed to S Zeitlin.

31 records · Page 2Linked 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↗
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

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↗
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

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

Guinea pig preproinsulin gene: an evolutionary compromise?

We characterized a clone carrying the guinea pig preproinsulin gene, which, in contrast to other mammalian preproinsulin genes, is highly divergent in its regions encoding the B and A chains of mature insulin. Blot hybridization analysis indicates that this gene is present in only one copy in the guinea pig genome and that other normal or mutated preproinsulin genes do not exist in this animal. Moreover, the position of introns in this gene and the homology of its 3' flanking region to the corresponding regions of other sequenced mammalian genes show that it has been derived from the common mammalian stock. The rapid evolution of the region encoding the B and A chains can be interpreted, according to our sequence-divergence analysis, as due to the fixation of both neutral and adaptive mutations.

Amino Acid Sequence↗
PubMed1980

Assessing coping behavior.

The Coping Inventory, an observation instrument based on Lois Murphy's longitudinal study of children's coping behavior, is described. The instrument assesses 48 kinds of coping behavior in order to develop a profile for use in educational and therapeutic planning. Examples and supportive technical data are given.

Adaptation, Psychological↗