Polyadenylation size

Transfection and expression of the GFP protein decreased exponentially in irradiated samples, but at a slower rate than the loss of supercoiled forms. This resulted in a target size considerably smaller than the size of the DNA molecule, only about one-third the mass. The map of this plasmid (Fig. 4) shows that the segment of the plasmid directly involved in transfection activity (which includes the CMV promoter, GFP gene, SV40 intron and polyadenylation signal) has a calculated mass of 1067.880 kDa. 

Researchers identified hnRNP proteins by first exposing cultured cells to high-dose UV irradiation, which causes covalent cross-links to form between RNA bases and closely associated proteins. Chromatography of nuclear extracts from treated cells on an oligo-dT cellulose column, which binds RNAs with a poly(A) tail, was used to recover the proteins that had become cross-linked to nuclear polyadenylated RNA. Subsequent treatment of cell extracts from unlrradiated cells with monoclonal antibodies specific for the major proteins identified by this cross-linking technique revealed a complex set of abundant hnRNP proteins ranging in size from 34 to 120 kDa. 

The human IGF-II gene is located downstream of the insulin gene, on chromosome 11. There are nine exons in the 30 kb human gene, but only six exons in the mouse and rat genes. Multiple promoters and polyadenylation sites give rise to transcripts of various sizes, regulated in a tissue-selective manner. Mature IGF-II is a neutral protein of 67 amino acids. 

Chapters 5 and 6 describe the preparation of plant and animal RNA. Chapter 6 also gives details on the further purification of the mRNA (i.e., polyadenylated) fraction from the total RNA. Chapter 7 describes the procedures necessary to separate this RNA by gel electrophoresis, and to transfer the separated fragments onto a nylon membrane. Chapter 8 describes a protocol for producing RNA dot blots on a nylon membrane. The protocols described in these chapters are those necessary to produce membrane filters for determining levels of expression of endogenous, or newly introduced, genes and the sizes of gene transcripts. 

For in-vitro synthesis of the compartment-specific phosphorylase forms polyadenylated RNA (mRNA) was isolated from cotyledons of either developing or germinating seeds and was used to program a reticulocyte lysate. When varying amounts of a mRNA preparation were applied to the translation mixture a decrease of the rate of total protein synthesis was observed at higher mRNA concentrations (Fig. 1). Although the mRNA concentration applied did not affect the size distribution of the translation products, for each mRNA preparation the optimal concentration was determined empirically. 

When in-vitro translation was performed using polyadenylated RNA from developing pea seeds the plastidic phosphorylase form (in its high molecular weight state) was the predominantly translated phosphorylase isozyme whereas the cytosolic counterpart was recovered as a very weak band of radioactivity (data not shown). Again, the apparent molecular weight of the labeled plastidic phosphorylase was lager by 11 kD than that of the purified enzyme form (Fig. 2C lane a). A complete conversion of the precursor to the size of the mature isozyme was achieved when the translation mixture (prior to precipitation with anti-plasti-dic phosphorylase IgG) was incubated with a stromal fraction (precipitate between 40-70% saturation of ammonium sulfate) of isolated pea chloroplasts (Fig. 2C lane b 50 pi stromal fraction per translation mixture, lane c 100 pi stromal fraction). The identity of the precursor was further confirmed by competition experiments (data not shown). 

A small plaque (S2) mutant of VSV, which is more efficient in inhibition of host cell protein synthesis than wild-type virus (Wertz and Youngner, 1970 1972) has been characterized (Davis and Wertz, 1980). This mutant makes two or three times the amount of viral mRNA in chick embryo cells than does wild-type virus, but synthesizes only about half as much viral protein. The mRNA transcribed by this mutant appears to be identical to wild-type mRNA in size, extent of polyadenylation at 3 termini, and extent of capping of 5 termini. It would appear, however, that the S2 mutant transcripts are associated with smaller polysomes than are the wild-type transcripts. Furthermore, this reduction in polysome size appears to be the result... 

Termination of in vivo polyadenylation or the in vitro reaction in nuclear extracts occurs over a defined range of lengths (e.g., 60-80 nt in yeast mRNAs, 200-300 nt in mammalian mRNAs) rather than at a precise number of nucleotides. What causes the termination of polyadenylation is not known. It is not an intrinsic property of poly(A) polymerase, although the in vitro reaction in the absence of nuclear extracts also results in a defined size distribution (5). 

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